Lipoprotein lipidomics in obesity-associated

metabolic and hepatic disorders

Gabriele Mocciaro
Wolfson College

Department of Biochemistry
University of Cambridge

This dissertation is submitted for the degree of Doctor of Philosophy

July 2021

1
Declaration
This dissertation is the result of my own work and includes nothing which is the
outcome of work done in collaboration except as declared in the Preface and specified
in the text. It is not substantially the same as any that I have submitted, or, is being
concurrently submitted for a degree or diploma or other qualification at the University
of Cambridge or any other University or similar institution except as declared in the
Preface and specified in the text. I further state that no substantial part of my
dissertation has already been submitted, or, is being concurrently submitted for any
such degree, diploma or other qualification at the University of Cambridge or any other
University or similar institution except as declared in the Preface and specified in the
text. It does not exceed the prescribed word limit.

2
Lipoprotein lipidomics in obesity-associated
metabolic and hepatic disorders
Gabriele Mocciaro

Abstract
Over the last few decades, obesity has reached epidemic proportions. Obesity is
strongly associated with insulin resistance (IR), hyperglycaemia, mixed dyslipidaemia,
and hypertension. These metabolic risk factors, grouped under the definition of the
Metabolic Syndrome (MetS), increase the risk of cardiovascular disease (CVD). Non-
alcoholic fatty liver disease (NAFLD) is one of the most common co-morbidities of
MetS. NAFLD ranges from simple steatosis to more aggressive forms, with the
potential to evolve to hepatocellular carcinoma and CVD. Lipoprotein dysmetabolism
is crucial to both MetS and NAFLD and few studies have investigated the circulating
lipidome, defined as the complete lipid profile in a specific tissue/biofluid, in these
conditions. The work contained in this thesis studied the lipoprotein remodelling
occurring in two cohorts; MetS cohort including 11 healthy people and 14 MetS
subjects and BioNASH cohort including 20 healthy people and 89 biopsy-proven
patients across the entire spectrum of NAFLD. To this end, we employed state-of-the-
art analytical techniques (mass spectrometry-based) along with molecular biology
assays to study the serum and lipoprotein lipidome of these patients.
In the MetS study, we found that the circulating lipidome of MetS was characterised
by a phospholipid (PL) dysmetabolism. The latter was driven, at least in part, by a
reduced activity of the enzyme lecithin–cholesterol acyltransferase (LCAT).
Dysfunctional LCAT could partly mediate the elevated CVD risk in MetS patients. Our
study demonstrated, for the first time, the link between reduced LCAT activity and
plasma lipidome in MetS.
In the BioNASH cohort, we observed a generalised PL and polyunsaturated fatty acids
(PUFA) depletion in NAFLD compared to the control group. By using fast protein liquid
chromatography, we isolated HDL and VLDL fractions where we performed lipidomic
analyses. As opposed to VLDL, the HDL lipidome was characterised by PL and PUFA
depletion. These changes have been reported in hepatic lipidomic studies of NAFLD
patients, thus suggesting a close link between peripheral tissues and the liver via HDL.

3
These results provide the basis for the study of HDL composition as a novel player in
the pathogenesis of NAFLD.
In conclusion, these data demonstrate how the study of lipoprotein metabolism in
obesity-related metabolic disorders can shed light on novel pathophysiological
mechanisms. Further efforts along these lines will clarify the role of LCAT in MetS and
CVD alongside establishing the contribution of the HDL lipidome to the liver
composition of NAFLD patients.

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Publications
1. Hall Z, Chiarugi D, Charidemou E, Leslie J, Scott E, Pelligrinet L, Allison M,
Mocciaro G, Anstee QM, Evan GI, Hoare M, Vidal-Puig A, Oakley F, Vacca M,
Griffin JL. Lipid Remodelling in Hepatocyte Proliferation and Hepatocellular
Carcinoma (Hepatology. 2020 May 27)

2. Mocciaro G, Bresciani L, Tsiountsioura M, Martini D, Mena P, Charron

M, Brighenti F, Bentley S, Harvey M, Collins D, Ray S. Dietary absorption
profile, bioavailability of (poly)phenolic compounds, and acute modulation of
vascular/endothelial function by hazelnut skin extract (accepted in Journal of
Functional Foods)

3. Hinz C, Liggi S, Mocciaro G, Jung SM, Induruwa I, Pereira MCDA, Bryant

CE, Meckelmann SW, O'Donnell VB, Farndale RW, Fjeldsted JC, Griffin JL. A
comprehensive UHPLC ion mobility QTOF method for profiling and
quantification of eicosanoids, other oxylipins and fatty acids. (Anal Chem. 2019
May 10)

4. Ducheix S, Peres C, Hardfeldht J, Fra C, Mocciaro G, Piccinin E, Lobaccaro JM,

Plateroti M, Griffin JL, Ntambi J, Moschetta A. Ablation of Stearoyl-CoA Desaturase
1 in enterocytes drives intestinal inflammation and tumorigenesis that are rescued
by dietary oleate. Gastroenterology 2018 Nov;155(5):1524-1538

5. Bhat S, Mocciaro G, Ray S. The Association of Dietary Patterns and Carotid

Intima-Media Thickness: A Synthesis of Current Evidence (Nutr Metab
Cardiovasc Dis. 2019 Dec;29(12):1273-1287)

6. Mocciaro G, Ziauddeen N, Godos J, Marranzano M, Chan MY, Ray S. Does a

Mediterranean-type dietary pattern exert a cardio-protective effect outside the
Mediterranean region? A review of current evidence. (Int J Food Sci Nutr. 2017
Oct 24:1-12)

Preprints (non peer-reviewed)

1. Seyres D, Cabassi A, Lambourne JJ, Burden F, Farrow S, McKinney H, Batista J,
Kempster C, Pietzner M, Slingsby O, Huy Cao T, Quinn PA, Stefanucci L, Sims
MC, Rehnstrom K, Adams CL, Frary A, Ergüener B, Kreuzhuber R, Mocciaro G,
D’Amore S, Koulman A, Grassi L, Griffin JL, Ng LL, Park A, Savage DB, Langenberg
C, Bock C, Downes K, Allison M, Vacca M, Kirk PDW, Frontini M. Transcriptional,
epigenetic and metabolic signatures in cardiometabolic syndrome defined by extreme
phenotypes. (doi: https://doi.org/10.1101/2020.03.06.961805)

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Dedication
To my best friend and partner Yasmine.

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Acknowledgments
First of all, I would like to express my deepest gratitude to my mentors Prof Julian
Griffin and Dr Michele Vacca for the tremendous help and support given to me
throughout my PhD journey. Their mentorship has been invaluable. I am grateful to Dr
Zoe Hall, Dr Antonio Murgia, Dr Sonia Liggi and Dr Ben McNally for helping me run
the LC-MS, with data processing and the training provided. In particular, I am very
grateful to Dr Murgia for having provided me with an excellent training on the LC
MS/MS system alongside the endless technical discussion and his support while he
had already taken up a different job. I am thankful to Dr Albert Koulman and Dr Ben
Jenkins for having let me use their lab space and mass spectrometers without any
restrictions. Furthermore, I am also grateful to Dr Jenkins for his extensive training on
the LC-MS Orbitrap system. I would also like to thank Dr Richard Kay for having run
the LC-MS proteomic analysis. I am indebted with Stefanie Neun, Damon Parkington
and his team for their great support in letting me use their plate readers for the
measurement of enzyme activity and ELISA. I am also grateful to Dr Mattia Frontini
and his team for the training provided and help with the recruitment of the healthy
volunteers (BioNASH cohort). A particular thank goes to my colleague and friend Dr
Luis Vicente Herrera-Marcos, whom thought me how to use the FPLC system and to
whom I owe most of my technical knowledge regarding the lipoprotein separation. I
am indebted to Dr Shivani Bhat for her help with English wording/editing of the
introduction chapter. I would also like to thank Steven Murfitt for all the help on the day
to day bench work at the Department of Biochemistry.

A special thanks go to my parents Carmelo and Giovanna, alongside my brother

Roberto. Their support and love have proven essential in shaping the person I have
become.

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Contents
Chapter 1. Background and rationale ................................................................................ 14
1.1 The Obesity Epidemic ................................................................................................ 14
1.2 The Metabolic Syndrome and Non-alcoholic fatty liver disease.......................... 14
1.2.1 Pathophysiology ................................................................................................... 17
1.2.2 Genetic factors of NAFLD ................................................................................... 17
1.2.3 Adipose tissue dysfunction ................................................................................. 18
1.2.4 Physiological role of insulin in lipid and glucose metabolism ....................... 19
1.2.5 Metabolic aspects of IR in MetS and NAFLD .................................................. 20
1.2.6 DNL and its drivers in NAFLD – the role of SREBP 1c & ChREBP............. 21
1.2.7 Lipoprotein metabolism ....................................................................................... 24
1.2.7.1 The exogenous pathway ............................................................................. 25
1.2.7.2 The endogenous pathway .......................................................................... 26
1.2.7.3 Reverse cholesterol transport (RCT) – HDL metabolism and
circulating lipoprotein remodelling enzymes ......................................................... 27
1.2.8 Dyslipidaemia in NAFLD and MetS ...................................................................... 29
1.2.8.1 Impaired VLDL turnover .................................................................................. 29
1.2.8.2 Increased atherogenic LDL particles – the role of sdLDL-C ...................... 30
1.2.8.3 Decreased levels of HDL-C ............................................................................ 30
1.2.9 NAFLD: a tale of hepatic lipid fluxes .................................................................... 32
1.2.10 Lipotoxicity and NASH progression .................................................................... 32
1.2.11 Complications of the MetS and NAFLD ............................................................. 36
1.2.11.1 Type II diabetes mellitus (T2DM) ................................................................. 36
1.2.11.2 Atherosclerotic cardiovascular disease (ASCVD) ..................................... 36
1.2.11.3 Chronic kidney disease ................................................................................. 37
1.3 Lipidomics as a tool to study obesity and its metabolic-related disorders............. 38
1.3.1 Lipoprotein lipidomics ............................................................................................. 43
1.3.2 The use of liquid chromatography-mass spectrometry in lipidomics and
metabolomics ..................................................................................................................... 44
Chapter 2. Materials and methods ..................................................................................... 47
2.1 Measurement of clinical biochemistry...................................................................... 47
2.2 Measurement of lipid levels by mass spectrometry .............................................. 47
2.2.1 Lipid extraction ..................................................................................................... 47
2.2.2 Lipid analysis by liquid chromatography-mass spectrometry ....................... 48
2.2.3 Esterified cholesterol analysis – LC-MS/MS ................................................... 51

