The role of osteopontin in non-alcoholic fatty liver disease

Doctoral thesis at the Medical University of Vienna for obtaining the academic degree

Doctor of Philosophy

Submitted by

Alexander Daniel Nardo, MSc

Supervisor:

Univ.-Prof. Dr. Thomas Stulnig

Christian Doppler Laboratory for Cardio-Metabolic Immunotherapy and

Clinical Division of Endocrinology and Metabolism, Department of Medicine III, Medical University of Vienna,

Vienna, Austria

Vienna, 12/2020

II

TABLE OF CONTENTS

Declaration ......................................................................................................................................... III

Table of contents ................................................................................................................................ IV

Abstract (English version) .................................................................................................................... V

Abstract (Deutsche Version)............................................................................................................... VI

Publication ......................................................................................................................................... VII

List of abbreviations .......................................................................................................................... VII

Acknowledgments ............................................................................................................................ XIII

III

DECLARATION

The research work described in this doctoral thesis has been carried out at the research

laboratories of the Clinical Division of Endocrinology and Metabolism, Department of

Medicine III, Medical University of Vienna, where I performed sample processing, gene

expression analyses, protein and lipid enzymatic assays. The animal experiments have been

carried out at the animal facility of the Medical University of Vienna by me and Nicole

Gabriele Sommer. General care of animals have been taken by the animal care-takers of the

animal facility. In vivo imaging of liver tumors has been carried out at the Department of

Biomedical Imaging and Image-guided Therapy, Division of Nuclear Medicine, Medical

University of Vienna. The analyses have been performed by Monika Dumanic under

supervision of Thomas H. Helbich. The processing of murine tissue samples for

histopathological evaluation has been performed by me and Helga Schachner at the

Department of Pathology, Medical University of Vienna. The histopathological evaluation of

paraffin-embedded liver sections has been performed by Georg Oberhuber at the Depatment

of Pathology, Medical University of Innsbruck. Mass spectrometry assessments of the

hepatic lipid species have been carried out by Daniel F. Markgraf and Michael Roden at the

Laboratory of gas-chromatography, mass-spectrometry and hormone-analytics, Deutsches

Diabetes Zentrum (DDZ), Düsseldorf (Germany). Quantification of plasma parameters have

been performed by Elisa Rivelles at the Department of Laboratory Medicine, Medical

University of Vienna. The study protocol has been designed by me, Thomas M. Stulnig and

Maximilian Zeyda.

IV

TABLE OF CONTENTS

Introduction ............................................................................................................................................. 1

Obesity: definition and epidemiology ................................................................................................. 1

Evolution of energy-storing tissues and their relationship with obesity ........................................ 1

Quantification of adiposity as a prognostic factor for obesity-related pathologies ....................... 2

Epidemiology of obesity .................................................................................................................. 3

Obesity and the metabolic syndrome ................................................................................................. 4

Physiological metabolic and endocrine roles of adipose tissue ...................................................... 4

Molecular adipose tissue pathophysiology in obesity and metabolic syndrome ........................... 6

Role of the immune system in the metabolic syndrome ................................................................ 7

The liver as a metabolic organ ............................................................................................................ 8

Hepatic glucose metabolism ......................................................................................................... 11

Hepatic lipid metabolism ............................................................................................................... 14

Hepatic insulin resistance .............................................................................................................. 18

The liver pathological evolution in the metabolic syndrome: from Non-alcoholic fatty liver (NAFL)

to Hepatocellular Carcinoma (HCC) ................................................................................................... 22

Non-alcoholic fatty liver (NAFL) .................................................................................................... 24

Non-alcoholic steatohepatitis (NASH) ........................................................................................... 27

Hepatic fibrosis .............................................................................................................................. 32

NAFLD-associated hepatocellular carcinogenesis ......................................................................... 36

The ambiguous, yet unresolved, role of osteopontin in NAFLD ....................................................... 40

Aim of the doctoral thesis ................................................................................................................. 47

Results ................................................................................................................................................... 48

Prologue ............................................................................................................................................ 48

Publication ......................................................................................................................................... 48

Conclusions ............................................................................................................................................ 63

References ............................................................................................................................................. 71

V

ABSTRACT (ENGLISH VERSION)

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and, due to the

lack of effective therapies, a leading cause of death worldwide. The steady increase in HCC

incidence in the last decades is associated with the onset of obesity as a nutritional

pandemic. Obesity leads sequentially to type 2 diabetes mellitus, hepatic steatosis and non-

alcoholic steatohepatitis (NASH), liver fibrosis and finally to HCC. However, research on this

causal chain of events has been limited due to the lack of suitable animal models, and no

mechanistic links between NASH and HCC development have been established yet.

Osteopontin (OPN) is a molecule believed to play a role in the development of NASH as well

as in the development, growth and metastasis of HCC. In obesity, OPN is up-regulated

mainly in adipose tissue, but also in the liver, and induces inflammatory and insulin-

desensitizing processes that ultimately lead to type 2 diabetes mellitus, fatty liver and NASH.

OPN has also been described in numerous publications as a factor involved in the growth

and metastasis of HCC in animal models and as an important biomarker for HCC in humans.

In certain pre-clinical models, however, the deletion of OPN was found to be tumor-

promoting. Therefore, using a recently established NASH-HCC mouse model, which allows

to investigate the described liver pathological evolution from fatty liver to HCC in an acquired

obesogenic and diabetic background, we want to evaluate the role of OPN in the

development of NASH-derived HCC and its suitability as a therapeutic target for the

treatment or prevention of HCC originally caused by fatty liver.

VI

ABSTRACT (DEUTSCHE VERSION)

Das hepatozelluläre Karzinom (HCC) ist die häufigste Form von Leberkrebs und auf Grund

des Mangels an wirksamen Therapien auch eine häufige Todesursache, die zunehmend ist,

weil ernährungsbedingter Einfluss der zu Fettleibigkeit führt ebenso zunimmt. Fettleibigkeit

führt zu Typ 2 Diabetes Mellitus, Fettleber und nicht-alkoholischer Steatohepatitis (NASH)

zu Leberfibrose und letztendlich zu HCC. Die Beforschung dieser Kausalkette war durch das

Fehlen eines geeigneten Tiermodells bisher jedoch beschränkt, daher konnte bisher keine

Verbindung zwischen Mechanismen bei der Entstehung von NASH und HCC hergestellt

werden. Ein Molekül, das sowohl bei der Entstehung von NASH als auch bei Entstehung,

Wachstum und Metastasierung von HCC eine Rolle spielen dürfte ist Osteopontin (OPN). Bei

Fettleibigkeit wird OPN v.a. im Fettgewebe, aber auch in der Leber hochreguliert und

induziert inflammatorische und insulindesensitivierende Prozesse, die letztendlich zu Typ 2

Diabetes Mellitus, Fettleber und NASH führen. OPN wurde aber auch in einer Vielzahl an

Publikationen als an der Wachstum und Metastasierung von HCC beteiligter Faktor in

diversen Tiermodellen sowie als wichtiger Biomarker für HCC im Menschen beschrieben. In

bestimmten Modellen jedoch zeigte sich die Deletion von OPN tumorfördend. Daher wollen

wir mit Hilfe eines seit kurzem etabliertes NASH-HCC Mausmodels, mit dem sich die

beschrieben Kette, insbesondere der Weg von der Fettleber zu HCC in einem erworbenen

adipösen und diabetischen Hintergrund beobachten lässt, die Rolle von OPN in der

Entstehung und seine Tauglichkeit als therapeutisches Target zur Behandlung bzw.

Verhinderung von dem ursprünglich von der Fettleber verursachten HCC evaluieren.

VII

PUBLICATION

Nardo A.D. et al., “Impact of osteopontin on the development of non-alcoholic liver disease

and related hepatocellular carcinoma”. Liver International (PMID: 32281248)

LIST OF ABBREVIATIONS

ABCB11, ATP-binding cassette, sub-family B member 11

ACBP, acyl-CoA binding protein

ACC, acetyl-CoA carboxylase

AdipoR1, adiponectin receptor 1

AdipoR2, adiponectin receptor 2

AKT, proteinkinase B

AMP, adenosine monophosphate

AMPK, 5' AMP activated protein kinase

APOC3, apolipoprotein C3

APPL1, adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1

APPL2, Adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 2

ATG7, autophagy-related 7

AT, adipose tissue

ATP, adenosine triphosphate

Bax, Bcl-2-associated X protein

BDL, bile duct ligation

Bim, Bcl-2-like protein 11

BMI, body mass index

VIII

CCL2, C-C motif chemokine 2

CCl4, carbon tetrachloride

CD36, cluster of differentiation 36

CD44, cluster of differentiation 44

CER, ceramides

CHREBP, carbohydrate-responsive element-binding protein

CoA, coenzyme A

CPT, carnitine palmitoyltransferase

Cyp7a1, cytochrome P450 7A1

C/EBP, CCAAT-enhancer binding protein

DAA, direct antiviral agents

DAG, diacylglycerol

DAMP, damage-associated molecular patterns

DDR, DNA damage response

DIO, diet-induced obesity

DNA-PK, DNA-dependent protein kinase

dnl, de novo lipogenesis

DNP, 2,4-dinitrophenol

DXA, dual energy X-ray absorptiometry

ECM, extracellular matrix

ER, endoplasmic reticulum

FABP, fatty acid binding protein

FAK, focal adhesion kinase

IX

FATP, fatty acid transporter protein

FAS, fatty acid synthase

FFA, free fatty acids

FOXO, forkhead box

GCK, glucokinase

GLP-1, glucagon-like peptide 1

GLUT2, glucose transporter type 2

GLUT4, glucose transporter type 4

GPAT, glycerol-3-phosphate acyltransferase

GRP78, endoplasmic reticulum chaperone (BiP)

GWAS, genome-wide association studies

GWAT, gonadal white adipose tissue

GYS2, glycogen synthase 2

G6PC, glucose-6-phosphatase

HBV, hepatitis B virus

HCC, hepatocellular carcinoma

HCV, hepatitis C virus

HFD, high-fat diet

HGP, hepatic glucose production

Hh, hedgehog

HIF, hypoxia-inducible transcription factors

HMGB1, high-mobility group box-1

HNRNPA1, heterogeneous nuclear ribonucleoprotein A1

X

HSC, hepatic stellate cells

IL, interleukin

INSR, insulin receptor

IRS2, insulin receptor substrate 2

IRTK, insulin receptor tyrosine kinase

JAK2, Janus kinase 2

JNK, c-Jun N-terminal kinase

KC, Kupffer cells

KDa, KiloDalton

KO, knock-out

K18, keratin 18

K8, keratin 8

LpL, lipoprotein lipase

LOXL2, Lysyl oxidase-like protein-2

LXR, liver X receptor

MAPK, mitogen-activated protein kinase

MEK, Mitogen-activated protein kinase kinase

MMP, matrix metalloproteinase

MoM, monocyte-derived macrophage

NAD, Nicotinamide adenine dinucleotide

NADPH, Nicotinamide adenine dinucleotide phosphate

NAFL, non-alcoholic fatty liver

NAFLD, non-alcoholic fatty liver disease

XI

NASH, non-alcoholic steatohepatitis

NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells

NK, natural killer

OPN, osteopontin

PAI-I, plasminogen activator-inhibitor 1

PCa, human prostate carcinoma

PCK1, Phosphoenolpyruvate Carboxykinase 1

PDGF, Platelet-derived growth factor

PDK1, 3-phosphoinositide-dependent kinase-1

PERK, PKR-like endoplasmic reticulum kinase

PKC, protein kinase C

PI3K, phosphatidylinositol-3-OH kinase

PNPLA3, patatin-like phospholipase domain-containing protein 3

PPAR, peroxisome proliferator-activated receptor

PUFA, polyunsaturated fatty acids

RIPK3, receptor-interacting serine/threonine-protein kinase 3

ROS, reactive oxygen species

RXR, retinoid X receptor

SMAD3, mothers against decapentaplegic homologue 3

SREBP-1, sterol regulatory element-binding protein 1

Stat3, Signal transducer and activator of transcription 3

STZ, streptozotocin

TAM, tumor-associated macrophages

XII

TG, triacylglycerols

TGF-β, transforming growth factor beta

TLR, toll-like receptor

TNF-α, tumor necrosis factor α

TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling

T2DM, type-2 diabetes mellitus

uPA, urokinase; URI, unconventional prefolding RBP5 interaction

VLDL, very low-density lipoproteins

WT, wild-type

XBP1, X-box binding protein 1

YAP, Yes-associated protein

XIII

ACKNOWLEDGMENTS

I thank Thomas M. Stulnig and Maximililan Zeyda for their great support and guidance as

mentors of this doctoral thesis, and Nicole Gabriel Sommer and Dagmar Lehner for their

outstanding contribution during the animal experiments. I further thank all the collaborators

who helped me gaining more deep insights on the molecular cues of the observed

phenotypes. Especially, I want to thank Michael Trauner, Thierry Claudel, Nicole Auer and

Victoria Kunczer (Hans Popper Laboratory of Molecular Hepatology, Division of

Gastroenterology & Hepatology, Medical University of Vienna) fort their contribution in the

process of manuscript and data revision prior to publication of the original research article

presented in this thesis.

I also thank my parents, my grandparents and my sister, who have always been by my side

and supported me throughout my academic career. They have been, and still are, my life-

mentors, and there are no words to properly express my gratitude to them.

Finally, I thank my wife, who is a great scientist. She not only helped me becoming a better

researcher, but also a better person.

And of course, I thank science, which made my life so beautiful and made me meeting my

wife!

INTRODUCTION

Obesity: definition and epidemiology

Evolution of energy-storing tissues and their relationship with obesity

Every living organism needs a defined amount of energy to fulfill the series of actions

necessary to its survival: the more complex the organism is, the more basal energy is

required to maintain physiological homeostasis. While autotrophic organisms rely simply on

sunlight, heterotrophic beings have to absorb nutrients, such as protein, carbohydrates and

fats as sources of energy. However, while light is daily present, the sources of heterotrophic

nutrition may be available only intermittently. Therefore, metabolic strategies aimed at storing

energy and compensate for the periods of famine have been adapted already in unicellular

organisms and became more refined and multifunctional in the course of evolution (Kadereit

et al, 2008). Nevertheless, energy-storing processes remained quite conserved throughout

the evolution tree (Ottaviani et al, 2011), and this further highlights their physiological

importance. In mammals, adipose tissue is the perfect example of energy-storing organ,

which most probably developed to fulfill this specific need. Nowadays, this marvelous

physiological property became almost futile in humans, due to the unlimited availability of

food in developed regions. Since adipose tissue cannot evaluate environmental food

availability, it stores all the energy that is taken up by absorption of nutrients and not

disposed via basal energy consumption and physical activities. Under overfeeding condition,

the energy depots undergo physical expansion to compensate the unbalanced uptake (WHO

| Obesity and overweight, 2017).

Overweight and obesity are defined by a state of overall positive energy balance and as an

excessive accumulation of visceral adipose tissue, which might impair health (WHO | Obesity

and overweight, 2017). The human body consumes energy via physical activity, basal

metabolism in the resting state and thermic effect of food (i.e. energy necessary to

metabolize the calories ingested) (Hill et al, 2012). When more calories than those consumed

2

are taken in, the remaining energy will be stored. Chronic excess of food intake leads to the

establishment of a significant energy storage, which eventually brings to an increased body

weight, with a specific enlargement of the fat-mass component (Swinburn et al, 2011). In

parallel with overfeeding, the today´s lifestyle of high- and middle-income countries, driven by

urbanization, industrialization, digitalization and the need of comfort, becomes always more

sedentary (Ladabaum et al, 2014; Ng & Popkin, 2012). Hence, the energy balance turns into

a pathologic positive energy imbalance, in which overeating increases the caloric input, and

the physical inactivity reduces the output.

Quantification of adiposity as a prognostic factor for obesity-related pathologies

Precise quantification of adiposity is critical for an accurate epidemiological determination of

the prevalence of overweight and obesity in a defined population. In parallel with that, it might

serve also as a clinical tool for longitudinal follow ups on the efficacy of obesity treatments

(Ponti et al, 2019). Adiposity can be clinically measured in direct and indirect ways. Indirect

methods such as body mass index (BMI) and waist-to-hip ratio are still widely used (Aune et

al, 2016; Song et al, 2013). The calculation of BMI consists in dividing the weight by the

square of the height of the tested individual, and is expressed in kg/m2 (Nuttall, 2015). BMI is

defined as an objective evaluation, since its precision relies only on the accuracy of the

devices employed to measure weight and height (Zierle-Ghosh & Jan, 2018). Other indirect

measurements of adiposity, such as waist-to-hip ratio, are less preferred because of the

potentially associated inter-operator bias (Zierle-Ghosh & Jan, 2018). Over the last 15 years,

significant effort has been invested in the optimization of BMI cut-offs to increase its accuracy

as risk predictor of clinically relevant metabolic disorders in different geographical

populations (Nishida et al, 2004; Ding et al, 2016). However, the need of differential cut-off

values makes systematic evaluations more complex and less reliable. Furthermore, BMI

does not distinguish between lean and fat mass and is therefore i) not able to identify

sarcopenic obesity; ii) inadequate to follow up changes in fat mass and distribution upon

3

treatment of obesity. Therefore, direct adiposity quantification methods, such as bioelectrical

impedance and dual energy X-ray absorptiometry (DXA), which allow for specific and

sensitive measurement of both total body- and visceral fat, have been developed (Padwal et

al, 2016). DXA technology is built on the principle that tissues with different density will

differentially affect the energy of a X ray beam passing through them (Brownbill & Ilich,

2005), and is therefore able to separately quantify fat mass, bone mass and lean mass.

Limitations of DXA application are mainly the higher costs when compared with BMI

calculation, and the possible bias in the positioning of the patients during the densitometry

scan (Brownbill & Ilich, 2005).

In summary, due to cost limitations, BMI stays the most widely used method for the

quantification of obesity and overweight. Nevertheless, it is important to bear in mind the

limitations of this indirect measuring tool, which shall be adapted mainly for epidemiological

studies on obesity, while direct methods, such as DXA, shall be preferred in clinical studies

aimed at evaluating the effects of obesity interventions on fat mass and distribution.

Epidemiology of obesity

Based on the last epidemiological data from 2016, overweight and obesity are a real social

plague, with 39% of the worldwide population being overweight (i.e. BMI > 25), and a third of

it being obese (i.e. BMI > 30) (WHO | Obesity and overweight, 2017; Global Burden of

Disease Study 2015 (GBD 2015) Obesity and Overweight Prevalence 1980-2015 | GHDx),

which account for a 50% and 80% increase in overweight and obesity prevalence since

1980, respectively (Chooi et al, 2019). Surprisingly, obesity raises faster in low-to-middle-

income countries due to the globalized distribution of cheap, low quality, energy-dense food,

high in fat and sugars (WHO | Obesity and overweight, 2017). The most important data

highlighting the magnitude of the health burden these new habits are causing is that the

prevalence of overnutrition-related deaths overcame the rate of undernutrition-related

mortality worldwide. Especially in third-world countries, these two phenomena are

4

furthermore concomitant and lead to a proper nutritional pandemic (WHO | Obesity and

overweight, 2017).

Obesity and the metabolic syndrome

Already in the early 90´s, visceral obesity was identified as part of a cluster of metabolic

disorders, referred to as metabolic syndrome (American Heart Association et al; Alberti et al,

2009). The metabolic syndrome is defined as a pattern of metabolic risk factors concurring

in the pathologic establishment of type-2 diabetes mellitus (T2DM) and cardiovascular

complications (National Cholesterol Education Program (NCEP) Expert Panel on Detection,

Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III),

2002). The exponential increase in metabolic syndrome prevalence in the last decades

correlates with a similar increasing burden of obesity worldwide. Indeed, abdominal obesity is

the prevailing risk factor for the syndrome, together with whole-body and peripheral insulin

resistance (Reaven, 1988), and systemic low-grade inflammation (Hanley et al, 2004; Hu et

al, 2004). Clinical studies demonstrated that mainly upper-body obesity, and specifically

visceral white adipose tissue expansion correlates with increased metabolic and

cardiovascular risks (Carr et al, 2004).

Physiological metabolic and endocrine roles of adipose tissue

Under lean/healthy conditions, adipocytes exert their storage duty by taking up free fatty

acids (FFA) and triacylglycerols (TG) from the diet, but they also act as endocrine cells

releasing adipokines, the most known being adiponectin and leptin. Plasma adiponectin

levels positively correlate with whole-body insulin sensitivity (Arita et al, 2012; Hotta et al,

2000). AdipoR1 and AdipoR2 are the main receptors for adiponectin and are expressed

principally in metabolically relevant organs, such as adipose tissue, skeletal muscles and

liver (Yamauchi et al, 2003). Once adiponectin binds to its receptors, the intracellular signal

5

cascade is transduced by APPL1 and APPL2 scaffold proteins, eliciting the activation of the

AMPK and MAPK pathways. Such signaling pathway results in the activation of cellular

functions aiming at energy production, and the inhibition of energy-consuming processes

(Ren et al, 2006; Nechamen et al, 2007; Deepa et al, 2011). In skeletal muscles, adiponectin

exerts its action mainly through AdipoR1, which induces an increase in fatty acid disposal

through oxidative pathways, and an increase in glucose uptake by enhancing GLUT4

membrane translocation (Yamauchi et al, 2001; Ceddia et al, 2005). Thus, adiponectin

signaling reduces lipid content in skeletal muscle cells and prevents insulin resistance, and is

therefore proposed as an insulin-sensitizing hormone (Yamauchi et al, 2001). Furthermore,

adiponectin modulates hepatic glucose production by inhibiting both gluconeognesis and

glycogenolysis (Fantuzzi, 2005). Thus, adiponectin signaling to the liver plays a key role in

maintaining blood glucose homeostasis. Adiponectin also favors hepatic lipid oxidation

through AdipoR2-mediated AMPK activation and prevents in turn mitochondrial dysfunction,

lipid peroxidation, hepatocellular lipotoxicity and therefore combats hepatic insulin resistance

(Xu et al, 2003a; Zhou et al, 2008). Constitutive adiponectin overexpression in ob/ob mice, a

model of obesity and insulin resistance, also proved autocrine effects of adiponectin in

adipocytes (Kim et al, 2007). Indeed, adiponectin stimulates adipogenesis, while preventing

adipose tissue insulin resistance and its consequent systemic metabolic complications (Kim

et al, 2007).

