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J Hepatol. Author manuscript; available in PMC 2021 July 01.
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Published in final edited form as:

J Hepatol. 2020 July ; 73(1): 149–160. doi:10.1016/j.jhep.2020.02.005.

Hepatic stellate cell activation promotes alcohol-induced

steatohepatitis through Igfbp3 and SerpinA12
Juan P. Arab1,2, Daniel Cabrera2,3, Tejasav S. Sehrawat1, Nidhi Jalan-Sakrikar1, Vikas K.
Verma1, Douglas Simonetto1, Sheng Cao1, Usman Yaqoob1, Jonathan Leon2, Mariela
Freire2, Jose I. Vargas2, Thiago M. De Assuncao1, Jung H. Kwon1, Yi Guo1, Enis Kostallari1,
Qing Cai5, Tatiana Kisseleva4, Youngman Oh5, Marco Arrese2, Robert C. Huebert1, Vijay H.
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Shah1
1Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
2Departamento de Gastroenterologia, Escuela de Medicina, Pontificia Universidad Catolica de
Chile, Santiago, Chile.
3Departamento de Ciencias Químicas y Biológicas, Universidad Bernardo O Higgins, Santiago,
Chile.
4Department of Surgery, University of California-San Diego, San Diego, CA, USA.
5Department of Pathology, Medical College of Virginia Campus, Virginia Commonwealth
University, Richmond, VA, USA.

Abstract
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Background/Aim: Steatohepatitis drives fibrogenesis in alcoholic liver disease. However, recent

studies have suggested that hepatic stellate cells (HSC) may regulate the parenchymal cell injury
and inflammation that precedes liver fibrosis, although the mechanism remains incompletely
defined. Neuropilin-1 (NRP-1) and synectin are membrane proteins implicated in HSC activation.
In this study, we disrupted NRP-1 and synectin as models to evaluate the role of HSC activation on
the development of steatohepatitis in response to alcohol feeding in mice.

Methods: Mice with HSC-selective deletion of NRP (ColCre/NRP-1loxP) or synectin (ColCre/

synectinloxP) vs. paired NRP-1loxP or synectinloxP mice were fed a control diet or the chronic/
binge alcohol feeding model. Several markers of steatosis and inflammation were evaluated.
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Corresponding Author: Vijay H. Shah, MD, Mayo Clinic, 200 First ST SW, Rochester, MN 55905, USA, Phone: (507) 255-6028/
Fax: (507) 255-6318, shah.vijay@mayo.edu.
Authors Contributions: JPA contributed study concept and design, acquisition of data, analysis and interpretation of data, statistical
analysis, and drafting of the manuscript. JIV, JL, MF, TMD, JHK, YG, EK, QC and YO contributed acquisition of data. UY
contributed acquisition of data and technical support. DC, TS, VKV, NJ-S, DS and SC contributed to acquisition and interpretation of
data. RCH, TK and MA contributed critical revision of the manuscript for important intellectual content. VHS contributed study
concept and design, critical revision of the manuscript for important intellectual content, and study supervision.
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Conflict of Interest: The authors have nothing to disclose.
 Arab et al. Page 2

Results: ColCre/NRP-1loxP mice showed less fibrosis, as expected, but also less inflammation
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and steatosis as assessed by Oil-Red O and Bodipy staining and hepatic triglyceride content.
Similar results were observed in the synectin model. Hepatocytes treated with supernatant of HSC
from ColCre/NRP-1loxP mice compared to supernatant from NRP-1loxP mice were protected
against ethanol-induced lipid droplet formation. An adipokine and inflammatory protein array
from the supernatant of HSC with NRP-1-knockdown showed a significant reduction in Igfbp3 (a
major insulin-like growth factor binding protein with multiple metabolic functions) and an
increase in SerpinA12 (a serine-protease inhibitor) secretion compared to wildtype HSC.
Recombinant Igfbp3 induced lipid droplets, triglyceride accumulation, and lipogenic genes in
hepatocytes in vitro, while SerpinA12 was protective against ethanol-induced steatosis. Finally,
Igfbp3 was increased, and SerpinA12 was decreased in serum and liver tissue from patients with
alcoholic hepatitis.

Conclusion: Selective deletion of NRP-1 from HSC attenuates alcohol-induced steatohepatitis

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through regulation of Igfbp3 and SerpinA12 signaling.

Graphical Abstract
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LAY SUMMARY
Hepatic stellate cells are known for their role in fibrosis (scarring of the liver). In this study, we
describe their role and mechanisms in the modulation of fat deposit and inflammation in the liver,
which occurs secondary to alcohol damage.

Keywords
alcohol; steatosis; steatohepatitis; neuropilin-1; Igfbp3; hepatic stellate cell; alcoholic liver
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disease; alcoholic hepatitis; insulin like growth factor binding protein 3; SerpinA12; vaspin; src-
kinase; integrin

INTRODUCTION
Alcohol-related liver disease (ALD) is a major cause of morbidity and mortality in the
United States [1, 2]. It encompasses a clinico-histologic spectrum of disease including fatty
liver, alcoholic hepatitis (AH), and alcoholic cirrhosis [3–5]. Steatosis is an early pathogenic

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lesion in the spectrum of ALD [6]. Steatosis is characterized by the accumulation of

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triglycerides, phospholipids, and cholesterol esters in hepatocytes. Alcohol consumption

regulates lipid metabolism by regulation of hepatic lipogenesis [7]. Understanding the
pathophysiological mechanisms occurring early in the disease process are needed to develop
new and innovative therapies to treat patients in the early stages of ALD [8, 9].