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 2.3 Measurement of apolipoprotein levels by mass spectrometry ............................ 51
2.3.1 Apolipoprotein extraction .................................................................................... 51
2.3.2 Apolipoproteins analysis by liquid chromatography-mass spectrometry .... 52
2.4 Lipoprotein separation by Fast protein liquid chromatography............................ 53
2.5 Statistical analyses ..................................................................................................... 54
Chapter 3. Lipidomic indices reveal reduced LCAT activity in patients with metabolic
syndrome ................................................................................................................................ 55
3.1 Abstract ........................................................................................................................ 55
3.2 Introduction .................................................................................................................. 56
3.3 Methods ........................................................................................................................ 58
3.3.1 Ethics and the MetS study cohort ..................................................................... 58
3.3.2 Sample collection and clinical biochemistry measurements ......................... 58
3.3.3 Measurement of lipid levels by mass spectrometry ....................................... 58
3.3.4 Measurement of protein levels by mass spectrometry .................................. 59
3.3.5 Circulating lipoprotein remodelling enzyme activity - fluorimetric assay ..... 59
3.3.5.1 Lp-PLA2......................................................................................................... 59
3.3.5.2 LCAT activity................................................................................................. 59
3.3.5.3 PLTP activity ................................................................................................. 59
3.3.6 Statistics ................................................................................................................ 60
3.4 Results .......................................................................................................................... 61
3.4.1 Clinical characteristics, serum lipidomic and apoprotein profile of healthy
and MetS participants.................................................................................................... 61
3.4.2 LCAT, but not Lp-PLA2, activity is reduced in MetS thus justifying the
changes in serum PC and LysoPC ................................................................................. 65
3.4.3 LCAT activity and its lipidomic proxies show an inverse correlation with
metabolic risk factors and positively correlate with HDL-C ......................................... 66
3.4.4 Discussion ................................................................................................................ 69
3.4.5 Limitations ................................................................................................................. 71
3.4.6 Conclusion ................................................................................................................ 72
Chapter 4. NAFLD is characterised by a reduced reverse polyunsaturated fatty acid
transport from peripheral tissues to the liver ..................................................................... 77
4.1 Abstract ........................................................................................................................ 77
4.2 Introduction .................................................................................................................. 79
4.3 Methods ........................................................................................................................ 82
4.3.1 Ethics and the BioNASH study cohort .............................................................. 82
4.3.2 Sample collection and clinical biochemistry measurements ......................... 82

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 4.3.3 Measurement of lipid levels by mass spectrometry ....................................... 82
4.3.4 Measurement of ApoA1 ...................................................................................... 83
4.3.5 Measurement of ApoB ........................................................................................ 83
4.3.6 Statistics ................................................................................................................ 83
4.4 Results .......................................................................................................................... 85
4.4.1 Clinical characteristics and whole serum lipidomic profile of healthy and
NAFLD patients .............................................................................................................. 85
4.4.2 HDL lipidomics of NAFLD patients suggest a reduced reverse PUFA
transport from peripheral tissues to the liver ............................................................. 89
4.4.3 VLDL lipidomics of NAFLD patients shows an enhanced SFA export from
the liver to peripheral tissues ....................................................................................... 94
4.4.4 Discussion ............................................................................................................. 98
4.4.5 Limitations ........................................................................................................... 102
Chapter 5. Conclusion ........................................................................................................ 116
5.1 Summary and conclusion ........................................................................................ 116
5.2 Future directions ....................................................................................................... 120

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Abbreviations

ACC Acetyl-CoA Carboxylate

ABCA1 ATP-Binding Cassette sub-family A member 1
ABCG1 ATP-binding cassette sub-family G member 1
ACL ATP-citrate lyase
ACN Acetonitrile
Akt Protein kinase B
Apo Apolipoprotein
AT Adipose Tissue
ASCVD Atherosclerotic Cardiovascular Disease
BMI Body Mass Index
CE Cholesterol Ester
CETP Cholesteryl ester transfer protein
Carbohydrate response element binding
ChREBP
protein
CKD Chronic kidney disease
CM Chylomicron
CVD Cardiovascular Disease
DG Diacylglycerol
DNL De Novo Lipogenesis
ER Endoplasmic reticulum
ESI Electrospray ionisation
FA Fatty Acid
FAS Fatty acid synthase
FADS1 Fatty Acid Desaturase 1
FADS2 Fatty Acid Desaturase 2
FC Free Cholesterol
FFA Free Fatty Acid
GC-MS Gas Chromatography-Mass Spectrometry
GLUT 4 Glucose transporter type 4
HDL High-Density Lipoprotein
HSPGs Heparan-Sulfate Proteoglycans

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 Homeostatic Model Assessment for Insulin
HOMA-IR
Resistance
HSL Hormone-Sensitive Lipase
IDL Intermediate Density Lipoprotein
INSIG1 Insulin induced gene 1
IR Insulin Resistance
LCAT Lecithin–Cholesterol AcylTransferase
LC-MS Liquid Chromatography-Mass Spectrometry
LDL Low-Density Lipoprotein
LDLR Low-Density Lipoprotein Receptor
LPL Lipoprotein Lipase
LysoPC Lysophosphatidylcholine
LysoPE Lysophosphatidylethanolamine
MAFLD Metabolic associated fatty liver disease
MetS Metabolic Syndrome
MS Mass Spectrometry
MUFA Monounsaturated Fatty Acid
NAFLD Non-Alcoholic Fatty Liver Disease
NASH Non-Alcoholic Steatohepatitis
National Cholesterol Education Program Adult
NCEP ATP-III
Treatment Panel III
OXPHOS Oxidative Phosphorylation
PC Phosphatidylcholine
PCA Principal Component Analysis
PE Phosphatidylethanolamine
PG Phosphoacylglycerols
Peroxisome Proliferator-Activated Receptors γ
PGC-1α
Coactivator-1α
PLA2 Phospholipase A2
PLTP Phospholipid Transfer Protein
Patatin-like Phospholipase domain-containing
PNPLA3
protein 3

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 Peroxisome Proliferator-Activated Receptors
PPAR α
α
PPARγ Peroxisome Proliferator-Activated Receptors γ
PUFA Polyunsaturated Fatty Acid
PyrC Pyruvate Carboxylase
QqQ Triple Quadrupole
RCT Reverse Cholesterol Transport
SCD1 Stearoyl-CoA Desaturase-1
sdLDL Small dense LDL cholesterol
SFA Saturated Fatty Acid
SM Sphingomyelin
SREBP1 sterol regulatory element-binding protein 1
T2DM Type 2 Diabetes Mellitus
TG Triacylglycerol
TM6SF2 Transmembrane 6 Superfamily Member 2
VIP Variable Importance in the Projection
VLDL Very Low-Density Lipoprotein
WAT White Adipose Tissue
WHO World Health Organization

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Chapter 1. Background and rationale

1.1 The Obesity Epidemic

Overweight and obesity are defined as excessive fat accumulation that can adversely
affect health. Body mass index (BMI), weight in kilograms per height in metres
squared, is widely used as a marker of body fat in the clinic. The World Health
Organisation (WHO) defines overweight as a BMI between 25 and 29.9 kg/m 2, and
obesity as a BMI greater than 30 kg/m2 (World Health Organization, 2018). Over the
last three decades, the steady increase in obesity has become a major public health
concern, which has now reached epidemic proportions (Ng et al, 2014). Worldwide,
1.9 billion adults are classified as overweight, of whom 650 million are obese (World
Health Organization, 2018). In England, 62% of the population is classified as
overweight and 24% as obese (Gov.UK national statistics, 2017). Obesity is strongly
associated with a cluster of cardiovascular risk factors [referred to as Metabolic
Syndrome (Eckel et al, 2005)] including hypertension, mixed dyslipidaemia, systemic
insulin resistance (IR), and hyperglycaemia, and many other “travelling companions"
like fatty liver, gout, and chronic-low-grade systemic inflammation. Obesity also
promotes life-threatening comorbidities including type 2 diabetes mellitus (T2DM),
cardiovascular disease (CVD), and cancers (e.g. liver, colon, breast, and prostate)
((Berrington de Gonzalez et al, 2010)).

1.2 The Metabolic Syndrome and Non-alcoholic fatty liver disease

The metabolic syndrome (MetS) and non-alcoholic fatty liver disease (NAFLD) are
obesity-associated metabolic disorders. Although they are clinically defined as
different diseases, several pathophysiological aspects overlap. Therefore, throughout
the introduction of this thesis, the two conditions are described together, with the
differences between them clearly specified.
The MetS is clinically defined by the co-existence of atherogenic dyslipidaemia,
increased apolipoprotein B lipoproteins [very low density lipoprotein (VLDL),
intermediate density lipoprotein (IDL), low density lipoprotein (LDL)], decreased high
density lipoprotein (HDL), elevated blood pressure and fasting glucose, together with
a pro-thrombotic state, and a pro-inflammatory state (chronic low-grade inflammation)

14
(Eckel et al, 2005). The real prevalence of MetS is difficult to estimate since differences
in inclusion criteria have led to varied results worldwide (Eckel et al, 2005). However,
in European countries, its prevalence is estimated to be between 10% and 30% (van
Vliet-Ostaptchouk et al, 2014). Although the MetS was clinically identified nearly a
century ago (Kylin, 1923), it was not until 1998 that a WHO panel proposed a set of
criteria for the MetS (Alberti & Zimmet, 1998) to establish an international standard.
Since then, different definitions have been proposed and among them, the most
commonly adopted definition was proposed as a joint statement of the International
Diabetes Federation Task Force on Epidemiology and Prevention; National Heart,
Lung, and Blood Institute; American Heart Association; World Heart Federation;
International Atherosclerosis Society; and International Association for the Study of
Obesity (Alberti et al, 2009). The latter defined the MetS when 3 out of 5 cardiovascular
risk factors [abdominal obesity, high blood glucose and triglycerides (TG), increased
blood pressure, and reduced HDL-C] are present (Table 1).

Table 1. Clinical risk factors and defining levels for MetS according to NCEP ATP-III.

*Waist circumference cut-offs are population specific. Here, the values used for the
European population are reported.

NAFLD encompasses a spectrum of disorders ranging from simple steatosis,

intrahepatic fat deposition (>5% fat content in the liver), to steatohepatitis (steatosis in
the presence of inflammation) (NASH), fibrosis, and cirrhosis, which can ultimately
evolve to hepatocellular carcinoma (HCC) (Vacca et al, 2015). The diagnosis of
NAFLD is through the exclusion of all the secondary causes of fat accumulation in the
liver (elevated alcohol consumption, presence of hepatitis B and/or C, drug abuse,

15
autoimmune liver disease, haemochromatosis or Wilson’s disease) and liver biopsy,
which is still considered the gold standard to differentiate fatty liver from NASH and to
stage fibrosis (Buzzetti et al, 2016). NAFLD is one of the most common liver disorders
worldwide with a global prevalence of 24% (Younossi et al, 2018). NASH related
cirrhosis is predicted to become the leading cause of liver transplantation within the
next two decades (Zezos & Renner, 2014).