On the other hand, circulating leptin levels reflect the amount of energy stored in the

organism, and therefore the individual’s fat mass (Considine et al, 1996; Licinio et al, 1997).

Interestingly, leptin exerts its anorexigenic action mainly by acting on the central nervous

system. As a matter of fact, the ObRa splice variant of the leptin receptor is predominantly

located at the blood-brain barrier and allows for leptin hypothalamic translocation (Bjørbæk et

al, 1998), while ObRb is expressed by neuronal cells and mediates leptin signaling through

activation of the JAK2-Stat3 pathway (Vaisse et al, 1996). Leptin signaling in the central

nervous system regulates principally food intake by modulating synthesis and systemic

release of anorexigenic and orexigenic mediators, inhibiting dopaminergic reward circuitries

6

elicited by ingestion of food, and inducing satiety (Robertson et al, 2008; Kelesidis et al,

2010). These effects are achieved not only by acute release of soluble mediators, but also

through long term rewiring of neuronal connections (Pinto et al, 2004). Furthermore, recent

observations proved that leptin neuronal stimulation elicits peripheral effects also via vagal

innervation of metabolic organs, such as hepatic VLDL secretion (Hackl et al, 2019).

Molecular adipose tissue pathophysiology in obesity and metabolic syndrome

The chronic positive energy imbalance in the obese state may cause a pathological lipid

uptake by adipocytes, which causes a significant increase in adipocyte death rate (Cinti et al,

2005). This phenomenon leads to the establishment of a pro-inflammatory local environment,

which in turn induces recruitment of macrophages, whose duty is to sequester lipid droplets

released by the dying adipocytes and to dispose of cell debris. Under these pathologic

conditions, macrophage become enlarged, multinucleated cells, defined as foam cells. Foam

cells actively release high levels of pro-inflammatory mediators, which in turn start a chronic,

systemic, low-grade inflammation. Hormonal cues also play a central role in the

establishment of this adipose tissue pro-inflammatory milieu. Indeed, leptin levels raise in

parallel with adiposity, and leptin induces both T lymphocyte polarization towards a pro-

inflammatory Th1 phenotype and monocyte activation with release of inflammatory cytokines

(Sánchez-Margalet et al, 2003; La Cava, 2017). On the other hand, adiponectin expression is

inhibited by inflammation, and TNF-α, the principal pro-inflammatory cytokine expressed by

the adipose tissue, is a direct repressor of adiponectin (Fasshauer et al, 2002). This might

explain the negative association between obesity and systemic adiponectin levels (Arita et al,

2012; Hotta et al, 2000). Thus, the obesity-associated adipose tissue inflammation, together

with the repression of the insulin-sensitizing adiponectin are proposed as the clues for the

establishment of adipose tissue insulin resistance (Xu et al, 2003b).

While insulin physiologically stimulates adipocytes to store lipids by inhibiting lipolysis, the

insulin-resistant adipose tissue of obese patients has an increased lipolytic rate, which

7

causes an elevation of FFA secretion in the circulation. FFA are eventually taken up by liver

and muscles, where further high lipid concentrations induce peripheral insulin resistance and

lipotoxicity, beyond the alterations of the overall muscular and hepatic metabolisms. The

general insulin-resistant state developed stimulates pancreatic beta-cells to increment insulin

synthesis and secretion. However, beta-cells are characterized by a baseline mitochondrial

activity which is up to 3-fold higher than any other cell type. Since the process produces

reactive oxygen species (ROS) as byproducts, an enhancement of mitochondrial activity by

increasing insulinogenesis drives through a pathogenic ROS synthesis, which in turn induces

IL-1β-mediated inflammatory responses (Maedler et al, 2002; Zhou et al, 2010). Pancreatic

lipotoxicity also occurs under constant hyperlipidemia (Lee et al, 1994), and it is further

worsened by hyperglycemia. The latter further inhibits beta-oxidation in beta-cells, and

induces even higher production of ROS (El-Assaad et al, 2003). The insulitis generated

under the here-described unbalanced metabolic conditions causes beta-cell death and

therefore predisposes to T2DM (Böni-Schnetzler et al, 2008).

The here-described chronic, low-grade inflammation that i) generates from the lipid-

overwhelmed adipocytes; ii) is exacerbated then by the foam cell-like macrophages that

invade the visceral adipose tissue; and iii) results in the subsequent lipotoxicity-induced

inflammation in liver, muscles and pancreas, is nowadays proposed as the conditio sine qua

non for the subsequent evolution of T2DM and cardiovascular diseases (Neuschwander-

Tetri, 2010). Therefore, the obesity-related para-inflammation seems to be the pivotal link

between obesity and metabolic syndrome (Hanley et al, 2004), which was even described by

some as a proper inflammatory disease (Donath & Shoelson, 2011).

Role of the immune system in the metabolic syndrome

The immune system comprises different cell types and processes, which aim to restore the

physiological homeostasis of the hosting organism upon insult. Obesity is induced by a

positive energy balance, which can be achieved simply by over-ingestion of nutrients.

8

Therefore, it is acceptable to postulate that the immune system gets activated upon changes

in amount of food consumed (Donath & Shoelson, 2011). Furthermore, it was also finely

demonstrated that some long-chain FFA induce pro-inflammatory responses through both

toll-like receptor (TLR)-dependent and independent pathways (Wen et al, 2011; Schaeffler et

al, 2009). Glucose and other sugars also activate systemically the immune system

(Deopurkar et al, 2010). Eventually, the mean of resolution becomes the etiology for the

worsening of the disease when chronically activated. Indeed, obesity secondary to chronic

over-eating, generates the activation of the immune response in a pro-inflammatory fashion,

which putatively predisposes to the development of symptoms of the metabolic syndrome.

The most studied example at the moment is IL-6. In a study conducted on healthy subjects, it

was demonstrated that upon physical activity, skeletal muscles produce and release IL-6

(Pedersen et al, 2001), which is thought to promote the intestinal secretion of GLP-1 and to

improve insulin secretion and glucose tolerance (Ellingsgaard et al, 2011). On the other

hand, increased levels of IL-6 are systemically measured in obese and T2DM patients and its

concentration in blood positively correlates with disease development. All in all, IL-6 might be

secreted during condition that alters the metabolic homeostasis (like sport) to cope with the

changes sensed by the organism, but its signaling becomes detrimental when it is chronically

produced. The immune system commitment in metabolic diseases is further strengthened by

the observed enrichment of pro-inflammatory T-helper lymphocytes in the blood and adipose

tissue of obesity mouse models and obese patients (Bertola et al, 2012). Hence, the adaptive

immunity plays also an active role in the establishment and development of the metabolic

syndrome, although its specific pathophysiological function has not been elucidated yet.

The liver as a metabolic organ

With an approximate mass of 1.5 kg, which makes up to 5% of the entire body weight, the

liver is the largest visceral organ in humans (Elias, 1949). Its size correlates with the myriad

of functions carried out within it, which span from synthesis, storage, uptake and secretion of

9

all the main macromolecules constituting the human body, detoxification/biotransformation of

endogenous and exogenous potentially harmful moieties, to the local and systemic immune

surveillance. The extreme functionality of the liver explains its high composition in

parenchymal cells. The overall structure is given uniquely by the blood vessels perfusing it,

and the overall coating mesothelial Glisson’s capsule (Goresky, 1963). The hepatic

circulatory system comprises both venous and arterial inputs: the portal vein carries oxygen-

poor, nutrient-rich blood, which reaches the liver after perfusing the upper digestive tract,

while the hepatic artery provides oxygen to the liver. Afferent venous and arterial blood

converges into sinusoids, the capillary structures that allow for fluids and solutes exchange

with the hepatic parenchymal cells. The blood is collected in central veins, which coalesce in

hepatic veins and empties into the inferior vena cava (Mitra, 1966; Kardon & Kessel, 1980;

Lautt & Greenway, 1987).

In contrast to other glands, the millions of hepatic functional units (generally identified as

lobules) are not delimited by connective tissue. Although debates are still open, the hepatic

lobule is generally defined as a segment of parenchyma perfused by a sinusoid supplied by

terminal afferent vessels and drained by small efferent vessels (Sasse et al, 1992).

Histologically, the lobule has a roughly hexagonal shape, which radially develops around a

section of an efferent central vein, and is delimited at the peripheral extremities by six portal

tracts. Each portal tract is composed of sections of a portal venule, an hepatic arteriole and a

bile ductule, which drains the bile produced by the hepatocytes of the lobule to the gall

bladder through the common hepatic duct (Sasse et al, 1992). This highly specialized

structure allows the lobule cells to permanently receive oxygen, nutrients and information

from the system, while granting the controlled release of macromolecules and signals to the

rest of the body, and the excretion of wastes.

Hepatocytes represent the predominant cellular population in the liver (Blouin et al, 1977).

Their high synthetic and metabolic activities are carried out by abundant intracellular

complexes (thousands of mitochondria, hundreds of peroxisomes and lysosomes), which fill

10

out the cytosolic space of these large, cuboidal cells (Weibel et al, 1969). The plasma

membrane of hepatocytes faces either other hepatocytes or the extracellular matrix (better

known as space of Disse), which separates them from sinusoids (Rappaport et al, 1954).

Hepatocytes bind each other thanks to any sort of interactions, including gap junctions for

direct and fast cytosolic exchange of small molecules (Kmieć, 2001). The flat interaction

surface between hepatocytes allows for the constitution of plate-like structures, typically

observed in liver histology (Ohtani et al, 2003). This homogenous hepatocyte-hepatocyte

organization is minimally interrupted by small invaginations of the cytosolic membrane, which

generates specific structures for the secretion and transport of bile acids through the hepatic

plate to the bile duct at the afferent extremity of the lobule: the bile canaliculi (Weibel et al,

1969). Cholangiocytes mainly reside at the luminal interface between canaliculi and the bile

ducts, and influence bile composition by adjusting the content of solutes and water

(Benedetti et al, 1996; Alpini et al, 1997). The sinusoids facing hepatocytes are mainly

composed by fenestrated endothelial cells lacking a basal layer (Blouin et al, 1977). This

unique structure ensures an optimal exchange of solutes and fluids with hepatocytes

(Schuppan et al, 1998). At the same time, endothelial cells efficiently clear the sinusoidal

blood from old and dysfunctional molecules through scavenger receptors recognition

(Smedsrød et al, 1994; Schuppan et al, 1998). Liver-resident macrophages, also known as

Kupfer cells, also inhabit the sinusoidal lumen and actively remove larger debris, pathogens

and senescent cells (Smedsrød et al, 1994; Wack et al, 2001). Hepatic macrophages mostly

account for the innate hepatic immune response and act as antigen-presenting cells for the

ignition of an adaptive immune response (Parker & Picut, 2005). On the other hand, stellate

cells surround sinusoids, partly occupying the space of Disse (Blouin et al, 1977). Apart from

their known vitamin A- and fat-storing duties, stellate cells get activated in response to

damage stimuli and play a major role in fibrotic responses (Friedman, 2008a).

The lobule complexity is not limited to the several cell types constituting it. Hepatocytes, as

well as the other hepatic parenchymal cells, show different phenotypes depending on their

localization along the hemodynamic afferent-efferent axis of each liver functional unit, a

11

functional organization defined metabolic zonation (Katz & Jungermann, 1976). Oxygen

gradient across the sinusoid has been initially postulated as the master modulator of the

different expression patterns and duties accomplished by hepatocytes at the periportal and

pericentral zones (Bartels et al, 1987; Koury et al, 1991; Jungermann & Kietzmann, 1997).

More recently, strong evidences have shed light on the pivotal role of the beta-catenin

(Benhamouche et al, 2006; Gebhardt & Hovhannisyan, 2009) and Hedgehog (Hh) (Matz-

Soja et al, 2013) pathways in the zonal paradigm. The demonstration that both pathways

influencing lobular zonation can be modulated by the hypoxia-inducible transcription factors

(HIF) (Lenart et al, 2007; Matz-Soja et al, 2014) contributed to link oxygen, beta-catenin and

Hh gradients. These observations constitute the foundation for the study of the implication of

dysfunctional zonation in liver diseases, such as non-alcoholic fatty liver (NAFL), non-

alcoholic steatohepatitis (NASH) and hepatocarcinogenesis.

Hepatic glucose metabolism

Sugars are essential sources of energy. Every human organ and tissue utilizes glucose, the

most abundant monosaccharide in nature, to carry out a conspicuous amount of enzymatic

processes. Thus, cells constituting each kind of tissue express surface transporters that

allow for the uptake of glucose, which in turn will be intracellularly biotransformed into energy

(Capaldo et al, 1999). In order to maintain systemic glucose homeostasis and to keep blood

sugar concentration within the physiological range, the liver – together with the kidneys, to a

lesser extent – is the organ in humans that not only utilizes glucose, but that is also able to

synthetize it and release it (Ekberg et al, 1999). Hepatocytes express insulin-independent

glucose transporters (GLUT2), which allow for facilitated diffusion of glucose, that is kept

inside the cell through phosphorylation by glucokinase (GCK) to glucose-6-phosphate, the

precursor for both processes of glycogen synthesis (storage) and glycolysis (energy

production) (Gomis et al, 2000; Landau, 2001; Moore et al, 2012). Carbon-3 compounds as

lactate and pyruvate, metabolites of the glycolytic pathway, can also be recycled in the

12

indirect pathway for glycogen synthesis, for which an intermediate glucose-1-phosphate is

synthesized and added to the spheroidal storage polymer thanks to the action of glycogen

synthase and branching enzymes (Shulman & Landau, 1992; Bollen et al, 1998). Under

hypoglycemic conditions, glucose is released by hepatocytes through glycogenolysis, a

process mediated by glycogen phosphorylase and debranching enzymes. Only liver and

kidneys, i.e. the organs able to dispose of glucose, express glucose-6-phosphatase that

further dephosphorylates glucose-6-phosphate to glucose, which can be then released from

hepatocytes and parenchymal renal cells into the circulation (Roden & Bernroider, 2003).

Hepatic glucose production (HGP) not only occurs through glycogenolysis, but also through

de novo synthesis from gluconeogenic precursors.

In the post-absorptive state, under resting condition, energy is extracted mainly by fatty acid

(FA) catabolism, and only partly by the glucose released by the liver through glycogenolysis

and glucose-6-phosphoneogenesis. In spite of what was believed for decades, in the early

post-absorptive phase gluconeogenesis accounts for more than 50% of HGP (Petersen et al,

1996). It was later discovered that the processes of glycogenolysis occur mainly upon longer

fasting times, i.e. during the night, while glucose uptake from meals and gluconeogenesis

play major role during daytime (Hwang et al, 1995). This concept is physiologically correct,

since depletion of energy stocks shall take place only when no other free forms of metabolic

fuel is available.

In the post-prandial state, the increased blood sugar, the incretins released by the stretched

intestine (glucagon-like peptide-1 and gastric inhibitory peptide) and brain-derived signals

induce the release of insulin from the pancreatic beta-cells (Roden & Bernroider, 2003).

Small increments in insulin levels are sufficient to stimulate systemically the expression of

insulin-dependent glucose transporters (GLUT4) for general glucose uptake. On the other

hand, higher levels of insulin, achieved through the direct hepatic perfusion by the portal

vein, are necessary to induce hepatic glycogenesis and inhibit HGP which gets reduced by

70%-to-80% when compared with fasted conditions (Taylor et al, 1996; Singhal et al, 2002).

13

Upon hypoglycemia, pancreatic alpha-cells release the insulin-antagonist hormone

glucagone, which enhances HGP, first by inducing glycogenolysis, and later by favoring

gluconeogenesis. Hence, upon glycemic fluctuation, the endocrine pancreatic-hepatic axis

reacts with a first, fast response mediated by mobilization of glycogen storages, and a

subsequent, late induction of gluconeogenesis and glycolysis. Following the concept of

hepatic autoregulation of glycogen stores, the high glycogen content at the early post-

prandial state might also induce glycogenolysis in an autocrine fashion (Cherrington et al,

1998).

Overall, the net effect of insulin action is to suppress hepatic glucose production (HGP).

Despite the direct interaction with the hepatocellular insulin receptor (INSR), insulin achieves

its systemic blood glucose-lowering duty manly through indirect ways. The first cue regarding

extra-hepatic effects of insulin on HGP was obtained by hyperinsulinemic-euglycemic human

studies (Lewis et al, 1996). In these studies, at comparable portal insulin concentrations,

peripheral insulin levels correlated with the degree on HGP inhibition (Lewis et al, 1996). It

was later understood that adipose tissue lipolysis, which is negatively modulated by insulin,

strongly influences hepatic gluconeogenesis. Indeed, both FFA and glycerol released by

adipocytes affect glucose synthesis in the liver. Glycerol is a gluconeogenic precursor, while

acetyl-CoA, the product of mitochondrial oxidation of FFA, activates pyruvate carboxylase

that transforms pyruvate in oxaloacetate, another substrate for gluconeogenesis (Perry et al,

2015, 2014, 2017). This hypothesis was further proved by the inability of insulin to suppress

HGP when its adipose effect is blunted (Lewis et al, 1997; Perry et al, 2015).

Conceptually, insulin modulates gluconeogenesis also intrahepatically. FOXO proteins are

transcription factors that mainly induce the expression of gluconeogenic factors, the most

relevant being PCK1 and G6pc (Puigserver et al, 2003; Nakae et al, 2001). Upon INSR

binding, insulin stimulates the activation of the AKT pathway that leads to the

phosphorylation and consequent cytosolic deportation of the FOXO proteins, inhibiting thus

the expression of the gluconeogenic enzymes (Nakae et al, 2001). The modulation of

14

transcriptional processes requires however longer time than what measured in vivo for the

effects of insulin on HGP (Petersen et al, 2017). Furthermore, only massive increments in

expression of PCK1 and G6pc have been shown to significantly influence gluconeogenesis

(Samuel et al, 2009). Therefore, PCK1 and G6pc physiological fluctuation in abundance

should not impact HGP, at least in the acute phase (Samuel et al, 2009).

On the other hand, insulin – together with glucose – has direct effects on hepatic glycogen

synthesis. In fact, maximal net glycogen storage is achieved through stimulation of

glycogenesis by insulin and suppression of glycogenolysis by glucose (Petersen et al, 1998).

Glucokinase (GCK) and glycogen synthase (GYS2) are the principal modulators in this

process: upon euglycemic/hypoglycemic conditions, GCK is bound and sequestered in the

nucleus by its repressor GKRP (Raimondo et al, 2015). Both insulin and glucose signaling

induces the dissociation of the complex (Agius, 2008). GYS2 can be activated by the

allosteric binding of Glucose-6-phosphate, the product of GCK enzymatic reaction (Agius,

2008; Carabaza et al, 1992), and inhibited by phosphorylation performed by the glucagon-

dependent cAMP-PKA pathway (Alemany & Cohen, 1986). The same phosphorylation

process impacts and activates glycogen phosphorylase, a key enzyme in the glycogenolytic

pathway (Alemany & Cohen, 1986).

Overall, the understanding of the fine and multi-organ regulation of HGP will be of key

importance to individuate and resolve the pathological changes that lead to the development

of metabolic diseases, such as insulin resistance and type 2 diabetes mellitus (T2DM).

Hepatic lipid metabolism

Lipids are a versatile and heterogeneous class of macronutrients. The amphipathic

properties of fatty acids and glycerophospholipds allow for the generation of single- and

bilayered membranes, such as micelles, liposomes and cell membranes. Triglycerides (TG),

the result of a full esterification process of a glycerol-3-phosphate molecule by three fatty

acids, are the deputed long-term lipid storage structures, while several lipid families, such as

15

diacylglycerols (Klein & Malviya, 2008), prostaglandins (Boyce, 2008) and oxysterols

(Bełtowski, 2008), act as signaling molecules. In the liver, lipids are eventually synthesized

through the process of de novo lipogenesis (dnl) and subsequently esterified to TGs for

energy storage, disposed for energy production through mitochondrial and peroxisomal

oxidative processes, or exported to other organs and tissue via very-low-density lipoproteins

(VLDL) (Neuschwander-Tetri, 2010). The liver also takes up circulating fatty acids deriving

from the diet by absorbing chylomicron remnants, and albumin-bound FFAs derived from

adipose tissue lipolysis.

FFA are taken up by the liver in an active way through transporters (FATP transporters

family) and translocases (the most known being CD36), or passively through diffusion(Berk,

2008). Once in the hepatocyte, fatty acids and fatty acyl-CoAs interact with fatty acid binding

proteins (FABP) and acyl-CoA binding proteins (ACBP), respectively. FABP and ACBP drive

them into metabolic compartments (beta-oxidation, energy storage, dnl) or into the nucleus

where they interact with and modulate the action of nuclear receptors and transcription

factors. This whole series of processes was evolutionarily adapted from hepatocytes to

rapidly clear intracellular FFAs, given their cytotoxic properties (Nguyen et al, 2008).