Hepatic stellate cells (HSC) are non-parenchymal cells of the liver with an established role
in fibrosis and tissue repair [10]. In response to steatohepatitis, quiescent HSC undergo
activation that drives fibrosis in the canonical injury to fibrosis pathway [11, 12]. However,
recent studies have suggested that HSC may also reciprocally regulate the parenchymal cell
injury and inflammation that precedes liver fibrosis [11]. This mechanism suggests
bidirectional hepatocyte-HSC crosstalk [13]. Thus, while the role of HSC in fibrogenesis is
well established, their contributions to hepatocyte lipid droplets and liver steatohepatitis are
not well understood.
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Neuropilin-1 (NRP-1) is a growth factor co-receptor implicated in HSC activation [14], and
its inhibition has been shown to ameliorate the recruitment and migration of HSC and block
liver fibrosis [14]. Therefore, we used mice with selective deletion of NRP-1 in HSC to test
the hypothesis that HSC activation through NRP-1 may regulate hepatocyte steatohepatitis
in response to alcohol feeding in mice. We also used an animal model with selective deletion
of synectin, as a second model of impaired HSC activation. Synectin, also known as GIPC,
is a scaffold protein, which links the transport of cell-surface receptors, including NRP-1, to
their intracellular signaling pathways. Like NRP-1, synectin has been implicated in HSC
activation [15]. Our results demonstrated that HSC are not only regulators of the fibrogenic
process but also mediate steatohepatitis in a model of ALD. Furthermore, we showed
specific mechanistic pathways involving insulin-like growth factor binding protein-3
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(Igfbp3, a major IGF-1 binding protein with multiple metabolic functions) and SerpinA12 (a
serine-protease inhibitor) by which HSC exert their lipogenic effects on hepatocytes.

MATERIALS AND METHODS

See the Supporting Methods for more details.

Generation of Col1Cre/NRP-1loxP mice

Neuropilin-1 floxed mice (NRP-1loxP) and synectin floxed mice (synectinloxP) mice [16, 17]
were crossed with ColCre mice [18, 19] to generate offspring with HSC selective deletion of
NRP-1 (ColCre/NRP-1loxP) or synectin (ColCre/synectinloxP). In all cases, control mice were
littermates carrying floxed genes (NRP-1loxP or synectinloxP) (Figure 1A–C and
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Supplementary Figure 1A–B). The efficiency of Cre recombinase in the ColCre mice was
assessed by crossing ColCre with Rosa26LacZ reporter mouse, where the LacZ gene follows
a STOP codon inserted between two loxP sites. In the ColCre/Rosa26LacZ offsprings, in Cre
+ cells, Cre recombinase deletes the STOP codon leading to the LacZ gene expression and

subsequent β-galactosidase protein expression. We then isolated hepatic stellate cells from
control or ColCre/Rosa26LacZ mice and performed X-gal assay to examine Cre efficiency
through β-galactosidase expression. HSC isolated from Cre− control mice contained no blue
cells (Supplementary Figure 1C). However, HSC isolated from ColCre/Rosa26LacZ mice

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were 74.24% blue, confirming a good recombination efficiency of the Cre (Supplementary
Figure 1D). Primary mouse hepatic stellate cells (mHSC) from ColCre/Rosa26LacZ mice
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were isolated, as described previously [20]. β-galactosidase (β-gal) staining was performed
as described previously [21]. Cells were washed with PBS and observed through an optical
microscope. β-gal staining was quantified using ImageJ.

Animal studies
All animal experiments were approved by IACUC, carried out in accordance with
institutional guidelines, and were conform to ARRIVE guidelines. Female C57BL/6J control
mice (NRP-1loxP or synectinloxP; n=6–9 each) and ColCre/NRP-1loxP or ColCre/synectinloxP
(n=6–9 each) mice were divided into four experimental groups receiving either the Lieber–
deCarli diet for 10 days followed by alcohol gavage (chronic/binge alcohol feeding model or
NIAAA model) [22] or control diet. Each group of animals were housed in transparent
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polycarbonate cages subjected to 12 h light/darkness cycles under a temperature of 21°C and

relative humidity of 50%. After ending the feeding course, mice were euthanized, and serum
and liver samples were collected and processed or stored at −80°C until analyses. Female
mice were used because they are more susceptible to alcohol-related liver injury [23].

Human Cells
Human primary HSC (ScienCell Research Laboratories, CA, USA) and HepG2 hepatocyte
cell line overexpressing ethanol-metabolizing enzyme cytochrome P450 2E1 (HepG2Cyp2E1)
were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum (FBS) and penicillin/streptomycin. Experiments were performed after
overnight serum starvation. Small interfering RNA (siRNA) for NRP-1 (SI00066787 and
SI02663206), integrin beta1, synectin, and SerpinA12 were purchased from Qiagen
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(Netherlands) or Thermo-Fisher (USA).

Biochemical determinations and compounds

Hepatic triglyceride content was assessed according to Carr et al. [24]. For the evaluation of
cellular triglyceride content, cells were grown in monolayer, resuspended in PBS with 0.5%
Triton X-100 and lysed with an ultrasound tip for evaluation with a commercial kit (Zen-bio,
USA), according to Beller M. (personal communication). Compounds used were murine or
human recombinant Igfbp3 (R&D systems, 0.3 μg/mL), Cytochalasin D (Sigma-Aldrich,
1:1000), PP2 (Sigma-Aldrich, 10 uM), recombinant SerpinA12 (R&Dsystems, 100 ng/mL),
Akt inhibitor (Tocris, Akti-1/2, 250 nM), PQ-401 (Tocris, 10uM) and AMPK inhibitor
(Tocris, Dorsomorphin dihydrochloride, 20 uM).
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Inflammatory proteins and adipokine arrays

Supernatant and lysate from control or NRP-1 knockdown (KD) human HSC samples were
subjected to human cytokines and adipokines antibody-based array (ARY024; R&D
Systems, USA) according to manufacturer’s instructions and quantitated using ImageJ-
software (NIH).

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Statistical analysis
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Results are expressed as the mean ± SD from three or more independent experiments. Two-
tailed Student’s t-test or ANOVA was used to test the statistical significance between groups
as appropriate. A P-value of less than 0.05 was considered as statistically significant.