Epidemiological studies have shown that despite the high prevalence of NAFLD, only
a subset of patients develops more aggressive forms of this disease. Specifically, 5 to
10% of patients with NAFLD develop NASH, 30% of these develop cirrhosis, and 1-
2% of these eventually develop HCC (Argo & Caldwell, 2009). The evolution of NAFLD
to HCC is an area of active research. NAFLD is also considered as the hepatic
manifestation of MetS, with a prevalence of 50% in patients with T2DM, 76% in obese
subjects, and 100% in those morbidly obese with T2DM (Vacca et al, 2015). The close
association with MetS is also represented by the fact that CVD is the leading cause of
mortality for people with NAFLD (Younossi et al, 2018). Of note, despite the strong
association of obesity with NAFLD, a subset of the population develops NAFLD in the
absence of obesity (Younossi et al, 2018). This population is referred to as “Lean
NAFLD”. The latter term, firstly described in Asia, has a prevalence in Europe and the
USA of up to 20% (Younossi et al, 2018). This condition is characterised by abdominal
obesity, IR, and elevated triglycerides when compared to matched controls without
NAFLD (Younossi et al, 2018).

Two recent position articles have proposed to re-define NAFLD into metabolic
associated fatty liver disease (MAFLD) to strengthen the metabolic nature of this
condition (Eslam et al, 2020a; Eslam et al, 2020b). This new definition highlights the
inadequacy of a biopsy centric approach to classify the disease. For example, high
variability in disease progression is well established. Specifically, some patients with
hepatic steatosis can develop steatohepatitis and revert to steatosis over a short
timeframe and although the progression to more severe forms of NAFLD spectrum is
more likely in patients with NASH, not all NASH patients will progress towards the end
stage of the disease (Eslam et al, 2020a). The new definition emphasises the crucial
role of metabolic impairment in this condition. Specifically, MAFLD defining criteria
include the presence of hepatic steatosis along with one of the three following
cardiovascular risk factors: overweight or obesity, T2DM, or normal weight with

16
evidence of metabolic dysregulation (Eslam et al, 2020a). In sharp contrast to the
NAFLD paradigm, MAFLD is no longer considered a disease of exclusion and elevated
alcohol consumption and other concomitant liver diseases can coexist with this new
definition (Eslam et al, 2020a).

1.2.1 Pathophysiology

Although the aetiology of the MetS is multifactorial, it is increasingly recognised that

sedentary lifestyles along with overnutrition are key players (Cornier et al, 2008). The
pathophysiological mechanism of the MetS has not been fully determined, yet
abdominal obesity and insulin resistance are believed to be the main drivers of the
MetS (Cornier et al, 2008). NAFLD is the result of the accumulation of hepatic fat. The
sources of fats contributing to NAFLD can be summarised as follows: a) dietary fatty
acids derived through the uptake of chylomicrons (CM), b) free fatty acids (FFA)
derived thorough lipolysis of TG stored within white adipose tissue (WAT), and c) fatty
acids (FA) newly synthesised within the liver through de novo lipogenesis (DNL)
(Vacca et al, 2015). The raised FA levels in the liver can then be 1) stored as lipid
droplets within hepatocytes, 2) secreted into the bloodstream as VLDL, or 3) oxidised
via the β-oxidation pathway (Vacca et al, 2015). However, fat accumulation on its own
is not sufficient to drive NAFLD progression towards its more aggressive forms. The
multifactorial nature of NAFLD has been recognised over the last two decades and its
pathophysiology is currently recapitulated by the multiple parallel hit hypothesis (Tilg
& Moschen, 2010). Under this hypothesis, the onset and progression of NAFLD are
driven by the synergistic action of multiple parallel factors including IR, lipotoxicity,
endoplasmic reticulum (ER) stress, mitochondrial dysfunction, adipose tissue (AT)
dysfunction, and genetic background. The following sections describe the role of
different risk factors characterising MetS and NAFLD.

1.2.2 Genetic factors of NAFLD

Recent studies are increasingly revealing the genetic factors involved in the onset and
progression of NAFLD towards more advanced forms of the disease (Brunt et al,
2015). Investigation of candidate gene analysis with a biologically plausible
pathogenic role for NAFLD, have shown the involvement of mutations in genes

17
including mitochondrial superoxide dismutase 2 (SOD2) (Al-Serri et al, 2012) and fatty
acid desaturase 1 (FADS1) (Wang et al, 2015). More recently, with the advent of a
non-targeted approach based on genome-wide association studies (GWAS), novel
genetic candidates have been associated with NAFLD specifically a) the Patatin-like
phospholipase domain-containing protein 3 gene (PNPLA3) on chromosome 22 and
b) the Transmembrane 6 superfamily member 2 gene (TM6SF2) on chromosome 19
(Brunt et al, 2015). PNPLA3 is involved in TG hydrolysis and acylglycerol transacylase
activity within the liver (Kumari et al, 2012), and a missense mutation I148M in
PNPLA3 (at SNP rs738409) leads to impaired hepatic triglyceride hydrolysis (He et al,
2010). Although this polymorphism has been extensively investigated, the mechanism
by which a mutation in PNPLA3 increases hepatic TG is yet to be fully elucidated.
Paradoxically, it has also been reported that carriers of this mutation have a reduced
CVD events (Ruschenbaum et al, 2018). A non-synonymous mutation Glu167Lys in
TM6SF2 (at SNP rs58542926) has been shown to influence hepatic TG content, with
carriers of the variant having a 34% higher liver fat content compared to those without
the variant (Zhou et al, 2019). Interestingly, none of the above mentioned SNPs are
associated with IR (He et al, 2010; Zhou et al, 2019).

1.2.3 Adipose tissue dysfunction

The last two decades have highlighted the underappreciated and dynamic role of AT
in obesity and its related disorders (i.e MetS and NAFLD) (Guerra et al, 2021). AT can
be classified into three “main” categories: 1) WAT, > 95% of total fat mass, 2) brown
AT (BAT), 1-2% of total fat mass, and 3) beige AT (BAT), located within WAT and with
the capability of turning into brown-like adipocytes following exposure to cold
temperatures or other stressors (Kahn et al, 2019). The anatomical localisation of
excessive WAT plays a key role in the onset of the MetS and NAFLD, with visceral
WAT surplus being more detrimental than subcutaneous WAT (Kahn et al, 2019). One
hypothesis behind the mechanistic link between obesity and the MetS and NAFLD is
the AT expandability hypothesis (Virtue & Vidal-Puig, 2010). The latter is based on the
idea that adipose tissue has a defined limit of expansion for any given individual
determined by genetic and environmental factors (Virtue & Vidal-Puig, 2010). At its
maximum storage limit, AT can no longer store more lipid, thus any additional surplus
is redirected into other tissues such as muscle, liver, and pancreas. This ectopic fat

18
deposition has detrimental effects leading to IR and apoptosis (Virtue & Vidal-Puig,
2010). Although the molecular basis regulating AT expansion capability is not fully
understood, the continuous increase in AT mass leads to adipocyte hypertrophy and
hyperplasia. These events lead to increased lipolysis as a means to counterbalance
the expansion and release of pro-inflammatory cytokines as a stress response, which
if protracted over time can lead to AT IR and adipocyte apoptosis (Virtue & Vidal-Puig,
2010).

1.2.4 Physiological role of insulin in lipid and glucose metabolism

Insulin plays a central role in glucose, lipid, and energy regulation, enabling the body
to efficiently respond to changes in nutrient levels during fasting and feeding states
(Boucher et al, 2014). In healthy subjects, the postprandial effect of insulin on insulin-
sensitive tissues can be generalised as follows. In the liver, insulin promotes the
formation of glycogen, through the activation of the enzyme glycogen synthase (GS)
via the serine/threonine-protein kinases (Akt) pathway (Samuel & Shulman, 2016). It
also suppresses glucose production through a) the reduction of gluconeogenic
enzymes (Edgerton et al, 2017), and b) indirectly, through the inhibition of AT lipolysis
(Figure 1). The latter is due to the inhibition of hormone-sensitive lipase (HSL), which
normally releases free fatty acids (FFA) and glycerol into the bloodstream, as a source
of energy for other organs. One of the mechanisms by which insulin inhibits HSL is
through the promotion of the cyclic AMP (cAMP) phosphodiesterase activity which
enhances cAMP hydrolysis with consequent reduction of cAMP depended PKA
activity, that is required for the HSL activation (Lan et al, 2019). Reduced HSL activity
results in a reduced FFA flux into the liver, normally taken up through the fatty acid
translocase/cluster differentiation protein-36 (FAT/CD36), which leads to decreased
hepatic acetyl-CoA levels, promoting the activation of pyruvate carboxylase (PyrC),
and therefore reduced PyrC activity and gluconeogenesis (Perry et al, 2015).
Furthermore, insulin suppresses the formation and export of VLDL into circulation
through a reduced transcription of apo-B100 mRNA, as well as genes encoding MTP
and APOC III via inhibition of the transcriptional factor forkhead box O1 (FoxO1)
(Altomonte et al, 2004; Kamagate et al, 2008) (Figure 1). The postprandial decrease
in VLDL export permits the preferential use of chylomicrons as a source of “fuel” from
the peripheral tissues, therefore sparing the liver FA pool for fasting conditions.

19
Furthermore, insulin directly co-modulates DNL by the activation of the transcriptional
factor sterol regulatory element-binding protein 1 (SREBP1) (Samuel & Shulman,
2016). SREBP1 regulates DNL through transcriptional upregulation of several key
genes involved in FA synthesis, namely acetyl-CoA carboxylase, fatty acid synthase
(FAS), and desaturation and elongation of FA through stearoyl-CoA desaturase 1 and
long-chain elongase 6 (ELOVL6), respectively (Postic & Girard, 2008) (Figure 1).
Insulin also promotes the TG-rich lipoprotein clearance through the activation of
lipoprotein lipase (LPL) (Panarotto et al, 2002), a key enzyme in the metabolism of
TG-rich lipoproteins. Moreover, insulin promotes glucose uptake in AT and skeletal
muscles through the upregulation of the glucose transporter type 4 (GLUT4) (Figure
1).