In humans, de novo lipogenesis occurs predominantly in the liver and secondarily in the

adipose tissue (Patel et al, 1975). Hepatic dnl provides lipids for energy and cellular

structures to every organ, while adipose tissue dnl aims to solely produce fat storages inside

adipocytes. In both organs, the synthesis of fatty acids is enzymatically defined by a series of

decarboxylation-condensation reactions, in which glucose or acetyl units are added to an

acetyl-CoA moiety, with NADPH being the hydrogen donor in these processes (Knight et al,

2005; Schadinger et al, 2005; Shen et al, 2011). Fatty acid synthase (FAS) is the rate-limiting

enzyme in the reaction and plays a crucial role in every single step of palmitate synthesis

(Smith et al, 2003). Both FAS activity and expression are enhanced by insulin and negatively

modulated by glucagon action and increased intracellular concentrations of fatty acids (Sul et

al, 2000). Homeostasis of fatty acid fluxes is mainly regulated at the transcriptional level, with

16

insulin and fatty acids modulating the activity of the nuclear receptor LXR (Yamamoto et al,

2007) and the SREBP-1 transcription factors (Dentin et al, 2005). While the overexpression

of the splice variant SREBP-1a induces the synthesis of both fatty acids and cholesterol,

SREBP-1c more selectively enhances GPAT and FAS expression and therefore uniquely

influences dnl (Eberlé et al, 2004). Together with dietary hormones and specific classes of

lipid, such as poly-unsaturated fatty acids (PUFA), LXR-alpha also controls the expression of

SREBP-1c in its RXR-LXR heterodimeric conformation (Barish, 2006; Zelcer & Tontonoz,

2006). LXR-alpha has furthermore direct control on CYP7a1, the rate-limiting enzyme in bile

acids synthesis and regulates therefore also cholesterogenesis (Gupta et al, 2002).

TG and phospholipid synthesis occurs on the cytosolic surface of microsomal membranes.

The glycerol-3-phosphate backbone is obtained either as byproduct of glycolysis or by

phosphorylation of glycerol released by adipose tissue lipolysis. The newly synthesized fatty

acyl-CoAs are consecutively added to it, sequentially generating 1-acylglycerol-3-phosphate,

diacylglycerol and finally triacylglycerol (Berg et al, 2007). TGs are temporarily stored in the

cytoplasm and can be later incorporated in VLDLs and released into the circulation or newly

hydrolyzed if FAs are needed for ATP production (Neuschwander-Tetri, 2010).

Non-esterified lipids can be alternatively redirected to oxidative catalysis for energy

production. Beta-oxidation is the main oxidative process taking place in the mitochondria

(Stryer, 1995). Fatty acyl-CoAs with less than 14 carbon molecules simply diffuse through

the mitochondrial membranes, while longer hydrocarbon chains are converted into

acylcarnitine by CPT-1, the rate-limiting enzyme of mitochondrial beta-oxidation (McGarry &

Brown, 1997). Each acylcarnitine molecule is internalized thanks to carnitine-acylcarnitine

translocases and reconverted to fatty acyl-CoA by CPT-2 at the inner mitochondrial

membrane (McGarry & Brown, 1997). Beta-oxidation consists of consecutive oxidative

reactions, each of them shortening the acyl-CoA molecule by two carbons and generating as

net product one acetyl-CoA, 5 ATPs and water (Lehninger AL, Nelson DL, 2005). All the

resultant acetyl-CoAs can be further fully oxidized to carbon dioxide in the Krebs cycle

17

(Nguyen et al, 2008).

Oxidation might also occur at the peroxisomal and microsomal level. Although significantly

less efficient, these processes are eventually necessary to maintain lipid homeostasis upon

overload conditions and to metabolize lipid species, such as very long chain FAs that cannot

be processed through beta-oxidation (Reddy, 2001; Nguyen et al, 2008). The transcription

factor PPAR-alpha, upon heterodimerization with RXR-alpha (Ahuja et al, 2003), is the

master regulator of FA disposal for energy production in the liver (Everett et al, 2000).

To maintain hepatocellular lipid homeostasis upon increased FFA input, the acetyl-CoAs

produced during mitochondrial beta-oxidation are eventually channeled in an alternative

pathway to generate acetoacetate and beta-hydroxybutyrate, the two water-soluble

components of ketone bodies (Randle et al, 1964). The net result of the incomplete

mitochondrial beta-oxidation, also defined as ketogenesis, is a significant lower ATP

production, but a 5-fold faster disposal of FA-CoA that allows to cope with the FFA overload

(Flatt, 1972; Reichard et al, 1974). On the other hand, ketogenesis might take place also

under starving conditions, when oxaloacetate is sequestrated from the Krebs cycle to serve

in gluconeogenesis and acetyl-CoA is therefore redirected into the ketogenic pathway

(Garber et al, 1974).

Hepatic TG storage and FA oxidation are clearly complementary processes, the former aims

to store energy while the latter disposes of it. FA oxidation and esterification rate is therefore

fine-tuned depending on the hepatocellular energetic state to provide homeostasis. GPAT,

the rate-limiting enzyme for TG synthesis, is inhibited by 5’ AMP-activated kinase (AMPK)

phosphorylation upon increased AMP concentrations. Activated AMPK also phosphorylates

and inhibits acetyl-CoA carboxylase (ACC), suppressing therefore the synthesis of malonyl-

CoA, a known suppressor of CPT-1 (Nguyen et al, 2008). The reduction in ACC metabolite

not only hinders FA synthesis, but also indirectly unleashes mitochondrial FA oxidation. The

same competitive behavior is observed at the transcriptional level; as described above, LXR-

alpha modulates the expression of lipogenic genes (Zelcer & Tontonoz, 2006), while PPAR-

18

alpha controls fatty acid shuttling and beta-oxidation (Schoonjans et al, 1996). Both factors

depend on the heterodimerization with RXR-alpha. The stoichiometric competition between

LXR-alpha and PPAR-alpha for RXR-alpha binding transcriptionally defines the rates of dnl

and mitochondrial beta-oxidation in an extremely efficient way that avoids simultaneous FA

catabolism and anabolism (Ide et al, 2003).

Hepatic lipases play a central role in lipid metabolism too. In fact, TGs stored in cytoplasmic

lipid droplets cannot be used directly and have to be first hydrolyzed to diacylglycerols and

monoacylglycerols for both energetic and exportation pathways. Microsomal lipases

hydrolyze stored TGs into non-esterified glycerolipids, which then undergo a new step of

esterification and VLDL incorporation inside the microsomal lumen (Gibbons et al, 2000).

The final packaging of TGs, cholesterol, phospholipids and apoproteins takes place in the

Golgi apparatus, from which budding vesicles will be released from the sinusoidal pole of the

hepatocyte into the blood stream. VLDLs represent indeed the principal source of FAs and

cholesterol for all the other tissues constituting the human body. With the process of hepatic

lipid export being clearly decrypted, present research focuses on how the entire pathway is

controlled at the molecular level. The whole picture has yet to be completed, but

nevertheless strong evidences claim as bottle-neck steps the synthetic rate of apoprotein

B100 (White et al, 1998; Adeli et al, 2001) and the magnitude of hepatic FFA uptake (Julius,

2003), while almost categorically excluding the influence of dnl (Julius, 2003; Schonfeld &

Pfleger, 1971).

Hepatic insulin resistance

Hepatic insulin resistance is a metabolic pathology caused by over-physiological liver lipid

accumulation that results in enhanced hepatic glucose production. Hepatic insulin resistance

is strongly related to non-alcoholic fatty liver disease (NAFLD) (Petersen et al, 2006), which

recently outrun viral hepatitis and became the most common liver disease in developed

countries (Browning et al, 2004; Smits et al, 2013).

19

To best understand the pathological mechanisms underlying the establishment and

development of hepatic insulin resistance, it is of great importance to first elucidate the

physiological action of insulin on the liver. In the postprandial state, insulin released by

pancreatic beta-cells interacts with and activates the hepatocellular surface receptor insulin

receptor tyrosine kinase (IRTK), which in turn phosphorylates insulin receptor substrate 2

(IRS2) (Cheng et al, 2010). The steric conformation of phosphorylated IRS2 allows for

binding with phosphatidylinositol-3-OH kinase (PI3K) (Hanke & Mann, 2009), and the further

sequential recruitment of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) and Akt

(Franke et al, 1997). Activation of Akt by 3-phosphoinositide-dependent kinase-1 (PDK1)

phosphorylation inhibits hepatic glucose production by dual inactivating-phosphorylation of

FOXO1 and glycogen synthase kinase-3beta. This dual inactivation leads further to the

nuclear exportation of the gluconeogenic transcription factor FOXO1 and the activation of

glycogen synthase. Hepatic insulin signaling also induces the lipogenic program through

synthesis and activation of SREBP1-c, the key modulator of dnl genes (Shimomura et al,

1999a), via the LXRalpha-C/EBPbeta pathway (Tian et al, 2016). Although Akt mainly

regulates hepatic glucose metabolism, the fairly normal gluconeogenesis observed in an

Akt1, Akt2 and FOXO1 triple knock-out mouse model suggests other alternative pathways

and molecular players to be involved in the physiological process (Lu et al, 2012). As

previously discussed, insulin anti-lipolytic effects on adipocytes makes nevertheless the most

prominent contribution to hepatic glucose metabolism (Perry et al, 2015).

Several metabolites have been correlated with hepatic insulin resistance. Although many of

them might act synergistically, major evidences point towards accumulation of diacylglycerol

(DAG) as the necessary and sufficient event for the onset of insulin signaling inhibition in the

liver. The significant accumulation of DAG species and their characteristic translocation to

the plasma membrane through a PKC-epsilon-dependent mechanism has been first claimed

to be the causative event in the establishment of hepatic insulin resistance in a rodent model

of NAFLD (Samuel et al, 2004)(Samuel et al, 2004). The translocation of DAGs to the

plasma membrane of hepatocytes is of paramount importance to inhibit insulin signaling and

20

direct action on the liver. Indeed, the sequestration of these metabolites in cytoplasmic lipid

droplets prevents PKC-epsilon activation and uncontrolled HGP even upon DAG overload,

thus dissociating NAFLD form insulin resistance (Cantley et al, 2013)(Cantley et al, 2013).

Successive studies in human subjects further corroborate these observations (Kumashiro et

al, 2011; Bergman et al, 2012) and ruled out correlations with other formerly proposed

etiological entities for hepatic insulin resistance, such as hepatic ceramide content,

inflammation and endoplasmic reticulum (ER) stress (Magkos et al, 2012). Despite

controversial evidences concerning the role of ceramides (CER) on hepatic and muscle

insulin resistance, CER 16:0 has been recently proposed as an inhibitor of mitochondrial fatty

acid oxidation, whose accumulation might cause a downstream lipid build-up and insulin

resistance (Raichur et al, 2014; Turpin et al, 2014). Nevertheless, clinical validation is still

pending.

Pathological lipid accumulation defines hepatic insulin resistance, and several molecular

routes can lead to the lipid-overwhelmed condition, from increased hepatic fatty acid uptake,

enhanced dnl and triglyceride synthesis, to decreased lipid oxidation and export.

Lipodistrophic patients and “fatless” rodent models best describe the principle of increased

lipid delivery to the liver as cue for hepatic steatosis and insulin resistance (Kim et al, 2000;

Savage et al, 2005). Further genetic polymorphisms that predispose to NAFLD have been

discovered in humans and assessed in animal models, such as gain-of-function variants of

FATP5, the major human hepatic FA transporter (Doege et al, 2008; Auinger et al, 2010),

and APOC3, inhibitor of lipoprotein lipase (LpL) (Kim et al, 2001; Petersen et al, 2010), both

requiring a positive energy balance to develop the pathologic phenotype. In the absence of

genetic predispositions, the FA translocase CD36 is eventually overexpressed upon increase

in circulating FFA, as observed in obese NAFLD patients (Bechmann et al, 2009). This not

only worsens hepatic steatosis, but also promotes hepatocellular apoptosis and NASH

progression to fibrosis (Bechmann et al, 2009). Increased lipid delivery to the liver is only

possible upon adipose tissue insulin resistance, as previously discussed.

21

Since also monosaccharaides can be substrates for dnl and TG synthesis, enhanced hepatic

glucose delivery might also impair insulin sensitivity. Skeletal muscles are the main organ for

glycogen synthesis and storage. Reduced mitochondrial function in myocytes causes a

pathological increase in intramyocellular lipid content, which might induce muscle insulin

resistance, resulting in defective glucose uptake (Roden et al, 1996; Petersen et al, 2003,

2004). The excess glucose will be redirected to the liver, where it will be converted into TGs

(Petersen et al, 2007). Since DAGs are intermediate products in and substrates for the

process of fatty acid esterification to TGs, unbalanced enzymatic activity, as overexpression

of the rate-limiting enzyme GPAT1, might also cause DAG accumulation (Nagle et al, 2007).

Rodent models of impaired or enhanced VLDL-mediated hepatic lipid export further

evidenced the pivotal role played by this process in NAFLD and insulin resistance (Singhal et

al, 2008; Shindo et al, 2010; Morán-Ramos et al, 2012).

Fatty acid oxidation is the most efficient way to lower lipid content and is therefore proposed

as the metabolic mechanism to be targeted to achieve NAFLD regression. Animal studies

demonstrated indeed that suppression of PPAR-alpha, the master regulator of hepatic

mitochondrial beta-oxidation, induces DAG accumulation and PKC-epsilon activation

(Neschen et al, 2007), while different approaches aiming at increasing overall energy

consumption are protective against NAFLD and insulin resistance (Jornayvaz et al, 2012;

Camporez et al, 2013). Observations in human subjects are still elusive, mainly because of

the lack of consensus on direct assessment methods of hepatic oxidative metabolism (Befroy

et al, 2014) and interspecies differences.

Major efforts have been invested in the last decades to uncover the reasons behind the

selective nature of hepatic insulin resistance. In fact, it has been initially counterintuitive to

observe insulin’s failure in suppressing gluconeogenesis, while it still induces dnl.

Esterification of circulating FFA taken up by the liver is the major hepatic pathway for lipid

synthesis and it is insulin-independent (Vatner et al, 2015). Furthermore, excess

monosaccharaides redirected to the liver upon skeletal muscle insulin resistance induce

22

SREBP1-c expression independently of insulin (Matsuzaka et al, 2004) and post-

translationally activate alternative lipogenic trasnription factors, such as CHREBP and LXRs

(Uyeda & Repa, 2006; Bindesbøll et al, 2015). Thus, hepatic dnl is regulated manly by insulin

under homeostatic conditions, but hyperglycemia shows that substrate fluxes strongly

influence it and might take over insulin control.

NAFLD and related insulin resistance are a real pandemic, and effective therapies are

needed to contrast these grave clinical and socio-economical burdens. Weight loss has been

shown to be an highly effective intervention in this regard, with just a 10% loss in fat mass

normalizing steatosis, NAFLD and hepatic insulin sensitivity in both obese and lean patients

with NAFLD (Petersen et al, 2005; Lim et al, 2011). The high rate of recidivism finally lays out

the need for canonical therapies. Clinical studies already demonstrated thiazolidinediones

effectiveness in improving hepatic insulin sensitivity through a proposed PPAR-alpha

mediated modulation of lipolysis in subcutaneous adipose tissue (Mayerson et al, 2002; Kim

et al, 2003; Prieur et al, 2013). Animal studies also showed 2,4-dinitrophenol (DNP) efficacy

in enhancing mitochondrial fat oxidation in hepatocytes, thus rescuing insulin sensitivity and

resolving hepatic steatosis (Samuel et al, 2004). The development of a liver-targeted DNP

allowed furthermore for minimal DNP toxic effects and increased therapy efficacy (Perry et

al, 2013). Since NAFLD and insulin resistance arise from a positive hepatic lipid input, future

therapies should focus on reducing hepatic lipid uptake or increasing lipid oxidation and

export.

The liver pathological evolution in the metabolic syndrome: from Non-alcoholic

fatty liver (NAFL) to Hepatocellular Carcinoma (HCC)

Hepatocellular carcinoma (HCC) accounts for approximately one million deaths per year and

is the principal form of liver cancer (El–Serag & Rudolph, 2007; Torre et al, 2015; Yang et al,

2019). Hepatitis B- (HBV) and C (HCV) virus infections are important risk factors for HCC,

accounting for a 5- to 100-fold increased risk for liver cancer (Fattovich et al, 2008;

23

Goodgame et al, 2003), which further positively correlates with the viral hepatitis-induced

liver fibrosis and/or cirrhosis extent (De Mitri et al, 1995; Haydon et al, 1995). Although oral

direct antiviral agents (DAAs) therapy for HCV and newborn vaccination against HBV are

effective in controlling the viral hepatitis burden and the related outcomes, nutrition-related

diseases significantly contribute to the continuously increasing prevalence of HCC worldwide

in the last decades. Metabolic syndrome plays a major role in this respect, with obesity and

type-2 diabetes mellitus (T2DM) paving the way for the establishment of non-alcoholic fatty

liver (NAFL), which potentially evolves to non-alcoholic steatohepatits (NASH), liver fibrosis

and finally HCC (Welzel et al, 2011).

NAFL is clearly associated with MS, irrespectively of the criteria applied for the diagnosis of it

(Chen et al, 2011; Anstee et al, 2013), and is estimated to become the underlying etiology for

HCC in the next years (Ertle et al, 2011; Anstee et al, 2013; Younossi et al, 2018). NAFL has

been described as a simple lipid accumulation in response to an increased flux of free fatty

acids (FFA) to the liver, not associated with hepatocellular damage (Marchesini et al, 2001).

Approximately 20% of patients manifesting simple steatosis further develop liver

inflammation, hepatocyte injury and fibrosis, a phenotype defined as non-alcoholic

steatohepatitis (NASH) (Rafiq et al, 2009; Ekstedt et al, 2006; White et al, 2012). NAFL-

related steatohepatitis mainly occurs when the rate of liver lipid uptake is higher than the

esterification potential of FFA into triglycerides (TG) (Wilson et al, 2016; Moylan et al, 2014;

Gadd et al, 2014; Krenkel et al, 2018). Under these conditions, non-esterified, mainly

saturated, FFAs may exert a lipotoxic effect and induce inflammation, insulin resistance and

hepatocyte cell death (Reaven, 1988; Neuschwander-Tetri, 2010). While the effect of NAFL

on hepatocarcinogenesis seems to be slightly relevant, NASH is a proper risk factor for the

development of fibrosis/cirrhosis and HCC (Adams et al, 2005; Marchesini et al, 2016; Burt et

al, 2015). Furthermore, a strong correlation between the magnitude of apoptosis and the

fibrotic stage in NASH was evidenced from both the clinical (Feldstein et al, 2003) and

experimental perspective (Nagata et al, 2010; Gregory, 2009). In order to maintain organ

homeostasis, apoptosis shall be counterbalanced by a comparable rate of proliferation.

24

Therefore, persistent hepatocyte programmed cell death in NASH and fibrosis might

paradoxically promote hepatic carcinogenesis (Guicciardi & Gores, 2005).

It is nowadays well accepted that HCC may also develop on a NAFL background in the

absence of evident steatohepatitis (Guzman et al, 2008) and/or fibrosis (Nzeako et al, 1996;

Brancatelli et al, 2002). However, when simply associated with NAFL, HCCs often remain

well differentiated despite a larger size (Paradis et al, 2009), while the more aggressive

phenotype of cirrhotic HCCs eventually involve the activation of hepatic stellate cells, which

are also known to play a pivotal role in pro-fibrotic processes (Bataller & Brenner, 2005).

Based on the above-reported data, it emerges that metabolic syndrome-associated

hepatocarcinogenesis is a complex, multistep pathophysiological process, largely unresolved

so far (Buzzetti et al, 2016; Anstee & Day, 2015). A fine molecular dissection of the metabolic

processes characteristic of each single pathologic manifestation in the developmental

evolution from NAFL to HCC is therefore crucial to uncover promising therapeutic targets and

strategies.

Non-alcoholic fatty liver (NAFL)

NAFL is the initial manifestation of non-alcoholic fatty liver disease (NAFLD) and is defined

by the accumulation of esterified lipids, in the form of macro- or microvesicles, in the

cytoplasm of a proportion of hepatocytes that exceeds 5% (Qayyum et al, 2012). Although

strongly associated with obesity, NAFL has been frequently reported also in lean subjects

(Tominaga et al, 1995; Younossi et al, 2018). Hence, metabolic derangements might also

occur in the absence of obesity, and factors underlying this pathophysiological phenomenon

need further investigation.

Increased hepatic fatty acid uptake is proposed as the paramount event concurring in the

establishment of liver steatosis. Although fatty acid transporters FATP1 and FATP5 are the

master-regulators of fatty acid influx in hepatocytes under physiological conditions (Doege et

25

al, 2006; Falcon et al, 2010), expression of the fatty acid translocase CD36 is directly linked

to obesity and the development of a fatty liver in both humans and diet-induced obesity

animal models (Koonen et al, 2007; Nassir et al, 2013; Bugianesi et al, 2005; Musso et al,

2009; Miquilena-Colina et al, 2011). CD36 transanctivation is mediated by the nuclear

receptor PPAR-gamma (Yang & Smith, 2007; De Souza et al, 2001), which has dietary fatty

acids as natural activating ligands (Forman et al, 1997; Dussault & Forman, 2000).

Therefore, under conditions of elevated circulating non-esterified fatty acids, a vicious circle

might ensue, in which initial enhanced fatty acid flux through FATPs causes PPAR-gamma-

mediated CD36 over-expression, which in turn provides more ligands for PPAR-gamma.

Since the liver is the organ with the highest resistance to lipotoxicity (after adipose tissue, the

only organ in which lipids should be stored), this mechanism might have evolved to

compensate for acute hyper-lipidemic conditions; however, it becomes detrimental for the

liver upon chronic over-feeding.

De novo lipogenesis is a hepato-specific function that allows the conversion of metabolized

sugars into fatty acids. As discussed previously, elevated levels of circulating glucose, as

observed after the ingestion of a carbohydrate-rich meal, activate the expression of dnl

genes, such as acetyl-CoA carboxylase and fatty acid synthase via SREBP-1c and ChREBP

transcription factors (Ameer et al, 2014). Clinical studies proved the involvement of dnl in the

establishment of NAFL by administering a radiolabeled palmitate-enriched meal to healthy

controls and metabolic syndrome patients with significant hepatic steatosis, and following the

metabolic fate of the experimental lipids (Lambert et al, 2014). The experiment reported a 5-

fold increased dnl in fatty livers when compared to healthy controls, although the principal

contributor to hepatic fat deposition remained uptake of circulating lipids (Lambert et al,

2014).