RESULTS
Impaired activation of HSC by selective deletion of NRP-1 reduces EtOH-induced fibrosis,
inflammation and steatosis in vivo.
HSC are established key regulators in hepatic fibrogenesis [25]. We previously showed that
NRP-1 is implicated in HSC activation and its blockage by a neutralizing antibody
ameliorated liver fibrosis in mice [14]. In this study, we used a genetic model of impaired
HSC activation by selectively deleting NRP-1 from HSC (Figure 1A–C and Supplementary
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Figure 1) to test whether impaired HSC activation could affect steatohepatitis in a model of
alcohol-induced liver disease. To validate the reduction in fibrosis in this model, we
performed Sirius Red staining from liver tissue sections. As expected, fibrosis was lower in
ColCre/NRP-1loxP mice fed with alcohol compared with the alcohol-fed control mice
(Supplementary Figure 2A). Also supporting this finding, the evaluation of fibrosis markers,
α-smooth muscle actin (α-SMA), collagen type I alpha 1 (Col1α1), and tissue inhibitor of
metalloproteinases-1 (TIMP-1) mRNA levels were lower in ColCre/NRP-1loxP mice fed with
alcohol compared with the alcohol-fed control mice (Supplementary Figure 2B–D). Based
on the hypothesis that HSC may regulate steatosis and inflammation that precedes liver
fibrosis, we determined if selective deletion of NRP-1 in HSC could protect against steatosis
and inflammation in response to alcohol feeding. Control mice fed with the chronic/binge
alcohol model developed significant steatosis compared with those fed with a control diet, as
observed by Oil-Red O staining, while ColCre/NRP-1loxP mice were protected against
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alcohol-induced steatosis (Figure 1D). These observations were consistent with a 50%
reduction in hepatic triglyceride content after alcohol feeding in ColCre/NRP-1loxP mice
compared to controls (p<0.05) (Figure 1E). Furthermore, the inflammatory markers tumor
necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and monocyte chemoattractant protein-1
(MCP-1) mRNA levels increased in response to alcohol feeding in control mice but not in
ColCre/NRP-1loxP mice (Figure 1F–H). Concordantly, CD68 staining from liver tissue
sections showed an increase in macrophage infiltration in response to alcohol in NRP-1loxP
mice, but not in the ColCre/NRP-1loxP mice (Figure 1I). More specifically, macrophage
markers CD68 and F4/80 increased with alcohol-feeding in NRP-1loxP mice, but not in the
ColCre/NRP-1loxP mice (Supplemental Figure 2F–G). Additionally, we assessed
inflammatory foci quantification with consistent results (Figure 1J). We validated these
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results in a second model of impaired HSC activation, produced by selective deletion of

synectin in HSC (ColCre/synectinloxP) obtaining similar results (Supplementary Figure 3A–
B). Thus, these approaches indicate that impairing the activation of HSC can protect against
alcohol-induced fibrosis, steatosis, and inflammation in mice models.

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HSC supernatant from ColCre/NRP-1loxP mice decreases lipid droplet formation in

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hepatocytes exposed to ethanol in vitro.

Based on the observation that selective deletion of NRP-1 from HSC protects from alcohol-
induced steatosis, we hypothesized that HSC could produce a NRP-1-dependent paracrine
signal to regulate hepatocyte steatosis. To test this hypothesis, we isolated and cultured HSC
from ColCre/NRP-1loxP and NRP-1loxP mice. The cell supernatant was collected and used to
treat primary murine hepatocytes in combination with different concentrations of EtOH (25
and 50 mM). Hepatocytes treated with HSC supernatant isolated from ColCre/NRP-1loxP
mice had fewer lipid droplets as compared to NRP-1loxP mice, as shown by quantitative
BODIPY staining (Figure 2A). We next used siRNA-mediated approaches to test our
hypothesis in human cells further. We performed indirect co-culture using a Transwell
device. We plated hepatocytes on the bottom plate and HSC on the insert (top). Previously,
we inhibited or not the expression of NRP-1 in HSC by using specifics siRNAs. Then, we
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exposed them to alcohol and assessed lipid droplets by Bodipy staining. Hepatocytes co-
cultured with NRP-1 siRNA HSC developed fewer lipid droplets (7.7-fold decrease in
Bodipy staining intensity comparing siRNA control vs. siRNA for NRP-1) (Figure 2B).
Next, we isolated and cultured primary human HSC to obtain supernatant from control HSC
and with NRP-1 KD. Treatment of HepG2Cyp2E1 cells with supernatant from NRP-1 KD
HSC decreased lipid droplet formation in response to ethanol (Figure 3A). In summary,
these experiments indicate that one or more soluble molecules secreted by HSC modulate
the development of steatosis in hepatocytes and support a bi-directional model of HSC-
hepatocyte crosstalk.

Insulin-like growth factor-binding protein 3 (Igfbp3) expression is decreased and

SerpinA12 is increased in supernatant and cell lysate from NRP-1 KD HSC.
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To evaluate possible mechanisms by which NRP-1 KD could influence the steatotic process,
we performed an adipokine and inflammatory protein array (Supplementary Figure 4). This
array showed a 4.4-fold reduction in Igfbp3 expression and a 2.4-fold increase of SerpinA12
from supernatant from NRP-1 KD HSC compared to WT HSC (Figure 3B). We confirmed
this finding by qPCR analyses for Igfbp3 and SerpinA12 from cells extracts and Western
Blotting (WB) for Igfbp3 in the conditioned medium. (Figure 3C–D). These experiments
confirmed that knockdown of NRP-1 induces Igfbp3 downregulation with a concomitant
SerpinA12 upregulation.

Igfbp3 is the main insulin-like growth factor (IGF) binding protein found in human serum
and has multiple metabolic functions either dependent upon, or independent from IGF [26].
Meanwhile, SerpinA12 is a serine-protease inhibitor which has been shown to modulate
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insulin resistance and endoplasmic reticulum stress [27]. Since these metabolism-related
molecules were the top positively and negatively regulated hits from the protein array, we
mechanistically tested their role in the development of steatosis in our model.