1.2.5 Metabolic aspects of IR in MetS and NAFLD

IR, a key feature of the MetS and NAFLD, results when cells from insulin-sensitive
tissues (liver, skeletal muscle, and AT) no longer adequately respond to insulin,
negatively impacting glucose and lipid homeostasis (Boucher et al, 2014). The action
of insulin on insulin-sensitive tissues in IR people can be generalised as follows. In
AT, insulin fails to regulate HSL and LPL with subsequent continuous release of FFA
(Lewis et al, 1995) and increased TG-rich lipoproteins (Brunt et al, 2015). The rise in
plasma FFA and glycerol is partially redirected into the liver and skeletal muscle. The
former primarily responds by a) increasing the synthesis of very-low-density
lipoproteins (VLDL) (Lewis et al, 1995), and b) transient upregulation of mitochondrial
β-oxidation which subsequently becomes dysfunctional with increased production of
reactive oxygen species (ROS) (Koliaki et al, 2015), and increasing TG in the liver.
Lastly, IR within the muscle results in defective uptake of glucose into cells, redirecting
glucose to the liver, and consequently increases substrates for DNL.

20
1.2.6 DNL and its drivers in NAFLD – the role of SREBP 1c & ChREBP

DNL is an important metabolic pathway to redirect energetic surplus coming from non-
lipid sources to a more energetically efficient form that can either be stored within the
liver or exported as VLDL. In mammalian liver, the state of positive energy balance
results in an accumulation of mitochondrial acetyl-CoA, which is at crossroads with
many metabolic pathways (Shi & Tu, 2015). The mitochondrial acetyl-CoA surplus is
transported into the cytosol, in the form of citrate and then converted back to acetyl-
CoA by the ATP citrate lyase, to start the DNL process (Postic & Girard, 2008). In the
cytosol, acetyl-CoA is carboxylated to malonyl-CoA by acetyl-CoA carboxylase, the
rate‐limiting, and first committed step in DNL (Harwood, 2005). Malonyl-CoA is
subsequently transformed into palmitic acid by FAS and is then subject to various
degrees of elongation and desaturation by ELOVL6 and SCD1 respectively (Mikkelsen
et al, 1985). In healthy subjects, fasting hepatic DNL contributes to about 10% of the
hepatic lipid pool (Sanders & Griffin, 2016), however, in IR/NAFLD patients, it is
responsible for over 25% of the total liver fat (Sanders & Griffin, 2016).
DNL has been increasingly recognised as a key player in the pathophysiology of
NAFLD, it is therefore crucial to understand the mechanisms behind its regulation in
health and disease. DNL is influenced by both metabolic and hormonal stimuli, such
as glucose and insulin, and finely controlled through transcriptional regulation of key
fatty acids synthesis enzymes. The two main transcription factors regulating DNL are
SREBP1c and carbohydrate response element-binding protein (ChREBP), which are
activated by insulin and glucose, respectively (Vacca et al, 2015). SREBP1c can also
be activated by SFA and the oxysterol sensor liver X receptor (LXR), whereas PUFA,
glucagon, and 5' AMP-activated protein kinase (AMPK) signalling have inhibitory
effects (Vacca et al, 2015). SREBP1c is mostly expressed in the liver and it is found
as an inactive precursor bound to the ER membrane, where it is associated with the
SREBP cleavage activating protein (SCAP) and the insulin-induced gene 1 (Insig1)
acting as an inhibitor of SRBP (Eberle et al, 2004). Insulin activates SRBP1c through
the Insulin/ phosphatidylinositol 3-kinases/protein kinase B (PI3K/Akt) pathway, which
eventually leads to the separation of Insig1 from the SRBP-SCAP complex. The latter
then relocates to the Golgi apparatus where it undergoes further maturation forming
the active SRBP1c form (Eberle et al, 2004). SRBP1c then migrates to the nucleus
where it binds to sterol regulatory element sequences of the DNL target genes

21
including ATP citrate lyase, ACC, and FAS. Of note, when SREBP1c is
overexpressed, in an attempt to maintain cellular homeostasis, it exerts negative
feedback on the insulin signalling pathway via the inhibition of insulin receptor
substrate 2, with subsequent inhibiting the PI3K/Akt pathway (Engelking et al, 2004).
The hyperinsulinemia observed in NAFLD therefore activates SREBP1c, which
promotes DNL, negatively feedbacks insulin signalling (leading to decreased glycogen
synthesis and increased gluconeogenesis) ultimately promoting hepatic fat
accumulation (Vacca et al, 2015).
ChREBP is activated by the postprandial increase in glucose levels, with a mechanism
not fully characterised (Denechaud et al, 2008). One proposed mechanism of
activation involves the dephosphorylation at serine residue 196 or AMPK
phosphorylation sites, which allows for its relocation to the nucleus and activation of
target genes containing the carbohydrate response element (ChoRE) (Uyeda & Repa,
2006). This mechanism is also in line with the inhibitory effects exerted by glucagon
during fasting, where increased levels of protein kinase A (PKA), promotes ChREBP
phosphorylation at serine residue 196 (Iizuka & Horikawa, 2008). ChREBP also
enhances the expression of liver pyruvate kinase (LPK), which converts
phosphoenolpyruvate to pyruvate thus promoting glycolysis, and the expression of
SCD1, favouring the conversion of newly generated SFA to MUFA (Benhamed et al,
2012).
The involvement of ChREBP in NAFLD has been reported in clinical and pre-clinical
studies. In leptin-deficient mice, a well-established model of obesity, liver expression
of ChREBP were remarkably increased, yet upon specific inhibition, the liver showed
a striking reduction of DNL gene expression alongside decreased hepatic steatosis
(Dentin et al, 2006). In a cohort of NASH patients, ChREBP expression was positively
correlated with the degree of hepatic steatosis but it was inversely related to IR
(Benhamed et al, 2012). The latter is attributable to the parallel upregulation of SCD1
by ChREBP, which avoids the accumulation of DNL products in the form of SFA but
rather as MUFA. The latter has shown to have little impact on liver IR as compared to
SFA (Musso et al, 2018).

22
 Fasting status

Postprandial status

Figure 1. Simplified figure of lipid and carbohydrate metabolism, in fasting and

postprandial status, in healthy subjects. Red lines = up-regulated pathway. Green arrows
= down-regulated pathways. GS = glycogen synthase, DNL = de novo lipogenesis, ACC =
Acetyl-CoA carboxylase, FAS = Fatty Acid Synthase, Elongation of very long chain fatty acids
protein 6, Stearoyl-CoA desaturase 1, FA = fatty acid, PyrC = pyruvate carboxylase,
FAT/CD36, fatty acid translocase/cluster differentiation protein-36, MTP = Microsomal
triglyceride transfer protein, Akt = serine/threonine-protein kinases, SRBP1c = Sterol
regulatory element-binding protein 1, ChREBP= carbohydrate response element-binding

23
protein, LPK= liver pyruvate kinase, FOXO1= forkhead box O1, GLUT 4 = Glucose transporter
type 4, GLUT 2 = Glucose transporter type 2, HSL = Hormone-sensitive lipase. Figure based
on (Samuel & Shulman, 2018), and done using the http://smart.servier.com/ image resource.

1.2.7 Lipoprotein metabolism

Cholesterol is an essential component of cell membranes, the precursor for the

synthesis of steroid hormones and bile acids, and the major sterol in animal tissues.
Its structure includes four linked hydrocarbon rings (steroid core), a hydrocarbon tail
linked to one end of the steroid, and a hydroxyl group linked to the other end (Tabas,
2002). Cholesterol is present in two forms, unesterified cholesterol (also known as free
cholesterol, FC) and cholesteryl esters (CE). The latter, the most abundant form in
blood plasma, is transported via lipoproteins due to their hydrophobic properties.
Lipoproteins are aggregates of lipids and proteins that allow the transport of
hydrophobic lipids in the bloodstream, but also transport endogenous proteins,
vitamins, hormones, and microRNA (Kuai et al, 2016). The outer part of the lipoprotein
is composed of a monolayer of phospholipids (i.e PC, LPC, PE), unesterified sterols
(i.e FC and oxysterols), and apolipoproteins, while the inner part is composed of non-
polar lipids (such as TG and CE) (Figure 2). Apolipoproteins provide stability to
circulating lipoproteins and play a crucial role in regulating metabolic processes via
activation/inactivation of enzymes as well as acting as a receptor-ligand (Cohen &
Fisher, 2013). Apart from apolipoprotein B (apo B), all the other apolipoproteins are
exchangeable within the different particles, although their abundance in different
fractions is relatively constant (Dominiczak & Caslake, 2011).
Lipoproteins show a great deal of heterogeneity, mostly based on their
physicochemical properties alongside the methodology used for their separation
(Scherer et al, 2011). Their size is widely spread ranging from 5 nm to over 100 nm.
Based on their density, lipoproteins are classified into five main fractions: chylomicrons
(CM), Very Low-Density Lipoprotein (VLDL), Intermediate Density Lipoprotein (IDL),
HDL, and LDL. They can be further differentiated, for instance, using immunoaffinity
isolation techniques. For example, Furtado et al., identified 16 HDL subspecies
(Furtado et al, 2018), and the role of the different sub-fractions are currently being
investigated in large cohorts.
Lipoprotein metabolism can be described through three pathways: a) the exogenous
pathway that describes lipid transport from diet to internal organs, b) the endogenous

24
pathway that describes lipid transport from internal organs to peripheral tissues) and
c) the reverse cholesterol transport (RCT)/HDL metabolism (endogenous pathway
from peripheral tissues to internal organs).

Figure 2. Simplified figure of a lipoprotein. TG = triglycerides, CE = cholesteryl esters, FC

= Free cholesterol, PL = phospholipids, Apo = apolipoprotein. Figure done using the
http://smart.servier.com/ image resource.