Increased hepatic dnl in patients with metabolic syndrome is associated with a raise in very-

low-density lipoprotein (VLDL) secretion as a mechanism to counteract excessive lipid

deposition in the liver (Fabbrini et al, 2009). However, VLDL secretion capacity seems to be

26

limited and reaches a plateau when hepatic fat content exceeds 10% (Fabbrini et al, 2008).

Hence, lipid disposal through excretion is not a viable strategy to prevent massive hepatic

steatosis and is not an appealing druggable target for NAFLD therapy.

Fatty acid catabolism is more efficiently achieved via mitochondrial beta-oxidation; in line

with that, obese patients with biopsy-proven NAFL show a 5-fold increased mitochondrial

activity when compared with lean healthy subjects (Koliaki et al, 2015). However, hepatic

mitochondrial flexibility has limited endurance: mitochondrial respiration decreases over time

upon chronic lipid overload, and is accompanied by a raise in reactive oxygen species

production (Koliaki et al, 2015). These events, paralleled by early lipotoxicity, hepatocellular

damage and apoptosis, are proposed as the committing steps towards the development of

non-alcoholic steatohepatitis (NASH), although the molecular pathogenesis of the NAFL-to-

NASH transition remains largely elusive.

Besides the great interest in uncovering molecular mechanisms driving the NAFL-to-NASH

evolution and the deriving fibrosis and hepatocarcinogenesis, the cause-effect relationship

between insulin resistance and NAFL has yet to be fully resolved. Strong evidences indicate

that selective hepatic insulin resistance is causative for the pathologically enhanced hepatic

lipid deposition and the consequent development of NAFL (Könner & Brüning, 2012). On the

other hand, various murine liver-specific genetic mutations leading to hepatic steatosis also

worsen hepatic insulin sensitivity (Dentin et al, 2006; Doege et al, 2008; Orellana-Gavaldà et

al, 2011; Knebel et al, 2012). Such genetic defects also cause significant body weight

variations in the animals, which might be the real etiological factor for the induction of insulin

resistance (Gruben et al, 2014). This hypothesis is corroborated by animal studies in which

body weight alteration, independently of NAFL, influences the insulin sensitivity state (Pamir

et al, 2009; Schattenberg & Galle, 2010). Hence, NAFL and insulin resistance surely

influence and enhance each other reciprocally, but most likely the increased lipid deposition

and subsequent chronic-low grade inflammation developed in adipose depots causes

adipose tissue insulin resistance (Samuel et al, 2010; Samuel & Shulman, 2012), that in turn

27

induces hepatic steatosis. NAFL will subsequently favor hepatic insulin resistance, which

together with lipotoxicity, oxidative stress and cell death predisposes to NASH.

Non-alcoholic steatohepatitis (NASH)

NASH is the inflammatory phenotype of NAFLD, with inflammation arising from dying

hepatocytes upon lipid overburden (Chalasani et al, 2018). At the present state, NASH can

be diagnosed only through examination of liver biopsies. Therefore, NASH prevalence is

inferred from available biopsy banks and is expected to affect 20% of NAFL patients, which

equals 3-6% of the world-wide population (Younossi et al, 2019, 2016). The inflammatory

pathophysiology of NASH is associated with a unique histological phenotype, which is

described as hepatocellular ballooning (Lackner et al, 2008). Hepatocellular ballooning is a

process characterized by significant hepatocyte enlargement, caused by a pathologic

intracellular accumulation of lipids, which in turn induces cytoskeletal injury and ER dilation

secondary to its malfunction (Lackner et al, 2008; Caldwell et al, 2010). Ballooning is

proposed to precede hepatocyte death, although ballooning hepatocytes have been also

reported to be viable and able to divide (Zatloukal et al, 2007). Furthermore, the cause-effect

relationship between hepatocellular ballooning and programmed cell death is still elusive and

is most probably mutual. As a matter of fact, upon injury causing ballooning, hepatocytes

over-express keratin 8 (K8) and keratin 18 (K18), whose functional hetero-dimerization

prevents apoptosis (Ku et al, 2007). However, if apoptosis is triggered, the acidic K18 is

degraded by pro-apoptotic caspases, and the loss of stoichiometric K8:K18 balance causes

the formation of undigestible K8 aggregates, the principal component of Mallory-Denk bodies

(Denk et al, 2000; Stumptner et al, 2007). These pathological events of misfolding, which

further involve other cytoplasmic proteins (Zatloukal et al, 2007), induce proteosomal

malfunction an ER stress, which subsequently promote apoptosis (Lee & Glimcher, 2009;

Solinas et al, 2006).

Based on these observations, apoptosis has been hypothesized as the primary cell death

28

process in NASH. To corroborate this hypothesis, Feldstein and colleagues applied TUNEL

staining to liver sections from NASH and NAFL patients and healthy subject, and observed

significant cell death only in NASH specimens (Feldstein et al, 2003). Furthermore, positive

caspase 3 and 7 staining in the same liver sections allowed them to conclude that the cell

death process implied was apoptosis (Feldstein et al, 2003). Successively, clinical trials on

NASH demonstrated that pan-caspase inhibitors normalize plasma ALT levels and are

therefore good candidates for NASH therapy (Ratziu et al, 2012; Shiffman et al, 2019).

Apoptosis can be triggered both by intrinsic and extrinsic cues. Intracellular pro-apoptotic

signaling requires permeabilization of the mitochondrial membrane and subsequent release

of cytochrome C, which further activates caspase 8-dependent apoptosis (Elmore, 2007).

Hence, persistent steatotic conditions might promote apoptosis in hepatocytes, since

accumulation of free fatty acids induces ER stress, ROS production and mitochondrial

dysfunction (Schattenberg et al, 2005; Malhi & Kaufman, 2011), as discussed previously. Of

note, free fatty acids can also induce intrinsic apoptotic signaling through JNK-mediated

activation of the pro-apoptotic factors Bax and Bim (Malhi et al, 2007). Alternatively,

apoptosis is triggered by extracellular mediators, such as TNF-alpha, binding trans-

membrane death receptors (Schattenberg et al, 2006). Also in this case, steatotic conditions

have been shown to sensitize to extracellular pro-apoptotic stimuli both in vitro and in vivo,

mainly through induction of death receptors expression (Ribeiro et al, 2004; Malhi et al, 2007;

Hui et al, 2004; Abiru et al, 2006).

It is important to precise, however, that upon TNF signaling, different patterns of post-

translational modifications might drive through the activation of alternative mechanisms of

programmed cell death. Indeed, TNF-induced activation of caspase 8 results in apoptosis,

while RIPK3 activation blocks apoptosis and promotes necroptosis (Yuan et al, 2016;

Newton et al, 2014). On the wave of these new observations, robust RIPK3 up-regulation

has been reported in NASH mouse models (Thapaliya et al, 2014), and inhibition of RIPK3

significantly improved liver injury (Gautheron et al, 2014). Interestingly, caspase 8 KO

29

worsened hepatic damage in the same animal models (Gautheron et al, 2014). Since

caspase 8 inhibition promotes RIPK3 activity (Kaiser et al, 2011), the authors of this work

concluded that necroptosis is key in NASH-related hepatocellular death, and that efficacy of

pan-caspase inhibitors in NASH may rely on inhibition of caspase 1 and the inflammosome,

rather than on the prevention of apoptosis (Schwabe & Luedde, 2018). Furthermore,

necroptotic processes better fit with the inflammatory phenotype of NASH, since necroptosis

is achieved through perforation of the cytosolic membrane, and the extracellular release of

cytosolic material is a well-known pro-inflammatory cue (Vucur et al, 2013). On the other

hand, apoptosis relies on autodigestion and subsequent efferocytosis, which is a sterile and

anti-inflammatory process (Ravichandran, 2011; Martin et al, 2012). It is worth to precise

that, in the case ATP resources in apoptotic cells are not sufficient to support the entire

apoptotic process, the cell fate switches to secondary necrosis, which is an inflammatory

mechanism of cell death (Malhi et al, 2006); this might happen also under conditions of

defective efferocytosis (Vucur et al, 2013). Hence, based on the latest discoveries, apoptosis

and necroptosis seem both to concur in the enhanced, although moderate, cell death in

NASH. This further complicates the understanding of NASH pathobiology, since the two

processes are supposed to be exclusive (Yuan et al, 2016; Newton et al, 2014; Kaiser et al,

2011).

Hepatocellular death is only one of the known triggers of hepatic inflammation, which is the

crucial determinant of NASH pathogenesis. The nature and amount of diet-related calories

consumed by an individual is the principal extra-hepatic factor influencing liver

steatohepatitis. As a matter of fact, fructose, widely used nowadays as food additive, has

been shown to be a risk factor for NAFL and NAFL-related fibrosis (Abdelmalek et al, 2010;

Ouyang et al, 2008) mainly by markedly enhancing hepatic de novo lipogenesis (Softic et al,

2016). The significantly higher danger for liver metabolic deregulations associated with

fructose when compared with glucose resides in the fact that, while glucose is metabolized

by every cell and tissue of the human organism, fructose is selectively metabolized by the

liver (Blanco & Blanco, 2017). Recent studies could further prove that fructose-related

30

hepatic steatosis and inflammation is achieved directly through induction of ER stress

(Charlton et al, 2011; Ren et al, 2012; Sapp et al, 2014), and indirectly through microbial

translocation following bacterial overgrowth-dependent impairment of intestinal mucosa

integrity (Rosas-Villegas et al, 2017; Kavanagh et al, 2013). The gut microbiota is further

influenced by liver-secreted bile acids, while at the same time microbial bile acid metabolism

in the intestine modifies and enriches the endogenous bile acid pool (Ridlon et al, 2014).

Thus, dysbiosis and altered bile acid pool, both observed in NASH, may promote liver

inflammation (Puri et al, 2018).

Dietary lipids also play important roles in NAFLD and hepatic inflammation; direct evidences

show that increased consumption and consequent hepatic accumulation of trans-fats (used

mainly at the industrial level to prolong the shelf-life of aliments) (Alisi et al, 2011),

cholesterol (Puri et al, 2007; Min et al, 2012), n-6 PUFAs (Araya et al, 2004) and oxidized

forms of linoleic and arachidonic acids (Feldstein et al, 2010; Santoro et al, 2014) is

causative for the establishment of steatosis, hepatic lipotoxicity and inflammation.

Since NAFLD is often associated with the metabolic syndrome, metabolic dysfunctions in

other organs, such as the visceral adipose tissue, might also influence hepatic inflammation.

As discussed previously, adipocyte synthesis and secretion of adiponectin is significantly

reduced in obesity and insulin resistance, and this affects the liver directly, since adiponectin

has anti-inflammatory and insulin-sensitizing effects on hepatocytes (Buechler et al, 2011).

Furthermore, animal model of obesity proved that increased circulating leptin levels in the

metabolic syndrome initially prevent hepatic lipid accumulation, but long-term hyper-

leptinemia induces liver inflammation and fibrosis (Procaccini et al, 2010).

At a glance, inflammation in NASH is proposed to initiate from liver-resident macrophages,

also known as Kupffer cells (KC) (Krenkel & Tacke, 2017a, 2017b), and from hepatic non-

immune cells, such as hepatocytes and hepatic stellate cells (Cai et al, 2017). The

inflammatory signaling cascade is elicited by pattern recognition receptors, the most known

being the Toll-like receptor- and the NOD-like receptor families, upon binding to exogenous

31

(i.e. bacterial components) or endogenous (i.e factors released by damaged or dying cells)

danger signals (Lotze et al, 2007; Kesar & Odin, 2014; Bieghs & Trautwein, 2014; Alegre et

al, 2017; Jo et al, 2016). Activation of pattern recognition receptors induces synthesis and

secretion of anti-apoptotic, pro-inflammatory and chemoattractive mediators, which ignite a

proper immune response (Krenkel & Tacke, 2017a, 2017b). As a consequence, circulating

monocytes and neutrophils are recruited to the liver and further promote hepatic injury

through release of pro-inflammatory cytokines and ROS (Xu et al, 2014; Baeck et al, 2012;

Reid et al, 2016). Under acute injury settings, when the noxious cue is cleared, recruited

immune cells undergo apoptosis and secrete factors promoting tissue repair (Soehnlein et al,

2017). However, in condition of chronic hepatic insult, such as NAFLD, the elicited innate

immune response becomes pathologic and causes a worsening of the disease (Hardy et al,

2016; Schuster et al, 2018).

Based on the central role of inflammation in NASH pathobiology, therapeutic fine-tuning of

the immune response in advanced NAFLD, and specifically promotion of “specialized pro-

resolving mediators” is proposed by many as a promising interventional solution (thoroughly

reviewed in (Schuster et al, 2018)). Indeed, induction of the alternative, anti-inflammatory

phenotype in Kupffer cells and recruited monocyte-derived macrophages might resolve

hepatic inflammation and prevent NASH malignant evolution (Tacke, 2017; Li et al, 2017a). It

must be considered, however, that the alternative macrophage phenotype is also associated

with late manifestations of NAFLD, such as hepatic fibrosis and carcinogenesis (Tian et al,

2019). Therefore, the timing and extent of such a therapeutic intervention shall be carefully

investigated.

Overall, modulation of hepatic inflammation in NASH seems a promising, yet intricate,

therapeutic strategy. At the moment, there is no pharmacotherapy available for NASH (for

and extensive overview of pharmacological therapies in NASH and related ongoing clinical

trials please refer to (Pydyn et al, 2020)). Nevertheless, astonishing results in the therapy of

NAFL and NASH have been obtained by simple life-style interventions (Pydyn et al, 2020).

32

Recent clinical studies demonstrated that 7-10% weight loss upon dietary calorie restriction

significantly improves NAS score in obese patients with metabolic syndrome, and that a body

weight loss of more than 10% resolves almost completely NASH (Vilar-Gomez et al, 2015;

Boden, 2009). Similar results were obtained in obese and non-obese NAFLD patients who

joined a followed physical activity program (Keating et al, 2012; Wong et al, 2018).

Therefore, life-style interventions are the only effective therapies for NASH so far, and they

are successful irrespectively of BMI. Unfortunately, adherence to life-style-modifying

programs is low, and alternative pharmacotherapies are urgently needed.

Hepatic fibrosis

Although induction of an inflammatory response is key in resolution of organ damage or

infection, chronic inflammation (as observed in NASH) causes pathologic tissue remodeling

and wound healing secondary to persistent injury of hepatic parenchyma, which predispose

to fibrosis. Since NASH-associated fibrosis is the principal risk factor for mortality in NAFLD

patients (Diehl & Day, 2017; Angulo et al, 2015; Vilar-Gomez et al, 2018), major efforts have

been invested recently to uncover the molecular mechanisms responsible for the

pathological deposition of extra-cellular matrix (ECM) in the liver, the paramount process

defining fibrosis severity. As a matter of fact, pathological expansion of hepatic ECM has

been proposed as etiology for NASH-related liver failure due to significant replacement of

functional parenchymal component, as also for the establishment of portal hypertension

through impaired elasticity of hepatic vessels (Schwabe et al, 2020).

Both clinical and experimental studies support a pivotal role for hepatic stellate cells (HSC) in

liver fibrogenesis (De Minicis et al, 2007; Marcher et al, 2019; Ramachandran et al, 2019;

Krenkel et al, 2019). HSC fibroblastic activation is a unique process, which ensues in

response to hepatic damage-related signals, causing a dramatic switch in both phenotypic

and metabolic properties of HSC (Friedman, 2008b). Following activation, HSC phenotypical

trans-differentiation from retinyl ester-storing cells to myofibroblasts is characterized by

33

induction of proliferative, contractile and pro-inflammatory processes (Puche et al, 2013).

The gene expression reprogramming that defines the initiation step of HSC activation also

mediates the synthesis of membrane receptors, which enhance HSC responsiveness to

extracellular fibrogenic signals (Tsuchida & Friedman, 2017). Extracellular cues lead

therefore to a perpetuation of the HSC myofibroblastic phenotype, which is defined by

enhanced collagen deposition and inhibition of ECM-degrading processes (Friedman, 2000;

Krizhanovsky et al, 2008; Schnabl et al, 2002). Hence, the concept of perpetuation of HSC

activation (Friedman, 2000) is crucial in the settings of chronic liver injury, and the molecular

pathways underlying it could be valuable therapeutic targets for fibrosis resolution.

Although the mechanisms of HSC activation seem largely conserved across different fibrotic

etiologies (e.g. drug toxicity, cholestasis, alcohol abuse and NASH) (De Minicis et al, 2007;

Marcher et al, 2019; Mederacke et al, 2013), the prevalent activating cues may vary

significantly depending on the type of injury preceding fibrogenesis. TGF-beta is a central

factor in HSC fibrogenic activation, which promotes inflammation, survival and perpetuation

of HSC activated phenotype, and induces collagen I and III synthesis through activation of

SMAD3, MAPK and YAP intracellular signaling cascades (Yu et al, 2019; Breitkopf et al,

2006; Friedman, 2008b; Khimji et al, 2008). Surprisingly, the type 2 immunity cytokine IL-13

also plays a crucial role in HSC pro-fibrogenic activation, since dual TGF-beta and IL-13

inhibition more potently resolved hepatic fibrosis in an experimental NASH model than TGF-

beta inhibition alone (Hart et al, 2017). Although misleading at first sight, over-physiological

type 2 immunity stimuli eventually favor exacerbation of wound healing processes(Hart et al,

2017), and therefore fibrosis. Platelet-derived growth factor (PDGF), IL-17 and angiotensin II

are further HSC-activating factors (Tsuchida & Friedman, 2017; Mannaerts et al, 2015;

Martin et al, 2016; Bataller et al, 2003; Meng et al, 2012), but their direct implication in

NASH-derived fibrogenesis has not been proven so far.

Metabolic deregulations also contribute to HSC activation in NASH-associated liver fibrosis.

A crucial phenotypic change occurring during HSC myofibroblastic differentiation is the

34

hydrolysis of intracellular lipid droplets (Blaner et al, 2009), a process that relies on activation

of autophagy in hepatocytes (Singh et al, 2009). Based on this information, Hernandez-Gea

and colleagues hypothesized that HSC activation also depends on autophagy, and proved

that by showing enhanced autophagic activity in activated HSC, and fibrosis resolution in

vivo upon HSC-specific knockout of ATG7, a master regulator of autophagy (Hernndezgea et

al, 2012). Although the pathomechanism underlying the implication of autophagy in HSC

activation seems to rely on the release of non-esterified lipids through hydrolysis of

intracellular retinyl ester droplets (Hernndezgea et al, 2012), the exact mechanism through

which these lipid moieties promotes HSC activation has yet to be elucidated.

The tight interconnection between ER stress and autophagic derangements often observed

in metabolic diseases (Rivera et al, 2014; Shigihara et al, 2014; Lee et al, 2017; Jung et al,

2015; González-Rodríguez et al, 2014) is key also in HSC activation. Indeed, induction of ER

stress through over-expression of X-box binding protein 1 (XBP1) in cultured HSC promotes

expression and secretion of collagen I, which can be inhibited by suppressing autophagy

through ATG7 knowckdown (Kim et al, 2016). PKR-like endoplasmic reticulum kinase

(PERK) is also activated upon ER stress and favors HSC activation by promoting the

degradation of heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) and the subsequent

destabilization of miR-18A (Koo et al, 2016). Moreover, oxidative stress is a hallmark of

NASH pathophysiology, which promotes HSC myofibroblastic transformation through both

paracrine (Puche et al, 2013) and autocrine (Novo et al, 2011) signaling pathways.

Under physiological conditions, wound-healing processes are mediated mainly by HSC and

by both liver-resident (KC) and monocyte-derived macrophages (MoM) (Friedman, 2008b;

Iredale et al, 1998; Troeger et al, 2012; Campana & Iredale, 2017). Upon injury, hepatocytes

release cytokines that activate HSC, chemo-attractants that promote the recruitment of MoM,

and damage-associated molecular patterns (DAMPs) that further stimulate HSC and KC pro-

inflammatory activation (Puche et al, 2013; Machado et al, 2015; Seo et al, 2016; Pinzani &

Marra, 2001). The recruited MoM, together with liver-resident KC, polarize into the classical,

35

pro-inflammatory macrophage phenotype as a result of the inflammatory milieu in the liver,

and secrete TGF-beta and PDGF, which further induce fibroblastic activation of HSC (Dooley

et al, 2001; Pinzani et al, 1989, 1991; Tacke, 2017; Pellicoro et al, 2014). MoM and KC

further release NF-kB, which acts as anti-apoptotic cue for activated HSC (Pradere et al,

2013; Tacke, 2017; Pellicoro et al, 2014).