Recombinant Igfbp3 increases lipid droplet formation, triglyceride content, and lipogenic
gene expression in hepatocytes in vitro.
To obtain further mechanistic insight regarding Igfbp3, we returned to our primary murine
hepatocyte model. The use of murine recombinant Igfbp3 in primary mouse hepatocytes

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increased lipid droplet formation, similar to that observed with EtOH, with a synergistic
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effect at low doses of EtOH (10 mM and 25 mM), as assessed by quantitative BODIPY
staining (Figure 4A). We next confirmed these results using a biochemical assay for cellular
triglyceride content. Cultured hepatocytes from wildtype (WT) mice were treated with 50
mM EtOH, Igfbp3 alone, EtOH plus Igfbp3, or vehicle. Hepatocytes treated with Igfbp3 had
an average of 104.3 ± 10.4 μg of triglyceride/mg of protein (p<0.05 compared to controls),
similar to hepatocytes treated with EtOH alone and to those treated with Igfbp3 plus EtOH
(Figure 4B). In summary, these experiments showed that recombinant Igfbp3 increases lipid
droplet formation and cellular triglyceride content in primary mouse hepatocytes.

Since Igfbp3 stimulates lipid droplets formation histologically, we next sought to evaluate
the effect of direct administration of recombinant Igfbp3 into in vitro primary mouse
hepatocytes to further assess canonical genes implicated in triglycerides biosynthesis [28].
Treatment of primary mouse hepatocytes with murine recombinant Igfbp3 increased
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expression of key genes for hepatic lipid homeostasis. Sterol regulatory element-binding
protein 1c (SREBP-1c), a key transcription factor in sterol and fatty acid biosynthesis,
showed a 4.2 ± 0.5 fold-increase in mRNA expression compared to control. We observed
similar results with other key genes associated with triglyceride synthesis (Figure 4C)
including acetyl-CoA carboxylase (ACC, 2.7 ± 0.5 fold), fatty acid synthase (FASN, 2.1 ±
0.2 fold), stearoyl-CoA desaturase 1 (SCD1, 6.4 ± 1.6 fold-increase), and diglyceride
acyltransferase 1 (DGAT1, 2.6 ± 0.2 fold-increase). These findings were consistent with our
evaluation of SREBP1-c and FAS protein levels by WB in response to Igfbp3 (Figure 4D).
Similar to primary mouse hepatocytes, treatment of HepG2Cyp2E1 cells with human
recombinant-Igfbp3 increased expression of key genes implicated in de novo hepatic
lipogenesis. SREBP-1c showed a 1.5 ± 0.2 fold-increase compared to control mRNA (Figure
4E). We observed similar results with ACC (1.9 ± 0.5 fold), FASN (1.4 ± 0.1 fold), SCD1
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(1.8 ± 0.4 fold), and DGAT1 (1.6 ± 0.2 fold). Consistently, SREBP1-c protein levels by WB
showed a reduction in hepatocytes treated with supernatant from HSC with siRNA mediated
KD of NRP-1 (Figure 4F). Additionally, we assessed genes implicated in fatty acid
oxidation observing a reduction in the enzymes carnitine-palmitoyl-transferase 1 (CPT1)
and the chain length-specific acyl-CoA dehydrogenases (long-chain [LCAD] and medium-
chain acyl-CoA dehydrogenase [MCAD]) by qPCR in hepatocytes treated with supernatant
from NRP-1 KD HSC (Supplementary Figure 5A). To assess reversal of protection against
alcohol-induced steatohepatitis by NRP-1 siRNA-mediated KD in HSC, we treated
hepatocytes with conditioned medium and alcohol in the presence or absence of recombinant
Igfbp3. Conditioned medium from NRP-1 KD HSC protected hepatocytes from alcohol
injury, and Igfbp3 abolished this protection (Figure 4G). Together, these data suggest that
Igfbp3 increases steatosis in hepatocytes by activation of key lipogenic genes and facilitates
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the accumulation of triglycerides.

Igfbp3 increases p-Akt through Src-kinase signaling in primary hepatocytes.

To evaluate how Igfbp3 exerts its pro-steatotic effects, we aimed to explore intracellular
pathways related to hepatic lipogenesis and growth (Akt, Erk, and IGF). We found that the
phosphorylated form of Akt (p-Akt) was induced 6.2-fold compared to control by the
administration of murine recombinant Igfbp3 in vitro in primary hepatocytes (p<0.05)

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(Figure 5A). As a specificity control, no significant changes were seen in the

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phosphorylation of the insulin-like growth factor 1 (IGF-1) receptor (IGFR) or the

extracellular signal-regulated kinase, Erk (Figure 5A). Additionally, we observed an
upregulation of lipogenic proteins (SREBP-1c [3.9 ± 0.4-fold comparing control vs. Igfbp3
+ PQ-401; p=0.0007] and FAS [6.3 ± 1.5-fold comparing control vs. Igfbp3 + PQ-401;
p=0.009]) in the presence of Igfbp3 regardless of pharmacologic IGF-1R inhibition
(PQ-401) (Supplemental Figure 6A). In order to determine whether Igfbp3-induced
lipogenesis depends upon its interaction with IGF1, we transduced primary mouse
hepatocytes with either wildtype (WT)-Igfbp3 adenovirus or adenovirus containing a
construct of an Igfbp3-GGG mutant protein (I56G, L80G and L81G, mutant IGF binding
sites), which expresses full-length Igfbp3 but has no binding affinity to IGF1 nor IGF1R.
Either Igfbp3 or Igfbp3 mutant construct induced key lipogenic genes expression (Figure
5B).
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The effect of Igfbp3 on Akt phosphorylation was blunted by the use of a specific Akt
inhibitor (Akti-1/2) (Figure 5C). Since Igfbp3 has been reported to signal in target cells
through integrin receptors [29], we tested p-Akt signaling in response to Igfbp3 in the
presence of a direct integrin receptor inhibitor (RGD peptide) and downstream inhibitors of
integrin receptors (Cytochalasin D, an inhibitor of actin polymerization; and PP2, a Src-
kinase inhibitor). Cytochalasin D, RGD peptide and PP2 blunted phosphorylation of Akt in
response to Igfbp3 (Figures 5D–F), while IGF1-R inhibitor (PQ-401) didn’t affect Akt
phosphorylation (Figure 5G), similar to the transfection with mutant Igfbp3-GGG protein
(Supplemental Figure 6C) suggesting that this effect is not mediated by IGF1. This effect on
p-AKT was correlated with an inhibition of the lipid droplet formation induced by Igfbp3, as
revealed by Bodipy staining (Figures 5H). Additionally, pretreatment of hepatocytes with
Cytochalasin D, Akt inhibitor, RGD peptide, and PP2 blunted the Igfbp3-mediated induction
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of key lipogenic genes (SREBP-1c, ACC, FASN, SCD1, DGAT1) by qPCR (Figure 5I) and
as evidenced by WB for SREBP-1c by RGD peptide, PP2 and an Akt inhibitor
(Supplemental Figures 6B and 7B). In total, these experiments indicate that Igfbp3 may
increase lipogenesis through integrin receptor and p-Akt signaling.