1.2.7.1 The exogenous pathway

The exogenous pathway begins in the small intestine (jejunum) once dietary lipids
have been hydrolysed, TG into FFA and monoacylglycerols (MAG), phospholipids (PL)
into FFA and lysophospholipids, and CE into FFA and FC, which are then absorbed
by the enterocytes via different mechanisms (Boren et al, 2012). Upon absorption,
these lipids are assembled to TG, CE, and PL. Subsequently, in the ER, a large cell
organelle acting as a major site of protein, lipid and steroid synthesis, carbohydrate
metabolism and calcium storage (Schwarz & Blower, 2016), the enzyme microsomal
triglyceride transport protein (MTP) lipidates the apolipoprotein B-48 (apo B-48) laying
the basis for CM formation. This process is tightly regulated by the action of apo B-48
on MTP (Kindel et al, 2010). Besides TG, CM also contains smaller amounts of other
lipids such as CE, FC, PL, and apolipoproteins such as Apo-AI, Apo-E, and Apo-Cs
(Kindel et al, 2010). Once assembled, CM are released from the enterocytes into the
intestinal lymphatic system, reaching the systemic blood circulation. Here, CM are
subject to the hydrolysing action of the LPL, enzyme located in the capillary surfaces
of peripheral tissues (i.e AT, skeletal muscle, heart), on CM TG. The released FA

25
relocates into tissues, leaving circulating CM with reduced TG, known as CM remnant.
The latter undergo further delipidation by hepatic lipase (HL), an enzyme-bound onto
heparan-sulfate proteoglycans (HSPGs) at the surface of hepatocytes and the
sinusoid endothelial cells (Stow et al, 1985), rendering the particles smaller. These
particles are then promptly taken up by the liver via a) the LDL receptor-related protein
(LRP), and b) the HSPGs, which also function alone as a receptor (Mahley & Ji, 1999).

1.2.7.2 The endogenous pathway

VLDL particles are the carriers of endogenous lipids, mainly TG, to peripheral tissues
as a source of “fuel”. These particles are assembled in the liver via a stepwise lipidation
of its structural protein apolipoprotein B100 (apoB100). The lipidation is carried out by
microsomal triglyceride transfer protein (MTP) in the ER (Adiels et al, 2008). This initial
step leads to the formation of a pre-VLDL particle, which subsequently undergoes
further lipidation, leading to the formation of a triglyceride-poor VLDL. The latter is then
further enriched in lipids in the Golgi apparatus and exported in circulation as a TG
rich VLDL (Adiels et al, 2008). VLDL particles, similarly to CM, are subject to TG
depletion by LPL in peripheral tissues (Boren et al, 2012). VLDL TG-depleted transfer
apoC-II and apoC-III to HDL, and in turn, accept apo-E. The presence of apo-E on
VLDL permits these particles to bind to high-affinity hepatic receptors and with this
mechanism, nearly 50% of apo-E VLDL are cleared from the circulation (Cohen &
Fisher, 2013). VLDL-TG are also exchanged for HDL-CE by the circulating enzyme
cholesteryl ester transfer protein (CETP) (Figure 2). These steps lead to a smaller and TG
depleted VLDL particle defined as IDL. The latter can be further delipidated by HL, further
reducing in size and forming LDL (Dominiczak & Caslake, 2011), or taken up by the liver
by LRP or HSPGs (Hurt-Camejo & Camejo, 2018). LDL is also subject to CETP activity,
leading to CE enriched LDL deriving from HDL (Shrestha et al, 2018) (Figure 2). Finally,
LDL is directed to the liver and internalised through LDLR (van de Sluis et al, 2017).

26
1.2.7.3 Reverse cholesterol transport (RCT) – HDL metabolism and circulating
lipoprotein remodelling enzymes

Reverse cholesterol transport (RCT) is the process by which HDL collects and
transports cholesterol excess from peripheral tissues to the liver, where it is directly or
indirectly (through transformation into bile acids) excreted into bile (Ouimet et al,
2019). RCT is a key player in cardiovascular protection since the accumulation of
cholesterol in macrophages is a defining step in atherosclerosis (Boren et al, 2020).
HDL is the smallest and densest of the lipoproteins, composed for nearly half of its
mass of lipids and half proteins (Kontush et al, 2013). HDL biogenesis starts with the
lipidation of the apolipoprotein AI (apoA-I), the most abundant apolipoprotein in HDL,
produced mainly by the liver and, to a lesser extent, by the small intestine (Figure 2)
(Kontush et al, 2013). ATP-binding cassette sub-family A member 1 (ABCA1), a
ubiquitous transmembrane protein, is responsible for the transfer of PL and FC to
apoA-1, which leads to the formation of a discoidal HDL or pre-β HDL (Ouimet et al,
2019). The latter is the preferential substrate of lecithin:cholesterol acyltransferase
(LCAT), an enzyme synthetized mainly by the liver and to a lesser extent by the brain
and testes (Jonas, 2000). LCAT circulates in plasma predominantly bound to HDL but
also LDL. In humans, LCAT catalyses the transfer of polyunsaturated fatty acids
(PUFA) from the sn-2 position of a PC to FC, generating a CE and LysoPC, with apoA-
1 as the main activator (Jonas, 2000) (Figure 2). As CE are more hydrophobic than
FC, they relocate to the core of HDL, thus reshaping the discoidal HDL into mature
spherical HDL(α) (Figure 2). Despite the crucial role of LCAT on HDL maturation, its
role in atherosclerosis is still debated. For example, while Dullaart et al. reported
increased activity of LCAT in MetS and positively correlated with carotid-intima media
thickness (Dullaart et al, 2008), the opposite was reported in patients with NAFLD
(Fadaei et al, 2018). Furthermore, while reduced levels of LCAT were found in patients
with ischemic heart disease independently of HDL levels (Sethi et al, 2010), a large
population study showed that LCAT mass was not associated with increased
atherosclerosis (Holleboom et al, 2010). Lack of association between LCAT mass and
CVD were also reported by Calabresi et al. (Calabresi et al, 2011), in a multicenter,
observational study including 540 participants. Studies on patients carrying genetic
LCAT deficiency and preclinical models have also yielded conflicting results (Ossoli et
al, 2016b), thus leaving its role in disease still debated.

27
Spherical mature HDL are further enriched in FC and PL by the ATP-binding cassette
sub-family G member 1 (ABCG1), which specifically acts on mature HDL (Gelissen et
al, 2006). Another important circulating enzyme, involved in lipoprotein remodelling, is
the phospholipid transfer protein (PLTP), ubiquitously expressed in human tissues (i.e
lung, thymus, and AT), but mainly synthetized by the liver (Huuskonen et al, 2001).
PLTP mediates the net transfer of PL from VLDL, IDL, and LDL, into HDL particle
during lipolysis to generate smaller HDL (HDL2) and can also convert larger HDL
(HDL3) particles to HDL2 and preβ-HDL (Zannis et al, 2015), as well as promote HDL
particle fusion creating HDL2 (Rye & Barter, 2014). Furthermore, PLTP is also capable
of transferring several lipids such as diacylglycerol, phosphatidic acid, sphingomyelin,
cerebroside and phosphatidylethanolamine, and α-tocopherol (Albers et al, 2012),
thus rendering it a non-specific transporter.
While the role of PLTP in lipoprotein remodelling and its relevance to CVD have been
investigated in murine models, the physiological role in humans is yet to be fully
understood.
HDL is also remodelled by CETP, which mediates the transfer of cholesteryl esters
from HDL to VLDL, IDL, and LDL in exchange for triglycerides (Shrestha et al, 2018).
Because of its ability to decrease HDL CE and increase VLDL CE, CETP has been
studied as a target for CVD prevention (Shrestha et al, 2018). Results from different
randomised clinical trials have failed to show any significant benefit on major coronary
events by CETP inhibition except for the Randomized Evaluation of the Effects of
Anacetrapib through Lipid Modification (REVEAL) trial (Group et al, 2017). Whether
inhibition of CETP activity is a viable option for CVD reduction is still debated.
In humans, HDL half-life is 2 to 4 days (Zannis et al, 2015), and its catabolism is
mediated through 1) liver uptake of HDL through the SR-BI; 2) liver uptake of
cholesterol coming from HDL and transported to the liver via apo-B lipoproteins (CETP
mediated exchange) by LDLR; 3) kidney uptake of lipid poor apoA-I, where it can be
filtered at the level of the glomerulus and then catabolized by proximal renal tubular
epithelial cells (Rader, 2006).

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1.2.8 Dyslipidaemia in NAFLD and MetS

NAFLD and MetS are characterised by increased VLDL levels, decreased HDL-C
(Speliotes et al, 2010) along with increased concentrations of atherogenic small sdLDL
particles, despite total LDL-C not necessarily being increased (DeFilippis et al, 2013;
Sugino et al, 2011; Toledo et al, 2006). Some of the mechanisms leading to the
dyslipidaemia observed in NAFLD and MetS are discussed below.

1.2.8.1 Impaired VLDL turnover

The increased plasma VLDL-TG levels observed in the early phases of NAFLD and
MetS are attributable to several factors. As already described, in the context of IR and
hyperglycaemia, the increased delivery of FFA from AT to the liver, coupled with
increased DNL (driven by SREBP1c and ChREBP activation), results in a net increase
of FA within the liver thus providing a surplus of substrate for VLDL synthesis. Besides
the increased VLDL-TG, IR also leads to qualitative changes in these particles.
Specifically, VLDL are particularly enriched in SFA TG and reduced in PUFA, this
being in line with increased liver DNL which ultimately leads to the production of the
SFA palmitic acid (Kotronen et al, 2009b; Roumans et al, 2020). Furthermore, in IR,
insulin no longer suppresses VLDL assembly and export, therefore leading to a
continuous release in the circulation of large VLDL1 (Sparks et al, 2012). Interestingly,
Borén et al., recently reported that in MetS nearly 46% of hypertriglyceridemia was
attributable to delayed catabolism and only 20% was explained by increased VLDL 1
secretion (Boren et al, 2015). The putative mechanism by which MetS causes delayed
VLDL1 catabolism was attributed to elevated levels of apoC-III in these particles.
ApoC-III exerts a powerful inhibitory effect on LPL which normally renders VLDL and
its delipidated forms, IDL, and LDL, more susceptible to clearance by the liver. Large
epidemiological studies also showed that elevated levels of apoC-III in other
lipoprotein fractions had a major role in modulating hypertriglyceridemia and CVD risk
(Jensen et al, 2018; Sacks, 2015). Furthermore, apoC-III inhibitors have been tested
in clinical trials showing promising results in reducing plasma TG levels in
hypertriglycaemic patients (Taskinen et al, 2019). Additionally, delayed TG turnover
can potentially be caused by the AT IR since LPL in AT is transcriptionally upregulated
by insulin (Wang & Eckel, 2009).

29
1.2.8.2 Increased atherogenic LDL particles – the role of sdLDL-C

Increased levels of sdLDL-C are a characteristic feature of NAFLD and MetS. The
main driver of this feature is attributed to elevated VLDL levels (Boren et al, 2020). In
this context, CETP is considered a key player exchanging TG from VLDL to LDL
particles in exchange for CE (Chapman et al, 2010). TG enriched LDL are then
delipidated by HL, which leads to smaller and denser particles (Diffenderfer &
Schaefer, 2014). Several lines of evidence have shown that sdLDL-C levels are a
stronger determinant of CVD risk than total LDL-C (Boren et al, 2020). Some of the
mechanism by which sdLDL-C are more atherogenic than larger LDL-C fractions
include greater propensity to penetrate the arterial wall due to smaller size (Berneis &
Krauss, 2002), delayed removal from circulation due to reduced LDL receptor affinity
(Taskinen & Boren, 2015), higher binding capacity for arterial wall proteoglycan (Flood
et al, 2004), susceptibility to glycation (Younis et al, 2009) and higher susceptibility of
PL and CE components to oxidation (Ivanova et al, 2017). The development of
automated enzymatic method assay for sdLDL is allowing the screening of these
particles in large population studies (Ivanova et al, 2017), rendering its use in the
clinical settings more feasible.