Concerning the process of ECM deposition and degradation, activated alpha-SMA-positive

HSC synthesize and secrete collagen type III and V, the main components of early

regenerative ECM (Kisseleva & Brenner, 2008; Bataller & Brenner, 2005; Mederacke et al,

2013). During the physiological resolution phase that follows liver damage, HSC get

deactivated and/or undergo apoptosis, while increasing their synthesis and secretion of

ECM-degrading matrix metalloproteinases (MMP), the most important being MMP1, MMP9

and MMP13 (Suzuki et al, 1990; Okamoto et al, 2005; Yata et al, 1999). Deactivated HSC

also express uPA, which enzymatically activates MMPs through hydrolysis of their N-terminal

domain (Leyland et al, 1996; Kim et al, 1997). However, upon chronic insult, the constant

hepatocyte cell death and the associated inflammatory background contrasts HSC

deactivation and in turn promotes HSC synthesis of collagen I and IV, which are significantly

more resistant to MMP-mediated degradation when compared with collagen III and V (Geerts

et al, 1990; Milani et al, 1990; Nakatsukasa et al, 1990). The same collagen fibrils deposited

in the damaged parenchyma promote HSC pro-fibrotic functions and survival trough

interaction with membrane-bound integrins on HSC. As a matter of fact, genetic or

pharmacological inhibition of integrin αV inhibits synthesis and proteolytic activation of TGF-

beta in HSC and significantly improves fibrosis in CCL4-treated mice (Tsuchida & Friedman,

2017; Henderson et al, 2013). Of note, chronically activated HSC negatively modulate MMP

synthesis, while up-regulating the uPA inhibitor PAI-I (Leyland et al, 1996; Andreasen et al,

2000). Activated HSC also up-regulate TIMP1 expression, which competitively binds to the

active site of MMPs and therefore inhibits collagen degradation (Hemmann et al, 2007;

Iredale, 1997). Hence, upon chronic HSC activation, fibrogenic processes prevail over

fibrogenolytic activities and this favors net collagen fibrils expansion over time. The action of

36

other HSC-expressed enzymes, such as transglutaminase and LOXL2, further introduce

crosslinks in the growing collagen fibrils, eventually hampering their degradation (Liu et al,

2016; Barry-Hamilton et al, 2010).

Advanced stages of fibrosis are further characterized by hypoxia secondary to decrease

oxygen availability for parenchymal cells, which are surrounded by thick fibrotic septa.

Hypoxic conditions not only promote neo-angiogenesis, mainly observed clinically in livers of

cirrhotic patients, but also further induce hepatocellular death. As reported in several chronic

inflammatory diseases, the over-physiological cell proliferation, necessary to cope with the

loss of functional parenchymal cells, enhances the incidence of mitotic errors and

accelerates aging processes, phenomena that are tightly linked to tumorigenesis and/or

organ failure (Ellis & Mann, 2012; Guicciardi & Gores, 2010, 2005).

NAFLD-associated hepatocellular carcinogenesis

Recently published meta-analyses corroborate previous observations concerning the

associations of obesity with increased risk of HCC development (Gupta et al, 2018), and

further prove that hepatocarcinogenesis associates with the hepatic fibrosis stage in NAFLD

patients (Kanwal et al, 2018; Kim et al, 2018). Hence, HCC arises as consequence and end

stage of a chronic pathologic chain of metabolic deregulations, a process that might last

decades. Nevertheless, HCC prevalence raises constantly, hand in hand with obesity and

metabolic syndrome prevalence (Siegel et al, 2016; Estes et al, 2018b, 2018a). These data

shed light on the limitations of diagnostic tools and therapeutic/surgical interventions

available at the moment, and highlight the need of better understanding when and how

transition from a stage of liver disease to next occurs. This is especially relevant in NAFLD-

derived HCC development, since HCC, together with hepatic cirrhosis, is the main cause of

liver-related death in patients with metabolic syndrome (Gupta et al, 2018). Therefore, first of

all, it is of paramount importance to identify molecular factors concurring in disease

development.

37

The implementation of holistic approaches in the investigation of genetic traits associated

with pathological states, such as genome-wide association studies (GWAS), allowed the

identification of genetic polymorphisms that might confer higher susceptibility to HCC in

NAFLD patients (Kozlitina et al, 2014; Dongiovanni et al, 2015; Liu et al, 2014; BasuRay et

al, 2017; Ehrhardt et al, 2017; Donati et al, 2017). Of them, the I148M polymorphism in the

patatin-like phospholipase domain-containing protein 3 (PNPLA3) locus has received the

most attention, since metabolic syndrome patients homozygote for this allele have a three-

fold increased risk for HCC (Singal et al, 2014; Romeo et al, 2008). Although this technique

might uncover previously unknown molecular factors that crucially influence disease etiology,

the reliability of GWAS studies is still controversially discussed, since mechanistic

explanations for the here-reported associations remain largely elusive, although significant

experimental effort has been invested in the last decade (BasuRay et al, 2019; Wang et al,

2019; Lindén et al, 2019; Mitsche et al, 2018). Although genetic variations might influence

NAFLD-associated hepatocarcinogenesis, environmental factors, such as dietary habits and

physical activity (as previously discussed) are the conditio sine qua non for NAFLD

establishment and malignant evolution. Therefore, since metabolic syndrome-associated

HCC develop on a NAFL/NASH background, all the molecular pathomechanisms identifying

these chronic liver diseases – inflammation, deregulated metabolism and chronic liver

regeneration – eventually affect hepatocarcinogenesis.

Generally speaking, tumors arise from events promoting genetic instability and consequent

unresolved DNA mutations, which cause pathologic gain- or loss of function in tumor

suppressor- and proto-oncogenes. In NASH, oxidative stress resulting from impaired

mitochondrial respiration (Begriche et al, 2013) and repressed anti-oxidant processes

(Masarone et al, 2018) is proposed as the principal cause for HCC-predisposing DNA

damages (Anstee et al, 2019). These hepatic metabolic dysfunctions might be further

enhanced by NASH associated inflammatory cues (Wolf et al, 2014). In line with that,

oxidative DNA damage has been copiously observed in liver samples of NASH patients

(Nishida et al, 2016; Seki et al, 2002), with the highest intensity in livers of NASH patients

38

with HCC (Tanaka et al, 2013). In both murine NASH models and HCC patients it has also

been shown that NASH-related up-regulation of the proto-oncogene unconventional

prefolding RBP5 interaction (URI) might also concur in the pathologic ROS production and

establishment of oxidative DNA damage through inhibition of the anti-oxidant NAD pathway

(Tummala et al, 2014). It is important to note, however, that ROS causing DNA damage

might be derived also from MoM and neutrophils invading the injured liver (Wiseman &

Halliwell, 1996; Kuper et al, 2000), a mechanism already described for other types of

gastrointestinal cancer (Yuan et al, 2017; Canli et al, 2017).

One has also to consider that DNA damage occurs regularly in cells, and although an

increased rate of DNA damage might enhance the likelihood of cancer-predisposing mutation

occurrence, carcinogenesis in NAFLD eventually relies also on concomitant malfunctions in

DNA damage response (DDR) mechanisms (Anstee et al, 2019). The most frequent DDR

deregulation reported in human HCC is the amplification of the genomic locus containing

DNA-dependent protein kinase (DNA-PK) (Collis et al, 2005), which plays paramount roles in

non-homologous end-joining processes (Cornell et al, 2015). Enhanced DNA-PK activity

promotes therefore non-conservative DNA repair, which in turn favors the perpetuation of

new mutations and HCC malignant transformation (Pascale et al, 2016).

Intestinal dysbiosis and impaired gut permeability, observed in both mouse and human

NASH (Zhu et al, 2013; Mouzaki et al, 2016; Le Roy et al, 2013; Miele et al, 2009; Volynets

et al, 2012), might also play a role in NAFLD-derived hepatocellular carcinogenesis. As a

proof of it, some classes of bile acids, as deoxycholic and lithocholic acid are able to induce

hepatocellular DNA damage (Jansen, 2007), and mutations in the bile acid export pump

ABCB11 have been found in >90% of progressive familial intrahepatic cholestasis cases with

diagnosed HCC (Knisely et al, 2006). However, molecular evidences linking intestinal

dysbiosis, altered bile acid pool and NASH-derived HCC are still missing.

Both innate and adaptive immune responses are activated in advanced NAFLD, and an

unbalanced immune response might also promote hepatic tumorigenesis. Although data are

39

still limited, NKp30-mediated suppression of natural killer (NK) cells has been reported in

patients with HCC (Hoechst et al, 2009). At the same time, enhanced hepatic invasion by

CD4+ regulatory T cell reflects a poor outcome for HCC patients (Fu et al, 2007; Gao et al,

2007). Taken together, these data suggest that suppression of immune response in HCC is

necessary to prevent clearance of tumor cells and to promote tumor progression (Hoechst et

al, 2009; Anstee et al, 2019). This hypothesis is further supported by animal studies, in which

suppression of cytotoxic CD8+ T lymphocytes favor NASH-derived hepatocarcinogenesis

(Shalapour et al, 2017), and by the fact that immune checkpoint inhibitors, together with anti-

angiogenic multikinase inhibitors, are the only effective treatments in advanced HCC so far

(Gerbes et al, 2018). Thus, these observations suggest that the whole tumor

microenvironment, composed of tumor cells, immune cells and neovascularizations, shall be

taken into consideration to design effective anti-tumoral treatment strategies. This concept

became even clearer when Ehling and colleagues demonstrated for the first time that hepatic

inflammation promotes neoangiogenesis independently of hypoxia in mouse models of

advanced fibrosis (Ehling et al, 2014). In this study, massive monocyte recruitment has been

observed upon fibrotic liver injury, and MoM localized primarily in site of neovascularization

(Ehling et al, 2014). The subsequent inhibition of monocyte infiltration, achieved through the

targeting of the chemoattractive cytokine CCL2, resulted in significant resolution of

neoangiogenesis (Ehling et al, 2014). Although this study did not investigate

hepatocarcinogesis, it proved the tight connection between immune and vascular

components in severe hepatic fibrosis, which is the pathological step preceding HCC

development. Based on that, Li et al. found that CCL2 is over-expressed in human HCC, with

higher CCL2 levels correlating with poorer HCC outcome (Li et al, 2017b). In this study, they

further characterized the tumor-associated macrophage (TAM) population in HCC mouse

models, and found that TAM are M2-like-polarized (i.e. immune-suppressive) macrophages,

which inhibit activation of CD8+ T cells and prevent therefore an effective anti-tumoral

immune response (Li et al, 2017b). Also in this experiment, both genetic and

pharmacological CCL2 inhibition prevented monocyte infiltration in the injured livers, and

40

inhibited HCC growth (Li et al, 2017b).

More recent and modern immunophenotyping techniques allowed Bartneck and colleagues

to identify a pro-inflammatory (M1-like) TAM population, which co-localizes with newly

formed blood vessels at the HCC border, and an immune-suppressing (M2-like) TAM

subpopulation inside the tumor mass in human HCC (Bartneck et al, 2019). Further anti-

CCL2 treatment in an HCC mouse model resulted in significant depletion of pro-inflammatory

TAM, resolution of neoangiogenesis and reduction of tumor volume (Bartneck et al, 2019).

Hence, these data further support the concept of targeting the tumor microenvironment to

cure HCC. Based on all these observations, CCL2 inhibition is now proposed as a promising

therapeutic strategy for HCC, which might be administered in combination with the already

approved immune checkpoint- and multikinase inhibitors (Tacke, 2018; Avila & Berasain,

2019).

The ambiguous, yet unresolved, role of osteopontin in NAFLD

The multifunctional protein osteopontin (OPN) was first cloned in the late 1980’s by Kiefer

and colleagues (Kiefer et al, 1989), and its expression has been observed primarily in

skeletal tissue, where it promotes bone mineralization by favoring the anchorage of newly

differentiated osteoclasts to hydroxyapatite on the bone surface (Reinholt et al, 1990). Under

physiological conditions, OPN is also expressed in kidneys (Ogbureke & Fisher, 2005),

elastic fibers of skin and aorta (Baccarani-Contri et al, 1995), and in immune cells, such as

macrophages, dendritic cells and T lymphocytes, where it promotes a type-1 immune

response (Ashkar et al, 2000; Shinohara et al, 2005, 2006).

SPP1, the gene encoding OPN, is located on chromosome 4 in humans, while on

chromosome 5 in mice (SPP1 secreted phosphoprotein 1 [Homo sapiens (human)] - Gene -

NCBI; Spp1 secreted phosphoprotein 1 [Mus musculus (house mouse)] - Gene - NCBI).

SPP1 genomic sequence remained conserved over time, as reflected by an 84% homology

41

with murine Spp1 open reading frame and a substantially unchanged promoter sequence

(Song et al, 2020). In line with these observations, both human SPP1 and mouse Spp1

genes give rise to five different transcript variants, although isoform sequences may vary

across species (SPP1 secreted phosphoprotein 1 [Homo sapiens (human)] - Gene - NCBI;

Spp1 secreted phosphoprotein 1 [Mus musculus (house mouse)] - Gene - NCBI). In humans,

these five alternative splicing products have characteristic tissue distribution and can be

differentiated by the presence/absence of exon 4 and 5, or by the presence of an alternative

starting codon and a longer, unspliced exon 3 sequence (Song et al, 2020; Briones-Orta et

al, 2017). Furthermore, the presence of two translational start sites on OPN mRNAs and the

consequent alternative events of OPN mRNA translation affect OPN cellular localization.

Indeed, while full-length OPN in secreted as extracellular signaling molecule via the

secretory Golgi pathway, a shorter translated version lacking the distal N-terminal domain is

retained intracellularly (Shinohara et al, 2008, 2006; Zohar et al, 2000; Zhu et al, 2004). The

acidic protein OPN is also a potential target for extensive post-translational modifications, the

most common being phosphorylations and O-glycosylations (Song et al, 2020; Boskey et al,

2012; Jono et al, 2000; Anborgh et al, 2011). This characteristic trait also explains the highly

variable molecular weight of OPN, which ranges between 45 and 250 KDa (Lok & Lyle, 2019;

Anborgh et al, 2011; Christensen et al, 2008; Kazanecki et al, 2007). OPN can be further

modified post-translationally by the proteolytic action of MMPs, thrombin and cathepsin D,

which generate characteristic cleaved products with distinctive interaction affinities for OPN

receptors, which consist mainly of transmembrane integrins and CD44 isoforms (Christensen

et al, 2010; Agnihotri et al, 2001; Uede, 2011).

Overall, OPN evolutional conservation, associated with its astonishing biochemical diversity

and its central role in cell-matrix interaction and signaling (Song et al, 2020; Denhardt et al,

2001; Zhao et al, 2018), suggest a cardinal role for OPN in several biological processes, the

deregulation of which is associated with various disease states (Zhao et al, 2018; Anborgh et

al, 2011; Clemente et al, 2016), and makes of it a potential therapeutic target for these

pathologies.

42

Under physiological conditions, adipose tissue and hepatic OPN expression levels are low.

However, significant OPN over-expression has been recorded in both obese humans and

mouse models of obesity (Bertola et al, 2009; Kiefer et al, 2008). Plasma OPN levels are

also elevated in obese patients, while contrasting results have been obtained in obese

mouse models in this regard (Kiefer et al, 2008; Riedl et al, 2008). Interestingly, adipose

tissue- and hepatic OPN over-expression in obesity has been proposed to favor macrophage

tissue infiltration and the ignition of a pro-inflammatory immune response in these organs

(Weisberg et al, 2003; Nomiyama et al, 2007a; Kiefer et al, 2010), which predisposes to the

classical, obesity-associated systemic low-grade inflammation, insulin resistance and hepatic

steatosis (Hotamisligil, 2006; Bertola et al, 2009; Sahai et al, 2004a). In line with this

hypothesis, both genetic and pharmacological OPN ablation resulted in significantly reduced

adipose tissue inflammation and macrophage recruitment, as well as improved liver steatosis

and insulin resistance in HFD-fed mice (Nomiyama et al, 2007a; Kiefer et al, 2010).

Therefore, OPN over-expression in adipose tissue and liver is proposed as a crucial

pathological event in the establishment of NAFLD, although molecular insights underlying

these putative mechanisms remain poorly understood.

OPN differential expression is implicated also in the pathophysiology of liver fibrosis. In

healthy livers, OPN expression is highest in ductular epithelial cells, while significant OPN

induction is observed immunohistochemically also in hepatocytes and Kupffer cells in both

human and murine liver fibrosis (Arriazu et al, 2017; Lorena et al, 2006; Urtasun et al, 2012).

Plasma OPN levels correlate with the fibrotic stage in humans, and this parameter is

considered a reliable biomarker for both metabolic-predominant- and alcoholic-predominant

liver cirrhosis (Sobhy et al, 2019; glass et al, 2018; Simão et al, 2015; Syn et al, 2012).

Importantly, liver-specific transgenic over-expression of OPN induces spontaneous fibrosis in

aging mice (Arriazu et al, 2017), which further underscores the centrality of OPN function in

hepatic fibrogenesis.

Mechanistically, it has been demonstrated that OPN secreted by hepatocytes and liver

43

macrophages activates HSC and promotes migration and collagen I production both in vitro

and in vivo (Arriazu et al, 2017; Xiao et al, 2012; Urtasun et al, 2012). This might be achieved

i) by enhancing TGF-beta-dependent pathways (Xiao et al, 2012), ii) through binding of

integrin avbeta3 and the consequent activation of the Pi3K-pAkt-NFkB pathway (Urtasun et

al, 2012), and iii) through the indirect induction of the same Pi3K-dependent signaling

cascade by transactivation of high-mobility group box-1 (HMGB1), whose expression, as for

OPN, correlates with liver fibrosis stage (Arriazu et al, 2017; Ge et al, 2018). In these

settings, OPN genetic and pharmacological inhibition halts collagen I deposition and induces

fibrosis resolution in vivo (Arriazu et al, 2017; Xiao et al, 2012; Urtasun et al, 2012; Ge et al,

2018; Coombes et al, 2016). At a variance with these observations, when applying the CCl4-

and the bile duct ligation models in WT and OPN-/- mice, Lorena and colleagues obtained

opposite results and postulated that OPN over-expression in hepatic fibrosis occurs as a

consequence of the liver injury and bears anti-fibrotic properties (Lorena et al, 2006). Thus,

the available data unanimously support a key role for OPN in hepatic fibrogenesis, although

it is still debated whether enhancement or repression of OPN signaling would be the

appropriate therapeutic strategy.

As for hepatic fibrosis, the association of OPN with tumorigenic processes has been

thoroughly demonstrated in various organs, although the precise role of OPN in tumor

establishment, growth and metastatic progression remains elusive. For what liver cancers

concerns, Gotoh and colleagues firstly reported a significant OPN mRNA and protein over-

expression in HCC specimens compared to paired adjacent non-tumor tissue (Gotoh et al,

2002). In this early cohort consisting of 30 patients, OPN positive staining was observed

predominantly at the tumor-stromal interface (Gotoh et al, 2002), and this further supports

OPN role in signaling and interaction with the surrounding tissue matrix. The same

immunohistochemical staining pattern has been observed in lung and breast cancers

(Coppola et al, 2004; Brown et al, 1994). Here, OPN seems to be expressed primarily by

macrophages located at the tumor edges, while OPN intratumoral positivity has been

proposed to represent extracellular OPN binding surface receptors on cancerous cells

44

(Brown et al, 1994). Hence, precise clarification of OPN cellular origin is necessary to

uncover its role in tumorigenesis. Furthermore, since both hepatocytes and hepatic

macrophages express OPN, its cellular origin in HCC might influence tumor phenotype. The

relevance of this concept has been underlined by Crawford and colleagues, who investigated

the role of host- and tumor OPN and their relation with tumor-associated immune cells in

mouse models of squamous cell carcinoma. The authors demonstrated that chemically-

induced skin tumors were arising faster and growing larger in OPN-deficient mice, while

metastases were strongly reduced in size in the absence of endogenous OPN (Crawford et

al, 1998). The same results were obtained in murine xenograft models, where the

tumorigenic potential of high- and low-OPN-expressing tumor cell lines has been compared

(Crawford et al, 1998). Overall, these studies highlighted the fact that host (i.e. stromal) OPN

expression is responsible for macrophage recruitment to the tumor mass, consistent with the

promotion of an immune anti-tumoral response, while tumor-expressed OPN shifts the

polarization of recruited, tumor-associated macrophages from a pro-inflammatory, M1-like, to

a M2-like phenotype, which eventually protects the tumor, favors matrix remodeling and

therefore promotes metastasis (Crawford et al, 1998).

Thus, OPN might exert a dual role in tumorigenesis, being tumor-suppressive at early stage,

while tumor-promoting in advanced tumor development. The fact that OPN expression

strongly correlates with tumor pathological stage in various malignancies (Coppola et al,

2004) further support the hypothesis of a role for OPN as promoter of metastatic tumor

transformation. A recent meta-analysis proved this concept to be true also in HCC, and

further supported the reliability of both tissue and plasma OPN levels as HCC biomarkers,

since they both positively correlate with overall survival, HCC size and stage (Sun et al,

2018).

In order to better understand how OPN promotes HCC transformation, Sun and colleagues

applied lentivirus RNA-interference to target OPN in HCCLM3 cells, an HCC cell line with

high invasive and metastatic potential. They proved this way that ≥ 80% OPN knock-down

45

(KD) is necessary to prevent in vitro invasion and in vivo metastases of HCC (Sun et al,

2008a), while only ≥ 95% OPN KD also halts HCC proliferation (Sun et al, 2008a). Further in

vitro examinations showed that OPN-mediated HCC proliferation and growth might be

mediated by the mitogen-activated protein kinase (MAPK) pathway, since HCCLM3

stimulation with OPN resulted in the activation of the MAPK downstream targets MEK and

ERK1/2, while ≥ 95% OPN KD completely inhibits this mitogenic pathway (Sun et al, 2008a).

On the other hand, gene expression analyses of HCCLM3 cells upon OPN KD revealed that

uPA and MMP2, both known downstream targets of OPN signaling, might be the molecular

effectors of OPN-mediated HCC metastasis (Sun et al, 2008a).

Hence, these data suggest that OPN expressed by cancerous hepatocytes is key for both

tumor growth and malignant transformation, although the two processes require significantly

different amounts of OPN. Nevertheless, the various signaling properties of OPN might

depend on the transcriptional isoforms expressed and on the pattern of post-translational

modifications characterizing OPN in different cell types (even within the tumor) and at diverse

stages of disease. Therefore, further research aiming at uncovering the biochemical changes

associated with OPN function during disease evolution is granted.