SerpinA12 treatment protects against ethanol-induced steatosis through the p-AMPK

pathway.
In contrast to Igfbp3, SerpinA12 was the most upregulated molecule in NRP-1 KD HSC
from the adipokine and inflammatory protein array (Figure 3B). To investigate the effect of
SerpinA12 on EtOH-induced steatosis, we treated primary mouse hepatocytes with murine
recombinant SerpinA12, with or without EtOH. As shown in Figure 6A, SerpinA12 was
protective against the lipid droplet formation induced by EtOH. With the aim to evaluate
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how SerpinA12 exerts its anti-steatotic effect, we assessed intracellular pathways related to
hepatic lipogenesis and growth. We found no differences in the phosphorylated forms of Akt
or Erk (Supplementary Figure 8B). Since the phosphorylated form of the 5’ adenosine
monophosphate-activated protein kinase (p-AMPK) has been shown to be reduced in the
presence of EtOH [30], we tested the protein levels of p-AMPK in response to EtOH with
and without SerpinA12. Indeed, EtOH decreased p-AMPK compared to control, but this
effect was lost in the presence of recombinant SerpinA12 (Figure 6B). Importantly, the

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effect of SerpinA12 on AMPK phosphorylation and the protection against EtOH-induced

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lipid droplet formation was blunted by the use of an AMPK inhibitor (Dorsomorphin
dihydrochloride) (Figure 6B–C). These observations correlated with reduced expression of
several key lipogenic genes (SREBP-1c, ACC, FASN, SCD1, DGAT1) as measured by
qPCR (Figure 6D). Concordantly, the siRNA-mediated knockdown of SerpinA12 in primary
human HSC followed by the use of this supernatant in HepG2Cyp2E1 cells in the presence of
alcohol correlated with increased expression of lipogenic genes compared to control (Figure
6E). In summary, these results indicate that SerpinA12 inhibits ethanol-induced steatosis in
hepatocytes and supports a model whereby Igfbp3 and SerpinA12 released by HSC have
opposing effects on hepatocyte steatosis.

Igfbp3 is increased and SerpinA12 is decreased in serum and liver tissue from patients
with alcoholic liver disease.
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To show disease relevance in humans, we evaluated the protein levels of Igfbp3 and
SerpinA12 from the serum of patients with alcoholic hepatitis (AH) and controls by WB.
Indeed, Igfbp3 expression was 5.5-fold higher in patients with AH compared with controls
(p<0.05), while SerpinA12 was 77% lower (p<0.05) (Figure 7A). Similar findings were
obtained by ELISA for Igfbp3 from NRP-1loxP and ColCre/NRP-1loxP mouse serum. We
observed an increase in Igfbp3 serum levels in NRP-1loxP alcohol-fed mice, which was
attenuated in ColCre/NRP-1loxP mice (Supplementary Figure 7C). Additionally, isolated
HSC from mice fed with control diet or ethanol showed consistent Igfbp3 and SerpinA12
mRNA expression levels (Supplementary Figure 7D). These data support our model,
suggesting that high Igfbp3 levels and low SerpinA12 levels correlate with human alcoholic
liver disease.
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DISCUSSION
The present study includes novel observations that advance our understanding of how HSC
interact with hepatocytes. These observations demonstrate, for the first time, that HSC
activation modulates EtOH-induced steatohepatitis. We find that the HSC-secreted
molecules, Igfbp3 and SerpinA12, mediate lipid droplet formation in hepatocytes. This
finding highlight novel signaling between HSC and hepatocytes. Mechanistically, we define
a previously unknown role for Igfbp3 in which it increases p-Akt through integrin receptor/
Src-kinase signaling in hepatocytes and promotes lipid droplet formation, triglyceride
content, and lipogenic gene expression. Furthermore, SerpinA12 treatment protects against
ethanol-induced steatosis through the p-AMPK pathway. Lastly, we show that Igfbp3 is
increased in serum from patients with alcoholic hepatitis while SerpinA12 is decreased,
demonstrating relevance to human ALD. Thus, these studies establish HSC as regulatory
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cells of the hepatocyte steatotic process in ALD, as summarized in Figure 7C.

HSC are activated during liver injury to orchestrate the fibrogenic response. However, HSC
not only play a role in fibrosis [31] but also, as suggested by our data, modulate
steatohepatitis [32]. Recently, it has been suggested that HSC produce cytokines and
chemokines that act as antigen-presenting cells [33]. It has also been shown that activated
HSC secrete IL-6 and TNFα, which further suppress the expression of HNF1α and SHP-1

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in hepatocytes and lead to more inflammation and fibrogenesis [34]. Thus, activated HSC
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may be considered as initiating cells that regulate crosstalk with hepatocytes rather than
simple target cells of liver injury in fibrogenesis.

Neuropilins (NRPs) are single-spanning transmembrane glycoproteins related to neuronal

development, angiogenesis, tumor growth, and progression [35, 36]. Additionally, NRPs
have shown to have a role in liver disease, through their actions as co-receptors for the
vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and
transforming growth factor-β (TGF-β) [37, 38]. NRPs are mainly expressed in HSC and
liver sinusoidal endothelial cells (LSECs) [39, 40], and are expressed in hepatocytes in
malignant conditions [41]. NRP-1, with its synectin binding partner, plays a crucial role in
fibronectin fibril assembly in tumor-associated myofibroblasts [42] and in the regulation of
PDGF and TGF-β signaling, which are involved in canonical pathways of HSC activation
and fibrosis [15]. Our data using both NRP-1 and synectin as models supports a role for
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HSC activation for driving the steatotic process through paracrine crosstalk with
hepatocytes.