1.2.8.3 Decreased levels of HDL-C

Low levels of HDL-C are a hallmark of MetS and NAFLD. One of the mechanisms by
which these conditions are associated with low HDL-C has been attributed to
hypertriglyceridemia. Specifically, elevated circulating VLDL levels facilitate the CETP
activity with the consequent net transfer of TG from VLDL to HDL along with CE
transfer in the opposite direction. CETP enhanced activity promotes reduced HDL-C
because of the CE removal per se, and increased HDL catabolic rate (Rashid et al,
2003). The latter is possible because increased HDL-TG content reduces its stability
and consequently facilitating the dissociation of apoA-I from HDL, which then leads to
faster apoA-I catabolism by the kidney (Rashid et al, 2003). Reduced TG hydrolysis in
VLDL decreases the formation of IDL and LDL which contribute to the maturation of
HDL (Rashid et al, 2003). The effect of NAFLD and MetS on the biogenesis and
maturation of HDL is still under investigation. In human liver cells, insulin promotes the
gene expression of apoA-I (Murao et al, 1998), thus raising the possibility that IR could

30
also drive a reduced production of HDL. Preclinical models show that hyperinsulinemia
decreases HDL-C biosynthesis through the reduction of hepatic ABCA1 functionality
(21402379). Taken together, these data strongly support the role of IR and
hypertriglyceridemia as drivers of the low HDL-C levels observed in NAFLD and MetS.
Besides low HDL-C levels, aspects of HDL functionality such as the cholesterol efflux
capacity (CEC), a measure of the capacity of HDL to accept cholesterol from
macrophages, is significantly reduced in MetS (Annema et al, 2016; D'Amore et al,
2018; Paavola et al, 2017) and NAFLD (Fadaei et al, 2018; van den Berg et al, 2018).

Figure 2. Simplified summary of key lipoprotein remodelling steps, with a focus on HDL
reverse cholesterol transport. SR-B1= Scavenger receptor class B type 1, ABCA1 = ATP-
binding cassette sub-family A member 1, LDLR = low density lipoprotein receptor, Apo A-1 =
apolipoprtein A1, LPL = lipoprotein lipase, HTGL = hepatic triglyceride lipase, FC =
unesterified cholesterol, PC = phosphatidylcholine, CE = cholesteryl esters, LysoPC =
lysophosphatidylcholine, PL = phospholipids, TG = Triglyceride, PLTP = Phospholipid transfer
protein, CETP = cholesteryl ester transfer protein. Figure based on (Rye & Barter, 2014), and
done using the http://smart.servier.com/.

31
1.2.9 NAFLD: a tale of hepatic lipid fluxes

Recently Azzu et al. described NAFLD in terms of derangement of the hepatic fat
fluxes equation (Azzu et al, 2020). In their equation, the pool of hepatic fat, [P], is
described by the following formula: [P] = ksyn/kdeg. ksyn is defined as the rate of fat
synthesis, expressed as the units of mass, including DNL, hepatic FFA uptake, and
lipoprotein uptake, whereas kdeg is the rate of fat degradation, expressed as fractional
removal over time, comprising hepatic fatty acid oxidation and export (Azzu et al,
2020). This equation frame describes the complexity and dynamic nature of NAFLD,
explaining for instance how even in absence of energy surplus, an increased k syn, if
not paralleled by equally increased kdeg, will result in hepatic fat accumulation and vice-
versa. The flux model is supported by experimental studies showing that both FFA
fluxes to the liver and intrahepatic TG levels correlate with VLDL secretion in healthy
people (Mittendorfer et al, 2016), whereas in NAFLD this correlation does not hold true
anymore (Adiels et al, 2006; Mittendorfer et al, 2016), likely pointing out to a reduced
kdeg. This model has also been used to interpret the role of genetic polymorphisms (i.e
PNPLA3 polymorphism I148M) that predispose to NAFLD (Azzu et al, 2020).

1.2.10 Lipotoxicity and NASH progression

Lipotoxicity refers to an abnormal ectopic fat accumulation in peripheral tissues,

leading to toxic lipid formation paralleled with cellular dysfunction and apoptosis
(Musso et al, 2018). The understanding of how lipotoxicity drives hepatic IR and
NAFLD progression has greatly benefited from studies of skeletal muscle physiology
and the pathophysiology of lipodystrophy. Lipodystrophy encompasses a
heterogeneous group of conditions characterized by a lack of and/or dysfunctional
WAT with consequent IR, NAFLD, and mixed dyslipidemia (Semple et al, 2011).
Skeletal muscle ectopic lipid accumulation is associated with muscle IR in healthy
young subjects (Krssak et al, 1999) and people with IR but otherwise healthy
(Perseghin et al, 1999). Animal models also support the role of skeletal muscle ectopic
fat as a driver of IR, with a mechanism involving DAG accumulation, lipids with
signalling functions, and precursor of glycerophospholipids, which inhibits the GLUT4
translocation to the plasma membrane upon insulin stimulation (Samuel & Shulman,
2012). To elucidate the role of ectopic hepatic fat on liver IR, while ruling out other fat

32
depots, studies in patients with severe lipodystrophy (Petersen et al, 2002) coupled
with murine models of lipodystrophy (Kim et al, 2000) have coherently shown the
presence of NAFLD in absence of excess visceral fat. The mechanism by which
ectopic hepatic fat promotes IR and NAFLD progression is still debated, but it likely
involves different pathways and multiple lipid species (Figure3) (Samuel & Shulman,
2018). A mechanism that has been coherently reported in both murine and human
studies involves DAG accumulation, which activates the intracellular enzyme protein
kinase C (PKC) isoform ε. Once activated, PKCε phosphorylates the hepatic insulin
receptor-1 at the inhibitory site serine with consequent inhibition to the insulin action
on its receptor (Mack et al, 2008). Rats placed on a 3 days high-fat diet show a
significant increase in liver DAG and PKCε activity, while knockdown of PKCε in the
liver was protected against lipid-induced IR (Samuel et al, 2007). Furthermore, obese
patients display increased hepatic levels of DAG (Magkos et al, 2012) as well as
increased activation of hepatic PKCε (Kumashiro et al, 2011). Ceramides, lipids with
a structural role in cell membranes, and bioactive properties have also been
investigated as putative mediators of lipid-induced hepatic IR and NAFLD progression
(Samuel & Shulman, 2019). The causative role of ceramides in skeletal muscle IR has
been widely reported, whereas in hepatic IR is still debated (Petersen & Shulman,
2017). Some human studies have reported increased levels of hepatic ceramides
across the IR-NAFLD spectrum (Chiappini et al, 2017; Gorden et al, 2015; Luukkonen
et al, 2016), while others did not (Kumashiro et al, 2011; Magkos et al, 2012; Ter Horst
et al, 2017). The mechanisms by which ceramides could lead to hepatic IR are not
fully characterised, yet two different pathways have been proposed, both acting
through inhibition of Akt, a key player in the insulin signalling pathway (Chavez &
Summers, 2012).
In the first model, increased hepatic ceramides activate the intracellular enzyme PKA
isoform zeta (ζ), which phosphorylates Akt (Powell et al, 2003) and this prevents the
recruitment of Akt to the plasma membrane upon insulin receptor activation (Stratford
et al, 2001). In the second model, ceramides activate the enzyme protein phosphatase
2A (PP2A), which leads to dephosphorylation and inactivation of Akt (Chavez et al,
2003). Furthermore, ceramides have also been implicated in the progression of
NAFLD, through the promotion of mitochondrial dysfunction and inflammatory
pathways (Pagadala et al, 2012). Despite the debate on the role of ceramides in

33
hepatic IR, its association with CVD (Hilvo et al, 2020), T2DM (Hilvo et al, 2018), and
its role in skeletal muscle IR, they remain at the centre of intensive research.
The type of FFA plays also a crucial role in promoting IR and NAFLD progression
(Musso et al, 2018). Different studies have shown that NAFLD is characterised by an
increased level of SFA, especially palmitic (C16:0) and stearic (C18:0) acids, and
decreased MUFA and PUFA FA (Chiappini et al, 2017; Puri et al, 2007; Sanders et al,
2018). SFA can exert hepatic lipotoxicity through different pathways. For instance,
SFA activates c-Jun N-terminal kinase (JNK), a member of the mitogen-activated
protein kinase (MAPK) family (Musso et al, 2018), and a key mediator of hepatic SFA
lipotoxicity (Solinas & Becattini, 2017). JNK promotes IR via the inactivation of insulin
receptor substrate-1 and its phosphorylation at the inhibitory site Ser-307 (Solinas et
al, 2006). JNK also impairs oxidative phosphorylation (OXPHOS) while promoting the
production of reactive oxygen species formation through the interaction with the outer
membrane mitochondrial protein SH3 domain-binding protein 5 (Sab). JNK also
promotes apoptosis through the activation of the pro-apoptotic protein p53 (Musso et
al, 2018).
Another mechanism by which SFA promotes lipotoxicity is through the promotion of
ER stress, a process by which ER homeostasis is perturbed leading to aberrant
accumulation of unfolded or misfolded proteins in the ER lumen (Han & Kaufman,
2016). Specifically, palmitic acid induces ER stress in hepatocytes by activating
protein kinase RNA-like endoplasmic reticulum kinase (PERK)/ Activating
Transcription Factor 4 (ATF4)/ C/EBP-homologous protein (CHOP) pathway (Wei et
al, 2006). Palmitic acid activates PERK, which leads to increased levels of eIF2α. The
latter promotes a pro-adaptive signalling pathway by the inhibition of global protein
synthesis and selective translation of ATF4, which upregulates the pro-apoptotic
transcription factor CHOP that subsequently induces apoptosis (Cao et al, 2012;
Salvado et al, 2015). Rodent studies have also shown that palmitic acid promotes
NAFLD progression enhancing inflammation through the activation of the toll‐like
receptor (TLR) 2 (Miura et al, 2013). TLR 2 belongs to a family of plasma membrane
receptors that recognise bacteria and virus, and once activated induce pro-
inflammatory cytokines triggering an inflammatory response (Kawai & Akira, 2007).
The discussion above highlights the crucial role of different lipids in IR and NAFLD
progression which is being increasingly appreciated due to the advancement in the
field of lipidomics.
34
Figure 3. Simplified figure of key pathways of lipotoxicity in hepatocytes. IRS-1 = insulin
receptor-1, PKCε = protein kinase C ε, Akt = serine/threonine-protein kinases, SFA = saturated
fatty acids, JNK = c-Jun N-terminal kinase, Sab = SH3 domain-binding protein 5, ROS =
reactive oxygen species, OXPHOS = oxidative phosphorylation, PP2A = protein phosphatase
2A, PKAζ = protein kinase A ζ, ER = endoplasmic reticulum, PERK = transmembrane protein
kinase RNA-like endoplasmic reticulum kinase, ATF4 = activating transcription factor 4, CHOP
= C/EBP-homologous protein. Dotted lines indicate possible but not mandatory steps. Figure
done using the http://smart.servier.com/ image resource.