An important step in this direction has been made by Xue and coworkers, who clinically

showed that tumor thrombin expression correlates with HCC poor outcomes only in HCC

patients with high hepatic OPN expression (Xue et al, 2010). Based on these observations,

subsequent in vitro thrombin administration to OPN-over-expressing HCC cell lines has been

proved to enhance HCC invasive phenotype, which could be later inhibited by mean of anti-

integrin-beta1 antibodies (Xue et al, 2010). Thus. Thrombin-cleaved OPN might be the

paramount form of HCC-expressed OPN, which promotes hepatocellular metastasis through

its well-exposed integrin-beta-binding domain and the activation of the integrin-beta1/FAK

pathway, which in turn has been already associated with metastatic processes (Shibue &

Weinberg, 2009; Itoh et al, 2004).

It has been later demonstrated that OPN might promote HCC metastasis by regulating

46

hepatocellular epithelial-to-mesenchymal transition (Bhattacharya et al, 2012) through direct

interaction with- and stabilization of vimentin (Dong et al, 2016). In line with these

observations, the combined evaluation of OPN and vimentin expression levels improves

HCC prognostic performances when compared to OPN alone (Dong et al, 2016).

47

Aim of the doctoral thesis

Although significant advances in the understanding of the pathophysiological role of OPN in

metabolism and inflammation have been achieved, the nature of NAFLD and the lack of

reliable mouse models to study the entire NASH-HCC evolution did not allow to unravel

OPN’s action in NAFLD-derived hepatocarcinogenesis. Based on the previously described

literature, I hypothesized that OPN signaling plays a detrimental role in NAFLD, and

therefore promotes NAFLD-derived hepatocarcinogenesis. This hypothesis could be tested

thanks to a recently described murine model, which spontaneously and sequentially

develops the full spectrum of NAFLD-related liver pathologies, strongly reflecting human

disease evolution (Fujii et al, 2013). Hence, the specific aims of this research project are:

To establish in the lab, in which the doctoral study has been carried out, the recently

described NASH-HCC mouse model (Fujii et al, 2013)

To characterize the established mouse model in order to define i) OPN expression

pattern throughout disease evolution, and ii) the optimal time points for the specific

investigation of hepatic manifestations in future studies

To apply the established NASH-HCC mouse model on wild-type and OPN-deficient

(Spp1-/-) mice in order to assess the role of OPN in the establishment and

development of NASH-associated hepatocarcinogenesis.

48

RESULTS

Prologue

I contributed to the following scientific article by reproducing in the animal facility of the

Medical University of Vienna the NASH-HCC mouse model utilized throughout the study. I

also performed the detailed characterization of the model and carried out the most of the

laboratory in vivo and ex vivo examinations. I further processed and analyzed the obtained

data, planned further experiments to test newly arisen hypotheses and wrote the final

manuscript.

Publication

49

Received: 15 October 2019 | Revised: 14 February 2020 | Accepted: 31 March 2020

DOI: 10.1111/liv.14464

OR I G I NAL AR T I C L E

Impact of osteopontin on the development of non-alcoholic liver

disease and related hepatocellular carcinoma

Alexander D. Nardo1 | Nicole G. Grün1 | Maximilian Zeyda1,2 | Monika Dumanic3 |

Georg Oberhuber4

Michael Roden7,8,9

Elisa Rivelles5 | Thomas H. Helbich3,6 Thierry

Claudel10 | Michael Trauner10

| Daniel F. Markgraf7 |

| Thomas M. Stulnig1

|

| 1Christian Doppler Laboratory for Cardio-Metabolic Immunotherapy and Clinical Division of Endocrinology and Metabolism, Department of Medicine III, Medical

University of Vienna, Vienna, Austria

2Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria

3Division of Nuclear Medicine, Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Austria

4Department of Pathology, General Hospital of Innsbruck, Innsbruck, Austria

5Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

6Division of Molecular and Gender Imaging, Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Austria 7German

Diabetes Center, Leibniz Center for Diabetes Research, Institute for Clinical Diabetology, Heinrich Heine University, Düsseldorf, Germany 8German Center of Diabetes

Research (DZD e.V.), München-Neuherberg, Germany

9Division of Endocrinology and Diabetology, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany

10Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology & Hepatology, Medical University of Vienna, Vienna, Austria

Correspondence

Thomas M. Stulnig, Third Department of Medicine, Hietzing Hospital, Wolkersbergenstrasse 1, Vienna 1130, Vienna, Austria. Email:

thomas.stulnig@meduniwien.ac.at

Present address

Alexander D. Nardo, Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology & Hepatology, Medical University of Vienna, Vienna, 1090, Austria

Thomas M. Stulnig, Third Department of Medicine and Karl Landsteiner Institute for Metabolic Diseases and Nephrology, Hietzing Hospital, Vienna, 1130, Austria

Funding information

FWF, Grant/Award Number: W1205-B09;

Federal Ministry of Economy, Family and

Youth; National Foundation for Research,

Technology and Development

Handling Editor: Helen Reeves

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the

original work is properly cited.

© 2020 The Authors. Liver International published by John Wiley & Sons Ltd

| 1620 wileyonlinelibrary.com/journal/liv Liver International. 2020;40:1620–1633.

Abstract

Background & aims: Osteopontin, a multifunctional protein and inflammatory cy- tokine, is

overexpressed in adipose tissue and liver in obesity and contributes to the induction of

adipose tissue inflammation and non-alcoholic fatty liver (NAFL). Studies performed in both

mice and humans also point to a potential role for OPN in malig- nant transformation and

tumour growth. To fully understand the role of OPN on the development of NAFL-derived

hepatocellular carcinoma (HCC), we applied a non-al- coholic steatohepatitis (NASH)-HCC

mouse model on osteopontin-deficient (Spp1−/−) mice analysing time points of NASH,

fibrosis and HCC compared to wild-type mice. Methods: Two-day-old wild-type and

Spp1−/− mice received a low-dose streptozo- tocin injection in order to induce diabetes, and

were fed a high-fat diet starting from week 4. Different cohorts of mice of both

genotypes were sacrificed at 8, 12 and 19 weeks of age to evaluate the NASH, fibrosis and

HCC phenotypes respectively.

50

| 1621 NARDO et Al.

1 | INTRODUC TION

Hepatocellular carcinoma (HCC) is the most common form of liver

cancer and the cause of approximately one million deaths yearly,

with an alarming mortality rate of 94%.1 Even though hepatitis B-

(HBV) and C (HCV) virus infections are major risk factors for HCC,

nutrition-related diseases strongly promote the increase in HCC

prevalence worldwide. This may be due to a chain of events starting

with obesity, metabolic syndrome, type-2 diabetes mellitus (T2DM)

and fatty liver and potentially leading to non-alcoholic liver steato-

hepatitis (NASH), liver fibrosis and finally HCC.

Non-alcoholic fatty liver (NAFL) will likely drive the increase in the

incidence of HCC in the next decades.2 Approximately 20% of patients

manifesting simple steatosis further develop non-alcoholic steato-

hepatitis.3 NASH mainly occurs when the rate of liver non-esterified

fatty acids (NEFA) uptake surpass its capacity for esterification into tri-

glycerides (TG).4,5 NASH, consequently, is a driver for the development

of fibrosis/cirrhosis and finally HCC.6 Hence, the study of hepatocar-

cinogenesis on a metabolic syndrome background is gaining significant

interest on the individual as well as public health level. However, un-

derlying molecular mechanisms are far from being understood.

Osteopontin (OPN; gene Spp1) is a multifunctional protein highly

expressed in activated macrophages and T-cells, but also in hepatic

stellate cells and hepatocytes. In obesity, OPN is vastly overex-

pressed in adipose tissue and induces infiltration and activation of

macrophages generating a pro-inflammatory environment, which

crucially contributes to the onset of insulin resistance.7 Hepatic

OPN expression is up-regulated in obesity8 and various models of

liver injury.9,10 Furthermore, OPN is involved in the pathogenesis

of NAFL associated with visceral obesity 11 and is a reliable bio-

marker for NASH/fibrosis in human non-alcoholic fatty liver disease

(NAFLD).12,13 Studies from others and our lab point to a pivotal role

of OPN in obesity-driven nutrition-dependent diseases including

high-fat diet-induced fatty liver 14-17 and thus suggest OPN as a

treatment target.

Also downstream in the proposed order of events, OPN is

highly upregulated in HCC and may even be evaluated as a po-

tential therapeutic target in HCC.18,19 However, a causal role of

OPN in the pathogenesis of NASH and NASH-derived HCC is still

not defined.To elucidate the impact of OPN on the sequential de-

velopment of NASH and derived HCC as occurring in metabolic

syndrome we took advantage of a novel mouse model,20 which

starting from hyperglycaemia recapitulates the development of

NAFL, NASH, fibrosis up to HCC. Applying this model to wild-type

(WT) and OPN-deficient (Spp1−/−) animals, we provide evidence

for a Janus-type role of OPN in various states of NASH-HCC pro-

gression eventually resulting in enhanced NAFL, NASH and fibro-

sis but also more highly differentiated HCC and improved overall

survival rate in Spp1−/− mice.

Key points

• Non-alcoholic fatty liver disease (NAFLD) is currently

driving the increase in hepatocellular carcinoma (HCC)

prevalence worldwide

• Dysregulated osteopontin (OPN) expression was associ-

ated with both obesity-derived metabolic syndrome and

HCC malignant development. However, a causal role for

OPN in the pathogenesis of NAFLD and NAFLD-derived

HCC has yet to be established

• In a mouse model of NAFLD-derived HCC, OPN defi-

ciency worsens hepatic steatosis and fibrosis, while im-

proving systemic inflammation, overall survival and HCC

outcomes

• Therefore, we propose OPN as a putative therapeutic

target in advanced, metabolic syndrome-associated

chronic liver diseases.

Results: Spp1−/− animals showed enhanced hepatic lipid accumulation and aggravated NASH, as

also increased hepatocellular apoptosis and accelerated fibrosis. The worse steatotic and

fibrotic phenotypes observed in Spp1−/− mice might be driven by en- hanced hepatic fatty

acid influx through CD36 overexpression and by a pathologi- cal accumulation of specific

diacylglycerol species during NAFL. Lack of osteopontin lowered systemic inflammation,

prevented HCC progression to less differentiated tumours and improved overall survival.

Conclusions: Lack of osteopontin dissociates NASH-fibrosis severity from overall survival and

HCC malignant transformation in NAFLD, and is therefore a putative therapeutic target only

for advanced chronic liver disease.

K E Y WO R D S

acute-on-chronic liver failure, fibrosis, lipoapoptosis, metabolic syndrome, non-alcoholic fatty

liver

51

1622 | NARDO et Al.

2 | METHODS Classification of Tumours of the Digestive System.21 Pancreas sec-

tion was formalin-fixed, paraffin-embedded and stained with haema-

toxylin/eosin to quantitatively evaluate Langerhans islets. Apoptosis

was assessed by terminal deoxynucleotidyl transferase-mediated

dUTP-biotin nick-end labelling (TUNEL) staining using the TUNEL

Andy Fluor™ 488 Apoptosis Detection Kit (Genecopoeia, Maryland,

USA) following product instructions. Hepatocyte proliferation was

assessed by BrdU immunostatining (Life Technologies, California,

USA). Nuclei were counterstained with Hoechst 33 342 and the pro-

portion of TUNEL positive cells was quantified using ImageJ soft-

ware (National Institute of Health) in an automated fashion.22

2.1 | Animals

All experimental procedures were approved by the institutional

animal care and use committees. Wild-type and Osteopontin knock

out (Spp1−/−, B6.129S6(Cg)-Spp1tm1Blh/J) mice on a C57BL/6J

background were purchased from Charles River Laboratories Inc

(Wilmington, Massachusetts, USA) and cohoused to minimize poten-

tial microbiome effects. Animals were treated as originally described

by Fujii and colleagues 20: two-day-old male newborns of both geno-

types received a single subcutaneous STZ (Sigma, Missouri, USA)

injection and were fed ad libitum a high-fat diet (HFD, 60 kcal%,

D12492; Research Diets, New Jersey, USA) starting at 4 weeks of

age. 14-hours fasting blood sugar (FBS) and body weight (BDW)

were assessed in 4-week-old mice, and four FBS-matched cohorts

per genotype were generated (n = 15). Of each group, a repre-

sentative subgroup of eight animals (FBS-matched between geno-

types) with lower alpha-diversity was used for molecular analyses.

Three-to-four littermate mice were housed together in wooden

bedding–containing cages in the presence of cage enrichment in a

light-controlled and temperature-controlled facility. At each time

point, animals were overnight fasted (dark cycle) and later sacrificed

by neck dislocation (light cycle). To assess in vivo proliferative poten-

tial of hepatocytes, all mice received an intraperitoneal injection of

5-Bromo-2′-deoxyuridine (BrdU) (Sigma) 2 hours prior to sacrifice.

Blood samples were drawn from the tail vein. Liver, subcutaneous

and visceral white adipose tissue (SWAT, GWAT), kidney and small

intestine samples were weighted, formalin-fixed and/or snap-frozen

for further analyses.

2.4 | Hepatic gene expression analyses

RNA was extracted from livers using TRIzol Reagent (Invitrogen,

California, USA). Complementary DNA obtained by reverse-tran-

scription was amplified using the appropriate, commercially avail-

able, gene expression assays (Life Technologies, California, USA).

Gene expression was normalized to ubiquitin C (Ubc) and analysed

by quantitative real-time RT-PCR on an ABI Prism 7000 cycler (Life

Technologies, California, USA).

2.5 | Western blotting

Liver samples were homogenized (Precellis 24, Bertin Technologies,

France) in RIPA buffer and centrifuged. Supernatants were collected

and assayed for protein concentration via BCA Protein Assay Kit

(Thermo Fisher Scientific, Massachusetts, USA). Fifteen milligrams

of protein/sample were separated by electrophoresis and blotted

on nitrocellulose membranes (Bio-Rad, California, USA), which were

then blocked and incubated overnight with a primary antibody to

OPN (AF808, R&D Systems, Minnesota, USA) 1:2000 diluted, and

beta-actin (A1978, Sigma, Missouri, USA) 1:10 000. After incuba-

tion with secondary antibodies, membranes were developed via the

Fusion FX Western Blotting and Chemi Imaging (Vilber Lourmat,

France), using the BM Chemiluminescence Blotting Substrate

(Roche, Switzerland). Bands intensity was quantified using ImageJ

software (National Institute of Health, Maryland, USA).

2.2 | Computed tomography

Liver X-ray computed tomography (Siemens Inveon µCT, by Siemens

Medical Solutions, Knoxville, USA) was used to non-invasively meas-

ure number and volume of liver tumours in isoflurane-anaesthetized

mice. Before the µCT X-ray examination each mouse was adminis-

tered 100µL of CT contrast medium intravenously (ExiTron™ nano

6000, Miltenyi Biotec GmbH, Germany) to improve the soft tissue

contrast. During the examination, the mice were placed on a heated

bed and the vital parameters (body temperature and respiratory

rate) were constantly monitored and a protective eye ointment was

applied. After completion of the study, mice were sacrificed by neck

dislocation and tissue samples were harvested as described above.

2.6 | Plasma biochemistry

Blood glucose was measured using a One Touch Ultra glucose meter

(LifeScan Inc, California, USA). Plasma alanine aminotransferase

activity (ALT) was determined using the Vitros 5600 technology

(Ortho Clinical Diagnostics, New Jersey, USA). Insulin was measured

using an ultrasensitive mouse insulin ELISA (Mercodia AB, Sweden),

serum amyloid P (SAP) by a commercially available ELISA kit (ALPCO

Immunoassays, New Hampshire, USA). NEFA concentration was

enzymatically assessed using a commercially available kit (Sigma,

Missouri, USA).

2.3 | Histological analyses

Liver sections from the left lobe were formalin-fixed, paraffin-em-

bedded and were stained with haematoxylin/eosin, or Sirius red to

assess liver fibrosis. Stained sections were evaluated by an expert

pathologist, blind to genotype and study period, following the WHO

52

| 1623 NARDO et Al.

2.7 | Liver biochemistry plasma alanine aminotransferase (ALT) was markedly elevated in the

Spp1−/− genotype (Figure 1D).

To evaluate the potential mechanisms of hepatic TG accumula-

tion in Spp1−/− animals, we measured the expression of genes playing

a pivotal role in hepatic lipid homeostasis. Surprisingly, expression

of Acaca was comparable between WT and Spp1−/− mice, while

Srebf1, Fasn and Scd1 where even significantly downregulated in the

Spp1−/− genotype (Figure 2A), indicating that the increased steato-

sis observed in Spp1−/− mice cannot be explained by enhanced de

novo lipogenesis (dnl). Also, expression levels of Ppara and Ppargc1a

did not differ between the two groups (Figure 2C), while Dgat1 and

Dgat2 enzymes were markedly downregulated in the Spp1−/− group

(Figure 2D). While the gene for the fatty acid transporter FATP4

(Slc27a4) was unchanged between genotypes (Figure 2B), gene ex-

pression of fatty acid translocase (CD36/FAT), a member of the class

B scavenger receptor family essential for fatty acid (FA) uptake and

lipid metabolism, was markedly increased in Spp1−/− compared to

WT animals (Figure 2B). Also, while inflammation was evaluated as

comparable between groups by liver histology (Figure 1B), gene ex-

pression analyses showed a non-significant and significant increase

in expression of the main hepatic inflammatory markers Tnf and Ccl2

in Spp1−/− mice respectively (Figure 2E). These data strongly empha-

size that increased liver lipid uptake by overexpression of the CD36

FA translocase could contribute to the increased hepatic steatosis

and inflammation in NASH-HCC-Spp1−/− animals. These differences

are observable only upon metabolic challenge (Figure S2A), meaning

that the lack of OPN expression does not influence hepatic lipid me-

tabolism at baseline.

TG concentration was enzymatically assessed using a commercially

available kit (Sigma, Missouri, USA), after chloroform-methanol lipid

extraction.

2.8 | Targeted lipidomics of

diacylglycerols and ceramides

Lipids were extracted, purified and analysed from frozen samples,

using lipid chromatography mass spectrometry as adapted from

Kumashiro et al.23 Briefly, tissue was homogenized in 20 mM Tris/

HCl, 1 mM EDTA 0.25 mM EGTA, pH 7.4, internal standards were

added and samples were centrifuged for 1 h (100 000 g, 4°C). Lipid

droplet, cytosol and membrane fractions were collected and lipids of

each fraction were extracted,24 followed by solid phase extraction

(Sep Pak Diol Cartridges; Waters, Milford, MA, USA). The resulting

lipid phase was dried and re-suspended in methanol. Lipid analytes

were separated using a Phenomenex Luna Omega column (1.6 µm

100A; Phenomenex, Torrance, CA, USA) on an Infinity 1290 HPLC

system (Agilent Technologies, Santa Clara, CA, USA) and analysed by

multiple reaction monitoring on a triplequadrupole mass spectrom-

eter (Agilent 6495; Agilent Technologies), operated in the positive

ion mode.

2.9 | Statistics

Data are presented as mean ± SEM, and two groups compared by

unpaired Student t-test, with a significance level of <0.05. When

more than two groups were compared, ANOVA with Tukey´s post

hoc analyses were performed. For the survival observations, Kaplan-

Meyer analysis was applied.

3.2 | Increased liver fibrosis in OPN-deficient mice

Hepatic fibrosis evolves from NAFL/NASH on a metabolic syn-

drome background. In line with the worse steatotic phenotype of

Spp1−/− livers, markedly increased liver collagen deposition and

myofibroblast activation were observed in Spp1−/− mice as shown

by Sirius Red staining and α-SMA immunostaining respectively

(Figure 3A,B). While WT mice just showed mild-to-moderate per-

isinusoidal and zone-3 fibrosis, fibrosis was also extended to the

portal and periportal area resulting in a higher fibrosis score in

Spp1−/− livers (Figure 3C). Only these livers also showed well-de-

fined bridging fibrosis and fibrotic septa (Figure 3A). In addition,

a significant increase in gene expression of pro-fibrogenic mark-

ers (Col1a1, Col 4a1, Timp1) was found in Spp1−/− mice (Figure 3D).

Of note, no changes in Tgfb expression were observed between

genotypes and time points (Figure 7C). Hepatocellular apoptosis

is implicated in the progression of fibrotic liver disease.25 Since

we and others have previously shown the anti-apoptotic poten-

3 | RESULTS

3.1 | Increased hepatic lipid uptake in NASH-HCC OPN-

deficient mice promotes liver steatosis

Spp1−/− and WT mice both developed comparable degrees of hyper-

glycaemia early after streptozotocin (STZ) treatment (plasma glu-

cose 378 ± 16 mg/dL and 348 ± 20 mg/dL, respectively), indicating

comparable efficiency of STZ administration. In line with that, the

magnitude of STZ-induced Langerhans islets damage was also com-

parable between genotypes (Figure S2B). Mice were sacrificed after

4 weeks of high fat diet (HFD) treatment to evaluate NAFL/NASH

phenotype. Body weight as well of weights of GWAT and SWAT and

plasma NEFA were similar (Figure S1). As shown by haematoxylin

and eosin-staining (Figure 1A) and the NAS score (Figure 1B) as well

as hepatic TG quantification (Figure 1C), there was a significantly

increased lipid accumulation in livers of Spp1−/− mice. Accordingly,

OPN,15,26 tial of we hypothesized that enhanced apoptosis in

OPN-deficient livers could contribute to the increased hepatic

fibrosis. Accordingly, terminal deoxynucleotidyl transferase-me-

diated dUTP-biotin nick-end labelling (TUNEL) staining of liver

sections revealed an increased proportion of apoptotic cells in

53

1624 | NARDO et Al.

FI G U R E 1 Lack of osteopontin

enhances hepatic steatosis in NASH/HCC

mice. A, Representative H&E-stained

sections of WT (left panel) and OPN

knock-out (Spp1−/−, right panel) NASH-

HCC livers. Scale bar = 100 µm. B, Non-

alcoholic fatty liver disease score (NAS),

qualitatively evaluated out of the H&E-

stained sections. NAS is the sum of the

scores attributed to the three hallmarks

of NAFLD, ie steatosis, hepatocellular

ballooning and inflammation. C-D, Liver

triacylglycerol content (C) and plasma

alanine aminotransferase activity (D).