Mechanistic data from our study suggests that HSC activation through NRP-1 exerts its
downstream effect through two molecules: Igfbp3 and SerpinA12. Igfbp3 is the main
insulin-like growth factor (IGF) binding protein found in human serum and has the potential
to either inhibit or enhance IGF actions in many cell types [26]. Our study highlights a novel
role for HSC-derived Igfbp3 directly promoting lipid droplet formation and de novo
lipogenesis and contributing to ethanol-induced steatosis. Moreover, we were able to show
that Igfbp3-induced steatosis, independent of IGF1, and p-Akt signaling can be blunted with
the inhibition of the integrin receptor/Src-kinase pathway, opening new possibilities for
therapeutic modulation. A recent study has pointed out HSC as the top source of Igfbp3 in
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the liver [43]. A decrease in plasma Igfbp3 levels has been shown in patients with
nonalcoholic fatty liver disease (NAFLD) [44], and lower levels have been associated with
poor outcomes in hospitalized patients with cirrhosis [45]. Another study has shown that
increased levels of Igfbp3 are a risk factor for steatosis and elevated ALT [46]. Globally, in
vivo data suggests that Igfbp3 is associated with hepatic insulin resistance and decreased
peripheral glucose sensitivity [47]. In summary, clinical data is mixed, and further studies
are needed to establish associations of Igfbp3 with steatosis and to clarify the biological
roles of Igfbp3 in glucose and lipid metabolism.

In our study, SerpinA12 was the most upregulated protein from the adipokine and
inflammatory protein array, comparing the supernatant from NRP-1 KD HSC and WT HSC.
Interestingly, we described a novel function for SerpinA12 as a protective factor for ethanol-
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induced steatosis through phosphorylation of AMPK. SerpinA12, also known as Vaspin, is a

serine protease inhibitor mainly produced by adipose tissue [48]. It has been previously
shown that SerpinA12 increases AMPK phosphorylation [27, 49]. The mechanism is not
completely clear, but it has been demonstrated that SerpinA12 acts in HepG2 cells by
binding to GRP78, a master regulator of the unfolded protein response [27]. SerpinA12 has
been shown to modulate insulin action by specifically inhibiting its target protease,
kallikrein-related peptidase 7 (KLK7) [50]. In murine models of NAFLD, a high-fat diet

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 Arab et al. Page 11

induces downregulation of SerpinA12 [51], and low SerpinA12 levels have been shown in
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patients with NASH [52].

Finally, our data from human serum samples showed that levels of Igfbp3 and SerpinA12
correlate with alcoholic hepatitis, suggesting that they could play a role in human alcohol-
induced liver disease. This is a very exciting finding because it provides insight into the
pathophysiology of ALD and opens the possibility for potential therapeutic targets aimed at
the aforementioned pathways. This finding has potential clinical relevance as it could be
speculated that increased levels of Igfbp3, as shown in patients with alcoholic hepatitis, may
serve as a marker of severity of the alcohol-induced liver injury, as has been proposed for
NAFLD [53, 54]. Also, modulation of serine protease inhibitor family, such as SerpinA12,
could be tested as a treatment option in in vivo models of ALD.

This study has limitations, most notably, the difficulty in proving causal-effect relationships
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of Igfbp3 and SerpinA12 in the attenuation of alcoholic steatohepatitis development. In

addition, Col-CRE deletion strategy could be non-specific for HSC. In conclusion, our data
support the concept that HSC are not only paracrine target cells of hepatocytes but also
regulate steatosis through the production of molecules that act on hepatocytes. Indeed,
activated HSC are able to modulate steatohepatitis through two key pathways that we
identified here, involving Igfbp3 and SerpinA12.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Financial Support Statement:

Author Manuscript

This work was supported by grant(s) NIH DK59615 and AA021171 (VHS), project FONDECYT #1200227 (JPA),
#1150357 & 1191145 (MA) and #11171001 (DC), the Clinical Core of the Mayo Clinic Center for Cell Signaling
in Gastroenterology (P30DK084567, Dr. L. Boardman). Arab JP was founded by an award from AASLD
Foundation (AASLD/LIFER Clinical and Translational Research Fellowship in Liver Diseases). Col1Cre mice were
kindly provided by Drs. Tatiana Kisseleva and David A. Brenner (UCSD), and mutant Igfbp3 by Dr. Youngman Oh
(VCU). The authors want to thank Mrs. Theresa (Terri) J. Johnson and M. Donna Felmlee Devine, M.S. for their
logistic support.

Abbreviations:
α-SMA α-smooth muscle actin

ACC acetyl-CoA carboxylase

AH alcoholic hepatitis
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ALD alcoholic liver disease

AMPK 5’ adenosine monophosphate-activated protein kinase

ASCL4 long-chain-fatty-acid-CoA ligase 4

Col1α1 Collagen type I alpha 1

DGAT1 diglyceride acyltransferase 1

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 Arab et al. Page 12

EtOH ethanol
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FASN fatty acid synthase

HepG2 hepatocellular carcinoma cells

HSC hepatic stellate cell

IGF-1 insulin-like growth factor 1

Igfbp3 Insulin-like growth factor-binding protein 3

IGFR insulin-like growth factor 1 receptor

IL-1β interleukin-1β

KD knockdown
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KO knockout

MCP-1 monocyte chemoattractant protein-1

NAFLD nonalcoholic fatty liver disease

NRP-1 neuropilin-1

NRP-1loxP Neuropilin-1 floxed mice

LSECs liver sinusoidal endothelial cells

PDGF-β platelet-derived growth factor-β

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RT-PCR reverse transcription PCR

SCD1 stearoil-CoA desaturase 1

siRNA small interfering RNA

SREBP-1c sterol regulatory element-binding transcription factor 1c

TGF-β transforming growth factor β

TIMP-1 tissue inhibitor of metalloproteinases-1

TNF-α tumor necrosis factor-α

VEGF vascular endothelial growth factor

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WT wildtype

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HIGHLIGHTS
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• HSC regulate hepatocyte steatosis that precedes liver fibrosis in ALD.