35
1.2.11 Complications of the MetS and NAFLD

1.2.11.1 Type II diabetes mellitus (T2DM)

T2DM is a multifactorial metabolic disorder characterised by dysregulation of glucose

and lipid metabolism caused by IR, impaired insulin secretion (pancreatic β-cell
dysfunction), or the coexistence of both (DeFronzo et al, 2015). T2DM is clinically
marked by increased fasting blood glucose, an increased presence of glucose in the
urine, or abnormal response to a glucose load. T2DM has reached pandemic
proportions, affecting 8.5% of adults worldwide, becoming a major public health
burden (Emerging Risk Factors et al, 2010). MetS and NAFLD are closely related to
T2DM since IR is a key feature of both conditions. Large prospective studies have
shown that people with MetS and NAFLD have a 5- and 3-fold increased risk for
incident T2DM, respectively (Cornier et al, 2008; Gastaldelli & Cusi, 2019). Similar to
the mixed dyslipidaemia described in the MetS and NAFLD, T2DM is also
characterised by increased circulating levels of large VLDL and decreased HDL-C
(Adiels et al, 2008). Furthermore, both conditions are well-established risk factors for
cardiovascular diseases (CVDs).

1.2.11.2 Atherosclerotic cardiovascular disease (ASCVD)

Cardiovascular disease (CVD) is the leading cause of death worldwide, with

atherosclerotic cardiovascular disease (ASCVD) being the most prevalent form of
CVD (Roth et al, 2017). Atherosclerosis is the process by which the tunica intima (the
innermost layer of arteries), accumulates fat with a simultaneous reduction of arterial
wall elasticity (Lusis, 2000). Epidemiological studies have shown that people with
MetS and NAFLD have a 2 and 3-fold increased risk of CVD, respectively (Mottillo et
al, 2010; Targher et al, 2016).
ASCVD is a multifactorial disease that begins early in life (Berenson et al, 1998).
Although some pathophysiological aspects of ASCVD are not fully understood,
evidence from genetic, epidemiological, and randomized studies of LDL-lowering
therapies, have shown that LDL-C has a causative role in this process (Ference et al,
2017). Besides LDL-C concentration, a mounting body of evidence is showing that the
LDL composition is crucial in determining its atherogenicity (Boren et al, 2020).

36
Atherosclerosis is hypothesised to result from the adhesion of LDL particles to the
tunica intima via proteoglycans (Boren et al, 2020). The retained lipoproteins show an
increased predisposition to oxidation by arterial-wall enzymes and oxidants. Oxidised
LDL exhibit pro-inflammatory activity which attracts monocytes and other immune-
competent cells and acts on the smooth muscle that further attracts other monocytes
(Boren et al, 2020). These monocytes then differentiate into macrophages and in an
attempt to clear up the LDL surplus, can give rise to foam cells, further triggering a
complex inflammatory process that develops atherosclerotic plaques. Recently, it has
been increasingly appreciated the role of hypertriglyceridemia and ApoC-III in the
onset and progression of ASCVD and CVD in general (Ginsberg et al, 2021). In a
prospective population-based study with a 10-year follow-up, Pechlaneret al. found
that ApoC-III was significantly and positively associated (hazard ratio/standard
deviation 1.38; 95% confidence interval [CI]: 1.17 to 1.63) with CVD, independently of
traditional risk factors such as HDL-C and non-HDL cholesterol (Pechlaner et al,
2017). Furthermore, in a multicentre, randomised, double-blind, placebo-controlled
trial involving more than 8,000 statin-treated adults with hypertriglyceridemia and
established cardiovascular disease or with diabetes, the use of a polyunsaturated fatty
acid (ethyl eicosapentaenoic acid (EPA), Icosapent Ethyl)) after a median follow-up of
4.9 year significantly reduced the cardiovascular risk in the icosapent ethyl group, as
compared with the patients in the placebo arm (Bhatt et al, 2019), thus further
supporting the importance of monitoring TG and ApoC-III as therapeutic targets in
CVD prevention.

1.2.11.3 Chronic kidney disease

Chronic kidney disease (CKD) is defined as persistent alterations in kidney structure,

function, or both, which impair health (Romagnani et al, 2017). The Kidney Disease
Improving Global Outcomes initiative classifies CKD based on glomerular filtration rate
and albuminuria (https://kdigo.org/wp-
content/uploads/2017/02/KDIGO_2012_CKD_GL.pdf). The global prevalence of CKD
varies between 7–12% (Hill et al, 2016). Several factors can contribute to the
pathogenesis of CKD, amongst them MetS and NAFLD and their components,
especially high blood pressure and hyperglycaemia, play a central role. Indeed, T2DM
is the leading cause of CKD, and subjects with MetS and NAFLD have a 2.5- and 2-

37
fold higher risk of developing CKD, respectively (Byrne & Targher, 2020; Singh & Kari,
2013).
There are several mechanisms by which T2DM and MetS increase the risk of CKD.
Hyperglycaemia promotes the reabsorption of sodium in kidneys via activation of the
sodium/glucose cotransporter 2, which after a cascade of events eventually activates
the renin-angiotensin system which causes dilatation of the afferent arteriole and
vasoconstriction of the efferent arteriole, ultimately increasing GFR (van Bommel et
al, 2017).
Prolonged and uncontrolled hypertension is also a well-established risk factor for CKD
(Romagnani et al, 2017). While the kidney can normally cope with transitionary
increased blood pressure (i.e during physical activity), persistently elevated blood
pressure leads to mechanical damage of kidneys’ glomeruli, a network of capillaries
that transport circulating blood within the kidney functional unit, nephrons, thus
impairing their function. Epidemiological studies have shown that obesity is associated
with CKD, with a mechanism that can be direct or indirect (i.e obesity-associated
hypertension) (Rhee et al, 2016). Some of the direct mechanisms include reduced
production of adiponectin and increased production of resistin and leptin promoting a
state of systemic inflammation, oxidative stress, and IR, all of which have a detrimental
effect on kidney structure and function (Camara et al, 2017).

1.3 Lipidomics as a tool to study obesity and its metabolic-

related disorders

The characterisation of the main lipid species and pathways involved in IR and NAFLD
has dramatically increased in the past decade due to progress in lipidomics, a well-
established but still expanding field, that aims to dissect the role of lipids and their
metabolic pathways on a large scale (Yang & Han, 2016). Before the advent of
lipidomics, researchers were limited to the study of major lipid classes, such as PL,
CE, FC, TG using thin layer chromatography, and total fatty acid composition by gas
chromatography mass spectrometry (GC-MS). The lipidomic field takes advantage of
several analytical techniques, most importantly chromatography coupled with mass
spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy (Griffin et
al, 2011).

38
Lipidomic studies are shedding light on the “lipidomic signature” of IR, MetS, and
NAFLD in blood plasma and tissues (i.e liver) (Chiappini et al, 2017; Kotronen et al,
2009b; Monnerie et al, 2020; Sanders et al, 2018). For example, while elevated
plasma TG levels partially mediate the increased risk of CVDs in MetS and NAFLD
(Zhang & Lu, 2015), lipidomic studies have shown that the composition of TG can help
better stratify the CVD risk (Sanders & Griffin, 2016). In a prospective population-
based study with a 10-year follow-up, Stegemann et al. found that TG (54:2) in
combination with cholesteryl ester (16:1), and phosphatidylethanolamine (36:5), and
traditional risk factors resulted in improved CVD risk prediction (Stegemann et al,
2014). Furthermore, Kotronen et al., examined the lipidomic composition of
lipoproteins showing that in healthy subjects with a wide range of insulin sensitivity,
serum TG from VLDL, with a lower carbon number and a double bond content
associated with IR, whereas those containing essential fatty acids were negatively
associated with IR (Kotronen et al, 2009b). Also, with a translational approach using
both pre-clinical models and human cohorts (interventional and observational),
Sanders et al., demonstrated the relevance of DNL in NAFLD which is partially driven
by a high refined carbohydrate diet (Sanders et al, 2018). More recently, Charidemou
et al., also demonstrated that a high protein meal can promote hepatic DNL in healthy
adults which poses a question on the safety of high protein diets for the treatment of
obesity (Charidemou et al, 2019). Other authors have also proposed lipidomic based
non-invasive biomarkers of NAFLD, but further studies are warranted to validate these
results (Guerra et al, 2021). Table 2, adapted form (Guerra et al, 2021), summarise
some of the key lipidomic findings in NAFLD. Taken together, these data clearly show
the clinical relevance of lipidomics is obesity and its related disorders, but also the
need for further studies to validate these results in people with different age, health
status and genetic background.