**P ≤ .01, ***P ≤ .001. Tissues were

harvested from 8-week-old mice (n = 8 per

group). See also Figures S1 and S2

NASH-HCC-Spp1−/− (Figure 2). Looking for potential molecular

drivers of the enhanced steatosis and fibrosis developed by NASH-

HCC-Spp1−/− mice, we performed targeted lipidomic analyses with

emphasis on diacylglycerols (DAGs) and ceramides (CERs) of livers

at the NAFL/NASH time point. Most of the DAG species were un-

changed between genotypes. Membrane DAG 18:1/18:1 signifi-

cantly increased in the absence of OPN (Figure 5C), as also DAG

16:0/18:2 in the lipid droplet compartment (Figure 5B). Membrane

CER 24:0 and especially 22:0 decreased in Spp1−/− livers (Figure 5F).

No significant increase in other ceramide species was observed in

Spp1−/− when compared with WT mice (Figure 5D-F). These data

support the hypothesis of a putatively lipotoxic accumulation of

specific DAG species driving the progression of hepatic disease in

NASH-HCC-Spp1−/− mice.

OPN-deficient mice compared to WT (Figure 4A,B). To assess

hepatocyte proliferation, the number of hepatocytes in replicative

S-phase was evaluated by BrdU immunostaining. The percentage

of proliferating hepatocytes was significantly higher in the livers

of Spp1−/− mice (Figure 4C,D), thus indicating a compensatory hy-

perproliferation of hepatocytes.

3.3 | Accumulation of specific diacylglycerol species during

NAFL in NASH-HCC-Spp1−/− mice

Hepatic lipid accumulation in NASH-HCC WT and Spp1−/−

mice seemed to rely on different mechanisms: increased lipo-

genesis in NASH-HCC-WT, but increased lipid uptake in

54

| 1625 NARDO et Al.

FI G U R E 2 Enhanced hepatic steatosis in Spp1−/− mice might be induced by increased CD36-mediated lipid uptake. A-E, Analysis of gene

expression regulating de novo lipogenesis (A), hepatic fatty acid uptake (B), beta-oxidation (C), fatty acid esterification (D) and inflammation

(E). *P ≤ .05, ***P ≤ .001. Tissues were harvested from 8-week-old mice (n = 8 per group). See also Figure S2

3.4 | Osteopontin deficiency prevents HCC

dedifferentiation

statistical significance (Figure 6F). The hampered HCC progression

observed in OPN-deficient mice and the specific overexpression

of OPN at the HCC time point in livers of NASH-HCC-WT animals

(Figure 7A,B) point toward a causative role for OPN in the dediffer-

entiation of HCCs harboured on a metabolic syndrome-background.

Given the central role played by OPN in hepatic inflammation and

carcinogenesis,27,28 and since the immunological milieu significantly in-

fluences tumorigenesis,29 we further analysed the expression pattern of

the principal inflammatory markers during disease evolution. Tnfa and

Ccl2 expression peaks at the fibrosis stage in Spp1−/− livers, decreasing

again later atthe HCCtime point(Figure 7C). Ofnote, the expression level

of the same genes increases over time in livers of WT mice and peaks at

the HCC stage (Figure 7C). The expression pattern of the pan-macro-

phage marker Emr1 follows the same trends, indicating altered macro-

phage recruitment in livers of Spp1−/− compared to WT NASH-HCC mice

(Figure 7C). Furthermore, we measured early and transient upregulation

of both specific pro-inflammatory and anti-inflammatory macrophage

markers (Itgax and Mrc1, respectively) in OPN-deficient livers. Only

Mrc1, an M2-like macrophage marker, is significantly overexpressed at

Already at 12 weeks of age, which corresponds to the liver fibrotic

stage, 87.5% of OPN-deficient animals developed liver tumours, which

were histologically defined as HCCs (data not shown). In the WT

group, just 1/8 of the mice showed HCCs at this time point (Figure 3E).

Mice were finally analysed at 19 weeks of age in order to eval-

uate hepatocellular carcinomas. CT scans showed widespread liver

tumours in both WT and Spp1−/− mice, but no significant differences

in tumour size and number were observed (Figure 6A-C). However, a

higher degree of tumour dedifferentiation in WT compared to Spp1−/−

animals was shown in histologic analyses, as indicated by a signifi-

cantly higher tumour grade in livers of wild-type mice (Figure 6D).

Expression levels of HCC markers revealed a marked overexpression

of Afp in tumours from WT compared to Spp1−/− animals. Moreover

when compared with the adjacent non-tumour tissue, its expres-

sion was upregulated only in WT but not in Spp1−/− mice (Figure 6E).

mRNA levels of Gpc-3 followed the same trend but did not reach

55

1626 | NARDO et Al.

FI G U R E 3 OPN protects against NAFL/NASH-induced hepatic fibrosis. A, Representative Sirius red (upper panels) and α-SMA (lower

panels) stained sections of WT (left panels) and Spp1−/− (right panels) NASH-HCC livers. Scale bar = 500 µm. B, Collagen deposition

(red signal) was quantified in an automated fashion. C, Fibrosis score, qualitatively assessed on H&E-stained liver sections by an expert

pathologist, blind to the genotype. D, Gene expression analysis of the main pro-fibrogenic markers. E, Tumour incidence at the fibrosis stage

(week 12). *P ≤ .05. Tissues were harvested from 12-week-old mice (n = 8 per group)

the HCC stage in WT livers (Figure 7C). These data strongly suggest

that the lack of OPN hampers HCC progression and dedifferentiation by

modulating the hepatic inflammatory kinetics.

NASH-HCC-WT mice as shown by increased serum amyloid P (SAP)

levels (Figure 8B). Hence, OPN promotes a systemic pro-inflamma-

tory milieu, which significantly reduces survival in non-alcoholic

fatty liver disease.

3.5 | Osteopontin deficiency reduces liver- related

mortality 3.6 | The worse liver condition in OPN-deficient mice is

the consequence of a better overall metabolic homeostasis

As described earlier, NASH-HCC mice started to die after week 11,

which corresponds to the liver pre-fibrotic/fibrotic stage.30 In the

present study, the mortality after 19 weeks in the WT group was

30% and significantly higher than in Spp1−/− mice (14%; P = .0085)

(Figure 8A). All dropouts showed symptoms of hepatic toxicity, such

as microvesicular steatosis (Figure S3) indicating acute-on-chronic

liver failure (ACLF) as described in patients with chronic liver dis-

ease.31 Mortality in clinical ACLF correlates with the magnitude of

systemic inflammation,31 which was significantly enhanced also in

NAFLD in metabolic syndrome is related to systemic metabolic

dysregulations. We wanted, therefore, to clarify the role of OPN

on metabolic homeostasis. At variance with Spp1−/− mice, some

NASH-HCC-WT animals at eight weeks of age (3 out of 8) showed

hepatic cytoplasmic and nuclear deposition of glycogen, symptom

of worse glycaemic control. STZ-treated mice exhibit hyperglycae-

mia throughout their entire life without differences in fasting blood

56

| 1627 NARDO et Al.

FI G U R E 4 Enhanced apoptosis and proliferation in Spp1−/− mice. A, TUNEL assay for the identification of apoptotic cells in WT (upper

panel) and Spp1−/− (lower panel) NASH-HCC livers. Hoechst 33 342 (blue signal, left panels) stains all nuclei, while apoptotic cells are

marked with green fluorescence (central panels). Right panels represent the merge of the two signals. Scale bar = 200 µm. Inserts at higher

magnification, scale bar 50 µm. B, Graph of the proportion of TUNEL-positive cells. C, BrdU staining for the identification of proliferating

cells. Sections were incubated with anti-BrdU antibody and counterstained with haematoxylin. Positive nuclei are depicted in red and

highlighted by black arrows. Scale bar = 50 µm. *P ≤ .05, **P ≤ .01. D, Analysis of BrdU-positive cells/25 mm2 area. Tissues were harvested

from 12-week-old mice (n = 8 per group)

sugar (FBS) between WT and Spp1−/− mice at 4 and 6 weeks of age

(Figure 8C). However, OPN deficiency was associated with slightly

though significantly reduced FBS (Figure 8C) and also with higher

numbers of intact Langerhans islets (Figure 8E) and increased plasma

insulin levels (Figure 8F) in 8-week-old NASH-HCC mice. Moreover

mRNA expression of FOXO1 was markedly reduced in NASH-HCC-

Spp1−/− livers (Figure 8D), and G6pc and Pck1 genes, which en-

code key gluconeogenic enzymes and are transcriptional targets of

FOXO1, were suppressed in OPN-deficient livers (Figure 8D, statis-

tically significant only for G6pc). Hence, higher insulin secretion and

downregulation of hepatic FOXO1 and its target genes in NASH-

HCC Spp1−/− mice could contribute to the beneficial effects of OPN

deficiency on glucose metabolism, as also to the detrimental effect

on hepatic lipid accumulation and toxicity.

and the forecasted future leading aetiology for HCC. Insights into

the establishment and development of such a sequela of pathophysi-

ological events may provide new interventional strategies focused

on blocking, or at least delaying, this yet uncontrollable process.

Unfortunately, the impossibility to sample patients´ livers at each

stage of the disease and the lack of an animal model that faithfully

mirrors the human pathological progression and aetiology made it

impracticable until today. In the current study, we used a recently

described mouse model, which sequentially develops NASH, fibro-

sis and HCC on a background of diabetes and obesogenic diet, to

finally assess the role of OPN, a putative prognostic and therapeu-

tic target for liver cancer, on the development of HCC in NAFLD. In

spite of OPN’s well-established inflammatory role, absence of OPN

even worsened hepatic steatosis and fibrosis, but reduced dedif-

ferentiation of HCCs and liver-failure-related mortality in mice on a

background of hyperglycaemia and high-fat diet. Even though OPN

exhibits some beneficial effects in early NAFLD stages, it promotes

dedifferentiation of HCC and organ failure probably through its pro-

inflammatory function. Most importantly, OPN ablation dissociates

NASH-fibrosis severity from overall survival and HCC malignant

4 | DISCUSSION

NAFL is a hallmark of metabolic syndrome, the committing step for

the potential further evolution of NASH, fibrosis and liver cancer,

57

1628 | NARDO et Al.

FI G U R E 5 Targeted lipidomic analyses at the NAFL/NASH time point. A-C, Total amount of individual DAG species in the (A) cytosolic,

(B) lipid droplet and (C) membrane fraction. D-F, Total amount of each individual CER species per cellular fraction. Black bars represent WT,

while grey bars Spp1−/− animals. *P ≤ .05, **P ≤ .01, ***P ≤ .001. Tissues were harvested from 8-week-old mice (n = 8 per group)

transformation. This study hence provides a basis for further trans-

lational research to target OPN in order to reduce liver-related mor-

tality in advanced NAFLD.

Deregulated hepatic NEFA influx covers a predominant role in

human NAFLD.32,33 An increased hepatic NEFA influx through spe-

cific membrane translocases, such as CD36, eventually induces en-

hanced hepatic steatosis also in the NASH-HCC mouse model in use,

upon genetic OPN deletion. Both experimental and clinical data show

that CD36 expression positively correlates with liver fat deposition

under conditions of elevated lipids and induces lipoapoptosis-depen-

dent inflammation and fibrosis.34,35 Elevated lipid uptake is not ex-

cluded by the downregulation of hepatic Dgat1 and Dgat2 expression.

Recently published data demonstrated that substrate flux, and not

Dgat expression, is the dominant regulator of hepatic TG synthesis.36

Moreover the concomitant increment in lipid uptake and reduction

in FA esterification potential also explains the significant accumu-

lation of DAGs in Spp1−/− livers. CD36 expression is controlled by

peroxisome proliferator-activated receptor gamma (PPARγ), and the

accumulation of linoleic acid, a known source of PPARγ activating li-

gands,37 might explain the significant Cd36 overexpression in Spp1−/−

hepatocytes. Furthermore, OPN was shown to positively impact on

JNK downstream pathways,38 and PPARγ activity can be inhibited

by JNK through phosphorylation of Serine 84.39 Therefore, lack of

OPN might reduce JNK-mediated suppression of PPARγ, resulting

in increased Cd36 expression. In a considerably less hyperglycaemic

model, which does not develop further fibrosis and HCC, it has been

shown that lack of OPN protects from HFD-induced hepatic ste-

atosis, due to the preservation of adipose tissue function in obesity,

hence preventing ectopic lipid accumulation in the liver.16,17 In con-

trast, the model used here is primarily hyperglycaemic but not obese

hence revealing the impact of hyperglycaemia in this process while

excluding a potential involvement of a dysfunctioning adipose tissue.

Several experimental and clinical studies also indicate that OPN is

involved in liver fibrogenesis.40,41 However, previous research did not

use models that replicate clinicopathological features of NAFL-related

fibrosis. In our model, hepatic fibrogenesis evolves from NAFL/NASH

on a diabetes background. The lack of OPN not only worsened the

steatotic and inflammatory phenotypes, but also enhanced hepatic

collagen deposition in NASH-HCC-Spp1−/− mice. We also provide ev-

idence of enhanced apoptosis and proliferation rate in Spp1−/− livers,

phenomena that tightly correlate with exaggerated tissue repair and

fibrogenesis. Overexpression of Timp1, a known inhibitor of the elas-

tin-degrading MMP12 and marker of tardive fibrosis,42 further con-

tributes to the more advanced fibrosis in Spp1−/− livers. While OPN

ablation was shown to reduce leptin-induced hepatic fibrosis in vitro

and in vivo,40 and ductal reaction and fibrosis in thioacetamide (TAA)-

treated mice,43 the NASH-HCC model, which closely mirrors the full

development of human disease up to HCC, reveals that NASH-fibrosis

may even exaggerate in the absence of OPN, when hyperglycaemia

and lipotoxicity are the driving forces.

Specific lipid metabolites are known to contribute to NASH-HCC

development. The significant accumulation of DAGs could be a cue

58

| 1629 NARDO et Al.

FI G U R E 6 Lack of OPN prevents HCC dedifferentiation. A, Representative in vivo liver X-ray computed tomography (CT) sections from WT

(left panel) and Spp1−/− (right panel) NASH-HCC mice. B, Number of tumours per liver and (C) tumour volumes were quantitatively evaluated

in an automated fashion. D, Tumour grading was histologically evaluated on H&E-stained liver sections by an expert pathologist, blind to

the genotype. E-F, Gene expression analysis of the HCC markers Afp (E) and Gpc-3 (F) in both tumour and non-tumour areas. Tissues were

harvested from 19-week-old mice. n = 3 per group for CT analyses, otherwise n = 8 per group. **P ≤ .01 compared to WT non-tumour area

for enhanced lipotoxicity-induced hepatocellular apoptosis in NASH-

HCC-Spp1−/− animals. The significant enrichment in DAG species con-

taining palmitic and linoleic acid in lipid droplets further confirms the

paramount effect of FA uptake as mechanism of increased steatosis in

NASH-HCC-Spp1−/− mice. The DAG and CER profiles obtained are in

line with previous studies 44,45 and confirm again the worse non-alco-

holic fatty liver disease phenotype developed in the absence of OPN. To

pinpoint the role of lipotoxicity on tissue-specific and systemic inflam-

mation, which is proposed by us as a key player in mortality and HCC

dedifferentiation, we plan to perform direct measures of FA uptake by

the liver and a detailed quantification of extrahepatic lipid distribution.

At 12 weeks of age, NASH-HCC-Spp1−/− mice harboured sig-

nificantly more spontaneous HCCs than WT. Previous reports on

NASH-HCC-WT animals just identified simple hepatic nodules at this

time point,20,30 suggesting OPN as a key tumour suppressor factor

in early HCC development. The increased proliferation rate probably

compensating for enhanced apoptosis may have paved the way to

early HCC development in Spp1−/− mice. Interestingly, at 19 weeks of

age, no significant differences in tumour size and number between

genotypes were measured. However, tumours harboured in OPN-

expressing, WT animals reached a significantly higher level of de-

differentiation compared with HCCs grown in OPN-deficient livers,

which remained well-differentiated. The specific overexpression of

OPN at the HCC time point in livers of NASH-HCC-WT animals may

hence be related to dedifferentiation of HCCs, and consequently

increase tumour and metastasis-related risks. Our data are in line

with previous publications asserting that OPN mainly promotes

late events in hepatocarcinogenesis, such as epithelial-to-mesen-

chymal transition (EMT), invasion and metastasis.46,47 Based on our

results, we also provide evidence that OPN induces HCC progres-

sion and dedifferentiation by modulating the hepatic inflammatory

kinetics. The significant overexpression of pro-inflammatory cyto-

kines during the fibrosis time point seems to induce macrophage

recruitment into the livers of Spp1−/− mice. As shown by the relative

expression of the main macrophage polarization markers, a compa-

rable amount of them are M1- and M2-activated, which may elicit

59

1630 | NARDO et Al.

FI G U R E 7 Expression patterns of OPN and other immunological markers. A, Gene expression assessment of OPN, black bars represent

WT, while grey bars Spp1−/− animals (n = 8 per group). B, Immunoblot against OPN at different time points in WT animals. C, Hepatic gene

expression analyses of Tnfa, Ccl2, Tgfb, Emr1, Itgax and Mrc1 at 8, 12 and 19 weeks. *P ≤ .05, **P ≤ .01. Tissues were harvested between the

8th and the 19th experimental week. (n = 8 per group)

anti-tumour processes. At the HCC time point, this recruitment is

significantly blunted in OPN-deficient livers, indicating a possible

resolution, or at least a good control of tumour development. In WT

mice, on the other hand, significant overexpression of pro-inflamma-

tory cytokines occurs later in time, at the HCC time point in parallel

with macrophage recruitment, at a time when OPN is also strongly

overexpressed. The selective overexpression of the M2-like macro-

phage polarization marker Mrc1 let us presuppose an accumulation

of tumour-associated macrophages (TAMs) 48 in NASH-HCC WT liv-

ers. Hence, the adverse hepatic immunological milieu in later HCC

development correlates with OPN expression, indicating again its

detrimental role in the malignant outcome of liver cancers.

Even though OPN deficiency in NASH-HCC mice induces stron-

ger steatosis, steatohepatitis and fibrosis, it significantly protects

against organ failure-related death, which closely resembles human

acute-on-chronic liver failure. Indeed, the development of acute on

chronic liver failure occurs in the setting of systemic inflammation,

the severity of which correlates with the number of organ failures

and mortality.31 As shown by plasma SAP levels of NASH-HCC mice

at the NAFL/NASH time point, systemic inflammation is dramati-

cally higher in WT mice, most probably because of the ectopic lipid

deposition and the consequent lipotoxicity and failure of non-meta-

bolic organs. Hence, OPN probably promotes liver failure due to its

pro-inflammatory action.

The liver has a fundamental role in maintaining hepatic and

whole body glucose and lipid homeostasis: it senses these met-

abolic moieties and a plethora of hormones and other molecular

mediators, integrates the signals and responds providing the right

balance between glucose and lipid uptake, synthesis, storage and

secretion. Our investigations show that lack of OPN protects

against the HFD-induced pancreatic lipotoxicity and improves

therefore the overall metabolism and the hepatic function in

60

| 1631 NARDO et Al.

FI G U R E 8 Spp1−/− mice show a

healthier overall metabolic homeostasis

and an improved survival rate despite

worse steatotic and fibrotic liver

manifestations. A, Kaplan-Meyer curve

showing survival of WT (red full line)

and Spp1−/− (black full line) mice. B,

Serum amyloid P (SAP) concentration

in the plasma of 8-week-old NASH-

HCC mice. C, Fasting blood sugar (FBS)

measured during the experimental week

4, 6 and 8. Black bars represent WT,

while grey bars Spp1−/− animals. D, Gene

expression analysis of the main genes

regulating hepatic gluconeogenesis. E,

Amount of intact pancreatic Langerhans

islets, manually counted on H&E-

stained, scanned sections. F, Enzymatic

assessment of plasma insulin levels.

*P ≤ .05, **P ≤ .01, *** P ≤ .001. n = 15

for metabolic assessments, n = 8 for

molecular analyses. See also Figure S3

Spp1−/− mice. Indeed, we showed that lack of OPN protects against

cytoplasmic and nuclear glycogen deposition in hepatocytes.

Since hepatocellular glycogen deposition is the principal clinical

manifestation of glycogenic hepatopathy in humans,49 we fur-

ther investigated glycaemic control in vivo, which was improved

in OPN-deficient animals. Moreover the fact that fasting blood

sugar showed significant improvement only after four weeks of

HFD and that the amount of intact Langerhans islets was com-

parable between genotypes before the HFD challenge suggests

that OPN deficiency did not interfere with initial STZ treatment

but improved beta-cell survival or recovery thereafter, maybe

by reduced lipotoxicity.50,51 On the other hand, upon sustained

hyperglycaemia and hyperlipidaemia, the more responsive OPN-

deficient liver gets more severely damaged by the lipid overload,

while protecting other organs, such as pancreas and possibly skel-

etal muscles, and preventing therefore systemic inflammation and

mortality. On the contrary of OPN neutralizing studies by means

of anti-OPN antibodies,15,19,52 completely depleting OPN action

disrupts the paradigm that a more severe NASH phenotype will

increase the risk of severe fibrosis development and in turn also

liver-related mortality.53-56 Hence our study emphasizes a central

role for osteopontin in NAFLD development and might guide fu-

ture studies on therapeutic interventions based on this multifunc-

tional cytokine.