• HSC’s derived Igfbp3 & SerpinA12 mediate lipid droplet formation in

hepatocytes.

• These results are recapitulated in samples from patients with alcoholic

hepatitis.
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Fig. 1. Impaired HSC activation by selective NRP-1 deletion reduces EtOH-induced steatosis and
inflammation in vivo.
Panel (A), Hepatic stellate cells isolated from ColCre/NRP-1loxP mice showed the absence of
signal for NRP-1 by western blot compared to NRP-1loxP mice (n=3–6; p<0.05). Panels (B)
and (C), represent α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (Col1α1)
mRNA expression respectively, in response to Transforming Growth Factor-β (TGF-β) and
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Cre recombinase containing adenovirus (AdCRE) in NRP-1loxP and ColCre/NRP-1loxP mice

(n=3–6; p<0.05). (D) Alcohol fed ColCre/NRP-1loxP mice exhibit less steatosis compared to
matched NRP-1loxP mice as assayed by Oil-Red O staining (intensity quantification in the
adjacent panel; n=6–9; 5x magnification; p<0.0001; one-way ANOVA). (E) HSC selective
deletion of neuropilin-1 inhibits triglyceride accumulation in the liver in response to chronic/
binge alcohol feeding (n=6–9; p<0.05; one-way ANOVA). (F-H) ColCre/NRP-1loxP mice
exhibit lower expression of inflammatory markers in response to alcohol as evidenced by
reduced TNFα, IL1β, and MCP-1 levels (n=6–9; p<0.05; one-way ANOVA). (I) Alcohol fed

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ColCre/NRP-1loxP mouse livers exhibit less infiltrative macrophages (arrowheads) compared

to matched NRP-1loxP mice based on CD68 staining (intensity quantification adjacent to
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representative images; n=6–9; 20X magnification; area 1.3mm2, p<0.05, one-way ANOVA).
(J) Alcohol fed ColCre/NRP-1loxP mouse livers exhibit less number of inflammatory foci
(arrowhead) per field compared to matched NRP-1loxP mice (representative images; n=6–9;
10X magnification, p<0.05, one-way ANOVA).
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Fig. 2. Supernatant from HSC with selective deletion or knockdown of NRP-1 prevents lipid
droplets formation in primary hepatocytes treated with ethanol.
(A) Supernatant from HSC from NRP-1loxP and Col1Cre/NRP-1loxP mice prevent lipid
droplets formation in primary hepatocytes treated with ethanol. Representative images of
primary mouse hepatocytes treated with supernatant of HSC from ColCre/NRP-1loxP or
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NRP-1loxP and different concentrations of ethanol (EtOH) (0, 25 and 50mM). (n=5–9, 63x).
Quantification of lipid droplets by BODIPY® 493/503 intensity evaluation. (n=6–9,
*p<0.05, one-way ANOVA). Hepatocytes treated with supernatant of ColCre/NRP-1loxP HSC
are prevented from the development of lipid droplets in response to EtOH. (B) Supernatant
from HSC with knockdown of NRP-1 by siRNA prevents lipid droplets formation in
primary hepatocytes treated with ethanol. Representative images of hepatocytes treated with
supernatant from NRP-1 KD HSC and different concentrations of ethanol (EtOH) (0, 25 and
50mM). (n=5–9, 40x). Quantification of lipid droplets by BODIPY® 493/503 intensity
evaluation. (n=6–9, *p<0.05, one-way ANOVA).
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Fig. 3. NRP-1 knockdown in human-derived primary HSC increases Igfbp3 while decreases
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SerpinA12 levels.
(A) Representative BODIPY® staining images and quantification from HepG2Cyp2E1 cells
treated with (I) medium, (II) control HSC supernatant, (III) siRNA mediated NRP-1
knockdown HSC supernatant. (III) shows decreased lipid droplet formation in vitro (n=6–9,
63X, one-way ANOVA). (B) Top ten down/upregulated molecules from adipokine and
inflammatory protein array from NRP-1 KD HSC supernatant compared to controls.
IGFBP3 and SerpinA12 were the most significantly decreased and increased proteins
respectively (n=3, p<0.05, unpaired t-test). (C) Igfbp3 mRNA and protein expression in
NRP-1 KD HSC is lower than human WT HSC on qPCR and WB (n=6–9, p<0.05, unpaired
t-test). (D) SerpinA12 mRNA expression in NRP-1 KD HSC is higher than human WT HSC
(n=6–9, p<0.05, unpaired t-test).
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Fig. 4. Recombinant murine-Igfbp3 and ethanol (EtOH) increase lipid droplet formation,
triglycerides, and lipogenic gene expression in primary mouse hepatocytes.
(A) Representative images and BODIPY staining from hepatocytes treated with basal
medium ± EtOH in different concentrations ± murine recombinant Igfbp3 (BODIPY® +
DAPI staining, n=6, 63x, one-way ANOVA). Treatment of primary mouse hepatocytes with
murine recombinant Igfbp3 and ethanol significantly increases lipid droplets formation in
vitro. (B) Quantification of triglycerides. Cultured primary mouse hepatocytes were treated
with Igfbp3, EtOH 50mM, Igfbp3 plus EtOH, or vehicle. All treated groups showed higher
cellular triglyceride content compared to control (n=6, *p<0.05 compared to control, one
way ANOVA). (C) Expression of lipogenic genes in response to recombinant Igfbp3
administration in primary mouse hepatocytes. Igfbp3 administration induces key lipogenic
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genes involved in hepatic lipid homeostasis (SREBP-1c, ACC, FASN, SCD1, and DGAT1)
(n=6, *p<0.05 compared to control, one way ANOVA). (D) Protein expression of SREBP-1c
and FASN by WB in response to Igfbp3. Igfbp3 administration increases both protein
expression (n=6, *p<0.05 compared to control, unpaired t-test). (E) In vitro human
recombinant IGFBP3 treatment to human hepatocytes induces the main lipogenic genes
involved in triglyceride de novo synthesis viz. SREBP-1c, ACC, FASN, SCD1, DGAT1
(n=6–9, *p<0.05, one-way ANOVA). (F) Supernatant from siRNA-mediated NRP-1