In summary, on a hyperglycaemic background resembling insuf-

ficiently controlled diabetes, OPN exerts not only detrimental but

also beneficial roles with respect to the development of NASH and

progression to HCC. OPN is likely involved in the intrinsic control

of excessive lipid uptake by the liver, and hence protects from li-

potoxicity, apoptosis and consequent fibrosis and hepatocyte pro-

liferation, which leads to differentiated HCC. On the other hand,

OPN promotes metabolic dysregulations observed in metabolic syn-

drome, dedifferentiation of HCC and organ failure probably through

its pro-inflammatory function. This study hence demonstrates a

bimodal Janus-type action of OPN, being tumour-protective at the

early stage while tumorigenic in the progressive phase. Furthermore,

OPN most likely plays a systemic role on inflammation and a hepatic

role on HCC malignant transformation in NAFLD. Now as the roles

of OPN in NASH-HCC development are established, further re-

search will focus on molecular mechanism of OPN action in early vs.

late stages of this devastating disease in order to elucidate potential

strategies for its prevention or treatment.

ACKNOWLEDG MENTS

We thank Ludwig Wagner, Anna Fenzl, Martina Hackl, Francesca

Bruschi, Matteo Tardelli, Nicole Auer, Victoria Kunczer, Clarissa

61

1632 | NARDO et Al.

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Kiefer FW, Zeyda M, Gollinger K, et al. Neutralization of osteopon- tin

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Kiefer FW, Neschen S, Pfau B, et al. Osteopontin deficiency pro-

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15.

16.

DECL AR ATION OF INTEREST

The authors declare no competing interests.

17.

ORCID

Alexander D. Nardo

Thomas M. Stulnig

18. https://orcid.org/0000-0003-1895-7161

https://orcid.org/0000-0003-3300-6161

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SUPPORTING INFORMATION

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Supporting Information section.

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Impact of osteopontin on the development of non-alcoholic

liver disease and related hepatocellular carcinoma. Liver Int.

2020;40:1620–1633. https://doi.org/10.1111/liv.14464 48.

63

CONCLUSIONS

With this thesis I have shown that lack of OPN improves overall survival and prevents HCC

dedifferentiation, although this genetic ablation worsens early hepatic manifestations in a

mouse model that closely mirrors human metabolic syndrome.

NAFL is a chronic and complex hepatic disease, caused predominantly by systemic

metabolic deregulations. Over the last decades, clinical investigations identified several

pathologic phenotypes, which characterize sequential stages of NAFLD, namely NAFL,

NASH, fibrosis/cirrhosis, and HCC. These manifestations are associated with unique clinical

severity levels and increased risks for liver-related mortality, and large-cohort studies on

patients´ natural history allowed to draw a causal line linking together the above-described

pathologic events. As a matter of fact, hepatic inflammation and necroinflammation correlate

with fibrotic stage in NASH patients (Argo et al, 2009; Pais et al, 2013), and NASH is

associated with a 7-fold-higher risk of fibrosis progression when compared with simple NAFL

(Kleiner et al, 2016; Singh et al, 2015). At the same time, multivariate analyses conducted on

large cohorts of NAFLD patients with long follow-up periods and systematic assessments of

national patient registries identified fibrosis stage as the only reliable predictor of hepatic

outcome (Ekstedt et al, 2015; Younossi et al, 2011; Angulo et al, 2015; Hagström et al,

2017).

It is still debated nowadays whether parameters defining NASH might also be used as

prognostic tools for severe liver outcomes in NAFLD, such as HCC and liver-related

mortality, since the above-described multivariate analyses tend to exclude these associations

(Ekstedt et al, 2015; Younossi et al, 2011; Angulo et al, 2015; Hagström et al, 2017). As Vlad

Ratziu pointed out recently, the relevance of multivariate statistical investigations shall be

carefully evaluated when the variables analysed are not strictly independent (Ratziu, 2017).

Therefore, univariate queries shall be preferred when evaluating the association of NASH

with HCC and liver-related mortality in NAFLD. In this regard, the extent of hepatocellular

ballooning, a pivotal hallmark of NASH, has been shown to predict severe hepatic outcomes

64

(Angulo et al, 2015), and patients with NASH develop earlier severe hepatopathies and have

significantly higher mortality than non-NASH patients over a 30-year follow up period

(Hagström et al, 2017).

Interestingly, our experimental results show that lack of OPN in a NASH-HCC mouse model

worsens the NASH and fibrotic phenotypes, while preventing NAFLD-related mortality and

HCC dedifferentiation (Nardo et al, 2020). Hence, these data propose OPN as a factor that

might dissociate early hepatic manifestations from severe liver outcomes. This hypothesis is

supported by a compelling list of evidence in other mouse models of human diseases.

Indeed, OPN has been shown to hamper squamous cell carcinoma growth, while OPN over-

expression promotes its metastatic transformation (Crawford et al, 1998). In the same way,

although OPN is over-expressed in human prostate carcinoma (PCa) and is regarded as a

predictor of disease outcome (Castellano et al, 2008; Thalmann et al, 1999), ablation of OPN

in a transgenic PCa mouse model prior to tumour establishment enhances tumorigenesis by

promoting a metastatic neuro-endrocine differentiation of the spontaneously harboured

neoplasms (Mauri et al, 2016). The duality of OPN action in acute/early vs chronic/late

disease development was further highlighted in allergies and autoimmune diseases (Weber

& Cantor, 2001; Xanthou et al, 2007). These results are in contrast with the observations

deriving from in vitro examinations and in vivo xenograft models, which only propose a pro-

tumorigenic role of OPN (Li et al, 2015; Song et al, 2016; Sun et al, 2008b). In this respect, it

has to be kept in mind that both in vitro and xenograft models represent conditions in which

tumors are already established, and only the worsening of the cancer phenotype (i.e.

metastasis, endothelial-to-mesenchymal transition, migration, invasion) can be investigated.

This important observation conciliates apparently conflicting findings, while further

suggesting that in vitro studies are less suited to study the duality of OPN effects. This

concept applies even more when trying to understand the cues behind the Janus-type

behaviour of OPN. Indeed, the site and cellular origin of OPN expression (Crawford et al,

1998), as also the transcriptional and post-transcriptional modifications characterizing OPN

in different pathophysiological conditions (Song et al, 2016; Xue et al, 2010) might influence

65

OPN action. Thus, now that OPN expression pattern and contribution to each stage of

NAFLD-associated hepatocarcinogenesis has been clarified (Nardo et al, 2020), more in-

depth investigations on the cellular origin and the biochemical properties of OPN in early vs

late and severe NAFLD stages is granted.

This study was designed to serve as a pioneering investigation of OPN function and

contribution in a chronic and highly complex metabolic disorder, whose pathological evolution

cannot be easily followed up in human patients (Ratziu, 2017). The new knowledge gained

with this study opens the way for a plethora of scientific questions, whose answers may help

us making a step further in therapy and prevention of one of the most deadly diseases

nowadays. At the same time, although OPN is highly conserved between humans and mice,

and evidence for the dualism of OPN action has been proven in a significant amount of

mouse models, many of them being considered the golden standards for the experimental

investigation of human diseases, the hypothesis that this phenomenon is unique to the

murine biology cannot be excluded yet. Therefore, future research should also more strongly

corroborate experimental data with clinical assessments in order to properly appraise the

reliability of murine models for the study of OPN.

OPN over-expression in obesity associates with hepatic insulin resistance (Bertola et al,

2009; Sahai et al, 2004a, 2004b), and genetic or pharmacological ablation of OPN improves

hepatic insulin sensitivity in metabolic syndrome mouse models (Kiefer et al, 2010, 2011;

Nomiyama et al, 2007b). Although no direct investigations of hepatic insulin-sensitivity state

have been performed in the present study, a significantly lower fasting blood sugar and a

more effective repression of hepatic Foxo1 and its downstream gluconeogenic targets in 8-

week-old NASH-HCC Spp1-/- mice let us suppose that, also in this case, the lack of OPN

improves hepatic insulin sensitivity. Nevertheless, insulin-tolerance and glucose-tolerance

tests, as also euglycemic-hyperinsulinemic clamp measurements should be performed to

unequivocally corroborate this assumption. This would be of crucial interest to clarify the net

effect and contribution of insulin on hepatic glucose and lipid production under diabetic

conditions. Indeed, if lack of OPN improves insulin-sensitivity in NASH-HCC mice, and

66

insulin stimulates Srebp-1c-dependent dnl in the liver, it is unclear why dnl is negatively

modulated in NASH-HCC-Spp1-/- mice. This might be simply explained by the fact that a

feedback loop regulating the expression of genes associates with de novo lipid synthesis

might exist, and therefore the higher hepatic fat content observed in the livers of Spp1-/- mice

induces a more effective transcriptional repression of the lipogenic pathway. On the other

hand, it could also be that circulating glucose controls hepatic dnl more potently than insulin

under conditions resembling human metabolic syndrome.

As a matter of fact, expression of the dnl master genes Acaca and Fasn is regulated not only

by Srebp-1c, which is controlled by insulin signaling, but also by ChREBP, whose activation

is glucose-dependent (Xu et al, 2013). Thus, ChREBP activation relies mainly on the rate of

cellular glucose uptake. While general glucose uptake is mediated by the insulin-dependent

glucose transporter GLUT4, hepatocellular glucose uptake in predominantly insulin-

independent and occurs through GLUT2 (Gottesman et al, 1983). Furthermore, NASH-HCC

mice have impaired insulin secretion and concomitant hyperglycemia, a condition that

supports a more prominent glucose signaling to the liver. Additionally, the insulin-dependent

pathway can be activated also by glucose, since ChREBP is a transcriptional modulator of

Srebp-1c (Shimomura et al, 1999b; Shin et al, 2016; Jeong et al, 2011). Our recent

observations seem also to support a primary role of glucose in hepatic dnl under

hyperglycemic and diabetic conditions, since NASH-HCC-Spp1-/- animals have lower fasting

blood sugar and lower dnl gene expression when compared with WT littermates. Hence,

further functional investigations will be necessary to assess the exact contribution of

circulating insulin and glucose on dnl under diabetic settings. Also, it would be of interest to

understand whether OPN therapeutic targeting might skew the paradox of hepatic partial

insulin resistance (i.e. impaired insulin-mediated inhibition of gluconeogenesis, but

maintained stimulation of dnl) to a full hepatic insulin resistance, which is proposed by many

as a better long-term condition for patients with a metabolic syndrome (Brown & Goldstein,

2008; Jelenik et al, 2017). In conclusion, these hypotheses challenge the accepted dogma of

partial hepatic insulin resistance and propose the ChREB pathway as a promising

67

pharmachological candidate for the treatment of hepatic insulin resistance in patients with

metabolic syndrome.

Secondary to a worse NAFL/NASH phenotype, NASH-HCC-Spp1-/- mice also developed a

more severe liver fibrosis when compared to WT NASH-HCC littermates (Nardo et al, 2020).

Previous observations in CCL4- and BDL-induced fibrosis and ischemia/reperfusion mouse

models corroborate our results (Lorena et al, 2006; Patouraux et al, 2014), and also

postulate that enhanced hepatocyte death caused by the missing anti-apoptotic effects of

OPN might be the cue for the worsening of the disease (Patouraux et al, 2014). On the other

hand, other experimental investigations showed improved hepatic fibrosis in OPN-deficient

mice, while worse fibrotic score in OPN-overexpressing transgenic mice (Arriazu et al, 2017;

Urtasun et al, 2012; Coombes et al, 2016). Although in the majority of the cases different

mouse models have been used, and this might explain the contrasting results, opposite data

have been obtained by different groups using the same CCl4 mouse model (Arriazu et al,

2017; Lorena et al, 2006; Urtasun et al, 2012). Therefore, although there is a consensus

about a significant OPN over-expression during hepatic fibrogenesis in both patients and

animal models (Arriazu et al, 2017; Lorena et al, 2006; Urtasun et al, 2012; Xiao et al, 2012;

Ge et al, 2018; Coombes et al, 2016), the role played by OPN in liver fibrosis remains

controversial. Although a positive correlation between OPN expression and fibrosis score

exists, this does not clarify the cause-effect relationship between the two events. Therefore,

further research should investigate whether OPN over-expression drives hepatic fibrosis, or if

OPN over-expression occurs upon fibrosis initiation as a promoter of anti-fibrotic processes.

Especially in NASH-derived fibrosis, it should be clarified whether the putative worsening of

hepatic fibrosis upon OPN KO is induced by the lack of direct anti-fibrotic effects of OPN, or

simply by the significantly increased lipotoxicity and inflammation caused by the lack of OPN

at the NALF/NASH time point. The use of inducible mouse models which would allow to

knock out OPN at selected stages of NAFLD development might be useful in pursuing this

biological question.

Oxidative stress is proposed as the principal noxious factor promoting DNA mutations that

68

predispose to HCC in NASH (Begriche et al, 2013; Masarone et al, 2018; Anstee et al,

2019). Interestingly, cellular senescence, a process characterized by sustained oxidative

stress (Muñoz-Espín & Serrano, 2014), not only promotes age-related hepatic steatosis

(Ogrodnik et al, 2017), but is also key in the pathogenesis of NAFLD (Aravinthan &

Alexander, 2016). Recent investigations by Gòmez-Santos and colleagues proved that OPN

is transcriptionally controlled by the senescence master regulator p53, and that while healthy

humans and wild-type mice show a positive correlation between age and OPN expression,

this correlation is lost in humans with senescence-driven NAFLD and OPN-deficient mice

older than 10 months (Gómez‐Santos et al, 2020). Further in vivo and in vitro experiments

also showed that lack of OPN causes a down-regulation of GRP78 and the consequent

induction of ER stress. This in turn promotes triglyceride accumulation in hepatocytes and

the over-expression of CD36, all finally promoting pathologic cellular senescence (Gómez‐

Santos et al, 2020).

Based on these latest news, the worse NAFL/NASH phenotype characterizing NASH-HCC-

Spp1-/- mice when compared with WT littermates might be driven by the incapacity of halting

senescence processes, which inexorably promote CD36-mediated lipid accumulation and ER

stress. In turn, this phenomenon might also account for the early onset of hepatic neoplasms

in NASH-HCC-Spp1-/- animals (Nardo et al, 2020). In order to test these hypotheses, a

detailed investigation of oxidative stress and histopathological assessment of senescence

markers should be performed in both WT and OPN-deficient NASH-HCC mice. Furthermore,

it should be also evaluated whether HCC harbored in NASH-HCC animals are characterized

by oxidative DNA damage and if this mutational pattern differs between the two murine

genotypes investigated.

If these hypotheses turned out to be valid, they could also explain the contrasting results

obtained in diet-induced obesity (DIO) mouse models, where lack of OPN was associated

with a significant improvement of the liver steatotic phenotype (Kiefer et al, 2010, 2011;

Nomiyama et al, 2007b). To better understand this statement, it is important to clarify the

differences between the experimental models used in previous and present studies. HFD-

69

feeding for 20-24 weeks (the model used in experiments showing a positive effect of OPN

inhibition on the hepatic phenotype) is a model of simple steatosis, which finely mirrors the

establishment of the obesity-induced metabolic syndrome, comprising weight gain, insulin

resistance, hyperinsulinemia and fatty liver. On the other hand, the preceding neonatal STZ

injection performed under our experimental settings as firstly described by Fujii and

colleagues (Fujii et al, 2013) induces an extra partial loss of pancreatic beta-cells, an event

that pathologically follows the beta-cell hyperfunction caused by insulin resistance in humans

and defines finally the progression from impaired glucose tolerance (IGT) to T2DM (U.K.

Prospective diabetes study 16: Overview of 6 years’ therapy of type II diabetes: A

progressive disease, 1995). The NASH-HCC mouse model used in the recent study is,

indeed, a model of progressive metabolic syndrome, defined by decreased insulin

production, low-grade systemic inflammation, ectopic lipid accumulation and fatty liver, which

evolves to HCC through fibrosis. Lower circulating insulin levels allow for a significantly

higher adipose tissue lipolysis, which explains why NASH-HCC mice don´t show an AT

expansion as conventionally happens upon HFD-feeding. The deriving systemic

hyperlipidemia will be therefore more elevated than in models of simple dietary intervention,

and the over-expression of FA transporters such as CD36 strongly defines hepatic FA inflow.

Based on this information, one can hypothesize that the severer hepatosteatotic injury

occurring in NASH-HCC mice compared to DIO animals might be characterized by

hepatocellular senescence, which progresses unrestrained in the absence of OPN and

drives therefore the worse phenotype of early hepatic manifestations in NASH-HCC mice. It

is also important to note that the improved hepatic phenotype in DIO mice upon OPN

ablation relies on improved gonadal white adipose tissue (GWAT) insulin sensitivity

secondary to lower GWAT inflammation (Kiefer et al, 2010, 2011; Nomiyama et al, 2007b).

Indeed, Spp1-/- DIO mice have enlarged GWAT compared to WT littermates, which indicates

better lipid storage capacity, and mainly reflects improved insulin-mediated inhibition of AT

lipolysis (Kiefer et al, 2010, 2011; Nomiyama et al, 2007b). Therefore, in DIO models, the

improved hepatic phenotype upon OPN inhibition relies on the otherwise detrimental effect of

70

OPN in adipocytes. In the NASH-HCC model used in the current study, the hypo-insulinemic

state obscures a possible effect of OPN in GWAT and allows to focus uniquely on the

hepatic role of OPN in metabolism and inflammation.

This information highlights again the complexity of therapeutically targeting OPN in metabolic

syndrome patients. Although OPN-based interventional strategies sound promising, the

tissue-specificity and the clinical indications for such a therapy shall be designed with

meticulous detail.

As conclusive remark, the most attractive biological observation emerging from the

presented study is that improving hepatic metabolic functions under metabolic syndrome

settings results in a more severe liver pathology, but also in a positive effect on survival due

to improved systemic conditions (Nardo et al, 2020). The metabolic syndrome is a metabolic

and inflammatory state, in which lipid toxicity plays a central role. Therefore, the best strategy

to improve survival (after behavioral intervention aimed at removing the origin of the injury) is

to first protect the lipotoxicity-sensitive organs, such as pancreas, kidneys, heart and

muscles. The development of a fatty liver in obese and pre-diabetic/diabetic patients and

animal models is an intrinsic mechanism, most probably adapted to achieve this systemic

protective role. However current therapies for NAFL and NASH are designed to improve the

hepatic phenotype, by targeting metabolic processes that induce lipid accumulation. Based

on the law of conservation of energy (Clapham & Nicholson, 2009), on which the first law of

thermodynamics is built, if the lipid overburden is not reduced at the source, decreasing

hepatic lipid content will inexorably cause ectopic lipid deposition, and while positive

outcomes might be recorded in the short term, worse systemic outcomes are almost

unavoidable on the long run.

Therefore, the final aim of future research in the gastroenterological field should be to

improve non-invasive diagnostic and prognostic tools for liver diseases, and to invest in

regenerative medicine projects aimed at reproducing functional hepatic units “in a dish”.

71

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131

APPENDIX

Alexander Daniel Nardo, M.Sc.

Obere Donau Strasse 17, 1020 Vienna, Austria

alexander.nardo@gmail.com

+436606308066

18/03/1989

Multidisciplinary, Endocrinology and Metabolism, Cancer, Inflammation, Animal studies

EDUCATION AND RESEARCH EXPERIENCE .

2018 – present: Research Associate. Characterization of bile acid metabolism and function in metabolic

syndrome- derived non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), hepatic fibrosis and hepatocellular carcinoma (HCC) mouse models

• Medical University of Vienna, Austria. Prof. Michael Trauner’s team

2014 – present: PhD Molecular Medicine (CCHD Ph.D. Fellowship). Thesis: Role of osteopontin on

the development of hepatocellular carcinoma in fatty liver disease

• Medical University of Vienna, Austria. Prof. Thomas Stulnig’s team

2014 (three months): Internship: Analysis of metabolites of wine by spectrophotometry and mass

spectrometry

• Laboratory for Flavors and Metabolites, Laimburg, Italy. Dr. Peter Robatscher’s team

2011 – 2014: M.Sc. Medical Biotechnologies, with honors (one year internship). Thesis: Gene

expression alterations in transit-amplifying keratinocyte subpopulations play a critical role in the development of psoriatic lesion

• University of Modena and Reggio Emilia, Italy. Prof. Carlo Pincelli’s team

2008 – 2011: B.Sc. Biomolecular Sciences and Technologies, with honors. Thesis:

Characterization of Pumilio 1 protein in mammal cells

• CIBIO/University of Trento, Italy. Prof. Paolo Macchi’s team

2010 (three months): Internship: Analysis and quality control of food products

• Laboratory of Organic Chemistry, Laimburg, Vadena, Italy. Dr. Peter Robatscher’s team

PROFESSIONAL SKILLS .

Project management: project structuring and scheduling, grant application- and animal studies ethical

application writing, national and international collaborations, staff and lab management

Experimental skills: mouse models of obesity, diabetes and metabolic syndrome-derived

hepatocarcinogenesis; molecular biology (DNA, RNA, protein and lipid isolation, qRT-PCR, IHC, IF, TUNEL, IP, WB, ELISA); cell culture

Computer skills: confident use of Image J, GraphPad, IBM SPSS, Mendeley, Office Pack, Adobe tools

132

Communications: talks [e.g. EASL International Liver Congress (Paris 2018), Young Scientist Association

Symposium (Vienna 2018)], posters [e.g. 12th

and 13th

Congress of the Central European Diabetes

Association/32nd

and 33rd

International Danube Symposium (Prague 2017, Krakow 2018), European Congress on Obesity (Vienna 2018)], Teaching [Basics in animal studies (Vienna 2017, CCHD Ph.D.

Program lab rotation), formal supervision of junior researchers]

Languages: Italian (native), German (fluent), English (fluent)

PUBLICATIONS .

Nardo A.D. et al., “Impact of osteopontin on the development of non-alcoholic liver disease and related hepatocellular carcinoma”. Liver International (PMID: 32281248)

Nardo A.D. et al., “Pathophysiological mechanisms of liver injury in COVID-19”. Liver International (https://doi.org/10.1111/liv.14730)