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knockdown HSC reduces the protein expression of SREBP-1c by WB (n=6, *p<0.05,

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unpaired t-test). (G) In vitro reversal of protection against alcohol of NRP-1 siRNA-
mediated knockdown in HSC. Hepatocytes treated with supernatant and alcohol +/−
recombinant Igfbp3. Hepatocytes treated with supernatant from NRP-1 KD HSC developed
fewer lipid droplets, and this protective effect was reverted in the presence of Igfbp3 (n=6,
*p<0.05 compared to control siRNA + Igfbp3, one way ANOVA).
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Fig. 5. Igfbp3 increases p-Akt through integrin receptor/Src-kinase signaling in primary

hepatocytes.
(A) The phosphorylated form of Akt is induced by the administration of murine recombinant
Igfbp3 in vitro in primary hepatocytes (n=6, p<0.05, one way ANOVA). (B) Transfection
with either WT Igfbp3 and Igfbp3 GGG mutant adenovirus (I56G, L80G and L81G, mutant
IGF binding sites), which expresses full-length Igfbp3 but has no binding affinity to IGF1
and does not bind to IGF1R) induced key lipogenic genes expression (n=3, p<0.05, one way
ANOVA). (C) Igfbp3-induced phosphorylation of p-Akt is blunted by Akt inhibitor
pretreatment (triplicates, n=9, p<0.05, one way ANOVA). (D) Igfbp3-induced
phosphorylation of p-Akt is blunted by Cytochalasin D (Cyt D) pretreatment (n=6, p<0.05,
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one way ANOVA). (E) Igfbp3-induced phosphorylation of p-Akt is blunted by RGD peptide
pretreatment (n=6, p<0.05, one way ANOVA). (F) Igfbp3-induced phosphorylation of p-Akt
is blunted by PP2 pretreatment (duplicates, n=6, p<0.05, one way ANOVA). (G) Igfbp3-
induced phosphorylation of p-Akt is not affected by IGF-1R inhibitor (PQ401) pretreatment
(n=6, p<0.05, one way ANOVA). (H) Representative microscopy images of lipid droplets
and Bodipy staining quantification from primary mouse hepatocytes treated with vehicle or
Igfbp3 ± PP2 or Akt inhibitor. Pretreatment with PP2 or Akt inhibitor protects against lipid

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 Arab et al. Page 24

droplet formation induced by Igfbp3 (n=6, p<0.05, 63x, one way ANOVA). (I) mRNA
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expression of lipogenic genes by qPCR. Igfbp3 administration to in vitro primary mouse

hepatocytes induces key lipogenic genes involved in hepatic lipid homeostasis and this effect
is blunted by Cytochalasin D, Akt inhibitor, and PP2 (SREBP-1c, ACC, FASN, SCD1, and
DGAT1) (n=6, *p<0.05 compared to control/vehicle, #p<0.05 compared to Igfbp3 alone,
one way ANOVA).
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 Arab et al. Page 25
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Fig. 6. SerpinA12 treatment protects against ethanol-induced steatosis in primary mouse

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hepatocytes and correlates with the p-AMPK pathway.

(A) Representative images and BODIPY® staining quantification show pretreatment with
SerpinA12 protects against lipid droplet formation induced by EtOH (n=6, 63x, p<0.05, one
way ANOVA). (B) WB and densitometry quantification shows that EtOH induces a
reduction in the phosphorylation of AMPK compared to control. SerpinA12 induces
phosphorylation of AMPK, even in the presence of EtOH, and this effect is blunted by the
presence of AMPK inhibitor (duplicates, n=6, *p<0.05, one way ANOVA). (C)
Representative images and BODIPY staining quantification show protection of SerpinA12
against EtOH-induced lipid droplet formation on treatment with SerpinA12 ± EtOH. This
effect was blunted by using an AMPK inhibitor (n=6, 63x, *p<0.05, one way ANOVA). (D)
EtOH induces key lipogenic genes involved in hepatic lipid homeostasis, and this effect is
reduced by SerpinA12. The protective effect of SerpinA12 is attenuated by an AMPK
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inhibitor (n=6, *p<0.05 compared to control, #p<0.05 compared to EtOH alone, one way
ANOVA). (E) Human hepatocytes treated with supernatant from human HSC with a siRNA-
mediated knockdown of SerpinA12 ± EtOH. EtOH ± supernatant from siRNA-mediated
knockdown of SerpinA12 increases expression of SREBP-1c, FASN, and SCD1 compared
to EtOH in siControl cells (n=9, *p<0.05 compared to control, #p<0.05 compared to EtOH
alone, one way ANOVA).

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 Arab et al. Page 26
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Fig. 7. Igfbp3 is increased while SerpinA12 is decreased in serum and liver tissue from patients
with alcoholic liver disease.
(A) Representative WB and densitometry quantification for Igfbp3 and SerpinA12 from
plasma from control subjects and with alcoholic hepatitis. Subjects with alcoholic hepatitis
have significative higher levels of Igfbp3 and significative lower levels of SerpinA12
compared to controls (n=9, p<0.05, unpaired t-test). (B) Schematic representation of the
proposed mechanism of hepatic stellate cells (HSC)-activation-induced steatosis. Activated
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HSC increase Insulin-like growth factor-binding protein 3 (Igfbp3) and reduces SerpinA12
secretion, which have a paracrine effect over hepatocytes. Igfbp3 increases p-Akt signaling
through integrin receptor leading to lipid droplets formation, triglyceride content, and
lipogenic gene expression; EtOH reduces p-AMPK, increasing lipogenesis. SerpinA12
protects against ethanol-induced steatosis through increasing p-AMPK.
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J Hepatol. Author manuscript; available in PMC 2021 July 01.