39
Table 2. Summary of key lipidomic studies in NAFLD

Number of
Study Diagnosis Tissue Main findings
participants

TAG, DAG, total cholesterol, SFA, n6: n3

ratio
↑ NAFLD and NASH

Puri et al TAG, FA 20:4n-6, TAG FA 22:6n-3

9 CT
2007 ↓ NAFLD and NASH
9 NAFLD Liver biopsy Liver
(Puri et al,
9 NASH
2007) PC, PE
↓ NAFLD

FC, LysoPC
↑ NASH

TAG, DAG, FFA and CE MUFA, CE, DAG,

PC, PE with 18:3 or 20:3 FA
↑ NAFLD and NASH
Puri et al
50 CT
2009
25 NAFLD Liver biopsy Plasma DAG, PC, PE, TAG SFA
(Puri et al,
50 NASH ↓ NAFLD
2009)

22:6n-3/22:5n-3 ratio in PC, PE

↓ NASH

FFA 16:0, SM 18:0/16:0, 18:1/18:0, PC 28:0,

LysoPC 20:2, 20:1, LPE P-16:0
↑ NAFLD and NASH

FFA 18:3, 20:2n6, SM 18:1/12:0, 18:2/14:0,

Barr et al 18:2/16:0, 36:3, PC 18:0/22:6
9 CT
2010 ↓ NAFLD and NASH
24 NAFLD Liver biopsy Serum
(Barr et al,
9NASH
2010) PC 14:0/20:4, 16:0/20:3, P-18:0/20:4,
LysoPC 18:1
↑ NASH

FFA 20:4, LysoPC P-24:0, P-22:0, O-20:0

↓ NASH

PC, PE, and PG, CER d18:0/22:0,

d18:1/16:0, d18:1/18:0, d18:1/20:0,
d18:1/22:0, d18:1/23:0, d18:2/20:0,
d18:2/18:0, d18:2/20:0, d18:2/21:0,
d18:2/22:0, d18:2/23:0; SM 36:1, PC 32:0,
32:1, 34:1, 34:3.36:1, 36:3, 36:4, 36:5, 38:3,
Anjani et al
38:4, 38:5, 38:6, 40:4, 40:5, 40:6, PE 34:1,
2015 24 CT
Liver biopsy Serum 34:3, 36:1, 36:2, 36:4, 36:5, 38:3, 38:4, 38:5,
(Anjani et 22 NASH
38:6, 40:4, 40:5, 40:6, 40:7, LysoPC 16:0,
al, 2015)
16:1, 20:3, 22:5, PG 36:1, 36:2, 36:3, 38:3,
38:4; PI 32:1, 34:1, 36:4, 38:4, 40:4, 40:5
↑ NASH

CER d18:1/24:0, SM 42:3

↓ NASH

40
 TAG, CE, CER, DAG 36:2, HexCER
d18:1/24:1, GlucCER d18:/24:1, d18:1/26:1,
PC 36:4, 38:4, PE 38:5, 38:4, 40:6, 40:5,
LysoPC 16:0, PI 36:1, 38:4, 38:3
↑ NAFLD and NASH

HexCER d18:1/24:1, d18:1/26:1, CER

m18:1/16:0, m18:1/26:1, GlucCer d18:1/26:1,
18:1/26:0, PC 32:0, PI 36:1
↑ Cirrhosis
Gorden et 31 CT
al 2015 17 NAFLD
Liver biopsy Plasma Total cholesterol, CE 18:2, 20:4, 20:3, TAG
(Gorden et 20 NASH
52:4, 52:3, DAG 36:2, CER d18:1/18:0,
al, 2015) 20 Cirrhosis
18:1/20:0, d18:1/22:0, d18:1/24:1, d18:1/24:0,
d18:0/18:0, d180/24:1, m18:1/16:0,
m18:1/26:1, m18:0/18:0, m18:0/20:0,
m18:0/22:0, m18:0/24:0, HexCER d18:1/24:1,
d18:1/26:1, GlucCer d18:1/24:1, d18:1/26:1,
18:1/26:0, SM d18:1/18:1, d18:1/18:0,
d18:1/20:0, d18:1/22:0, d18:1/24:0, PC 32:0,
34:3, 36:4; 38:6, 38:5, 38:4, 38:3, 40:6, PE
36:4, 38:6, 38:5, 38:4, 40:6, 40:5, LysoPC
16:0, PI 36:1, 38:4, 38:3
↓ Cirrhosis

SFA (14:0, 16:0, 18:0)

Chiappini
7 CT ↑ NASH
et al 2017
39 NAFLD Liver biopsy Liver
(Chiappini
15 NASH PC, PE, PI, PS PC/PE, SM
et al, 2017)
↓ NASH

Total CER, LactCER 24:1, HexCER 22:0,

Liver 24:0, 24:1; dhCER 16:0, 22:0, 24:1
Apostolopo ↑ NASH
ulou et al
2018 7 CT
Liver biopsy
(Apostolop 7 NAFLD dhCER 20:0
oulou et al, 7 NASH ↑ NAFLD
2018) Serum
Total dhCER, dhCER 16:0, 22:0, 24:1
↑ NASH

Tiwari-
Heckler et TAG, SM, PC
28 CT
al 2018 ↑ NAFLD and NASH
25 NAFLD Liver biopsy Plasma
(Tiwari-
42 NASH
Heckler et LysoPE
al, 2018) ↓ NAFLD and NASH

TAG 54:2, 48:1, 48:2, 50:1, 50:2

Sanders et
↑ NAFLD
al 2018 663 CT
Ultrasound Plasma
(Sanders 233 NAFLD
TAG 52:3, 52:4, 56:7, 56:6, 54:4, 56:8
et al, 2018)
↓ NAFLD

41
 CER d18:0/16:0, d18:0/18:0, d18:0/20:0,
d18:0/22:0, d18:0/24:0, d18:0/24:1, DAG SFA
(16:0, 18:0), MUFA (18:1), PUFA (18:2), TAG
Plasma
SFA (16:0, 17:0, 18:0), MUFA (18:1), PUFA
(18:2, 20:3, 20:4)
↑ NAFLD

Total dhCER, TAG, DAG, CER d18:0/18:0,

d18:0/20:0, d18:0/22:0, d18:0/24:0,
d18:0/24:1, LPC 26:0, PI 18:0/22:5, CE 18:0,
> 20 DAG, > 40 TAG
↑ NAFLD and NASH

PC 15-MHDA/18:2, PC 15-MHDA/22:6, PC
17:1/18:2, 18:1/22:6, CE 22:5
↓ NAFLD and NASH
Ooi et al
50 CT
2021
110 NAFLD Liver biopsy Total CER, CE, THC, CER d18:1/16:0,
(Ooi et al,
16 NASH d18:1/18:0, d18:1/20:0, d18:1/22:0,
2021)
d18:1/24:0, GM3 d18:1/20:0, PC 28:0, 31:0,
PC O-40:7, PS 38:4, CE 18:3, FC
Liver ↑ NAFLD

SM 37:2, d18:2/20:0, PC 17:0/18:2,

18:1/18:2, 39:5, 17:0/22:6, PC P-38:5, PE
18:1/22:6, PE P-18:1/22:4, 20:1/22:6
↓ NAFLD

Total DHC, DHC d18:1/18:0, d18:1/22:0,

d18:1/24:0, SM d18:0/16:0, PC 36:0,

↑ NASH

PC 16:1/20:4, 38:6, PC 15-MHDA/20:4, PE

16:0/20:4, 38:5, PI 38:5,
↓ NASH

CT, control; NAFLD, non-alcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; DAG: diacylglycerol;
TAG: triacylglycerol; SFA: saturated fatty acids; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty
acids; FC: free cholesterol; CE, cholesteryl esters; FFA: free fatty acids; SM: sphingomyelin; PC:
phosphatidylcholine; LysoPC, lysophosphatidylcholine; PI: phosphatidylinositol; PS: phosphatidylserine; PE:
phosphatidylethanolamine; LysoPE, lyso phosphatidylethanolamine; CER: Ceramides; dhCER, dihydroceramides;
DHC/Hex2Cer, dihexosylceramide; THC, trihexosylceramide; GM3; GM3 ganglioside; LactCER, lactosylceramide;
GlucCER, glucosylceramide. Table adapted from (Guerra et al, 2021)

42
1.3.1 Lipoprotein lipidomics

Although several studies show the whole plasma “lipidomic signature” under different
conditions (Fernandes Messias et al, 2017; Kulkarni et al, 2013a; Puri et al, 2009), the
changes reported do not provide information on the origin of each lipid species. For
example, for CVD risk it is more relevant knowing which compartment circulating
cholesterol is partitioned to among the lipoproteins (HDL-C vs. LDL-C) rather than the
total amount. This is likely to happen to other lipid species, and therefore to have a
better understanding of the biological function of circulating lipid species, it is important
to study the lipoproteins fractions in isolation.
Lipoproteins are commonly separated according to their density through
ultracentrifugation (UTC). UTC is one of the first methods described to separate
lipoprotein and it is still considered the gold standard in the lipoprotein field. UTC can
be performed with a sequential floatation, separating CM, VLDL, IDL, LDL, and HDL
(Havel et al, 1955), or with a single step density gradient centrifugation, allowing
further speciation (HDL2 and HDL3) (Chapman et al, 1981). There are several
limitations with this method including the technical expertise required, the amount of
time needed, the large volumes of sample (1 mL) required, possible loss of
apolipoprotein from fractions, and the lack of parameters specifying the density cut-off
between lipoproteins. Besides UTC, several other separation methods have been
described, including electrophoretic mobility (Noble, 1968), fraction precipitation
(Warnick et al, 1982), immunoaffinity for apolipoproteins (Cheung & Albers, 1984) and
particle size, achievable through size exclusion chromatography (SEC) (Innis-
Whitehouse et al, 1998). Each method has its drawbacks; it is therefore important to
decide which separation technique adopts based on the question the investigator aims
to answer.
SEC has been used in this work because of a) the low sample volume required, and
b) it has been validated against UTC (Han et al, 2012; Innis-Whitehouse et al, 1998)
while having a higher throughput. On the other hand, SEC does not allow to separate
chylomicron from VLDL, thus being not recommended for post prandial lipidomic
studies. IDL are distributed between the VLDL and LDL fractions, and albumin co-
elutes with HDL, thus contributing to the lysophosphatidylcholine pool found in these
fractions. Because my project does not investigate the meal impact on lipoprotein
metabolism and I did not study the LDL remodelling, the impact of albumin on HDL

43
lipidome might affect only a single lipid class (LPC) (Wiesner et al, 2009), and thus
considered an acceptable drawback.
Here, it is briefly reported the principle of separation for the SEC only as it is the
method adopted for this work, but the detail of the different methods can be found in
comprehensive reviews (Hafiane & Genest, 2015; Krauss, 2010).
SEC separation is based on the capacity of the column, packed with fine, porous
beads made of agarose, to retain the smaller particles due to their ability to diffuse into
the pores and let elute first the larger particles, which are bigger than the pores so do
not diffuse through them (Osei et al, 2015). The technical details are reported in the
method section (Chapter Two).

1.3.2 The use of liquid chromatography-mass spectrometry in

lipidomics and metabolomics
Liquid chromatography (LC) is an analytical technique that allows the separation of
different compounds based on their affinity with two phases, one stationary (solid) and
one eluent (liquid) while passing through a chromatographic column. The LC system
consists of a series of pumps (from 1 to 4) which lead a solution of a mixture of solvents
(mobile phase) through a chromatographic capillary column (stationary phase). Before
entering the column, the mobile phase will be mixed with the sample solution through
a valve system. Once inside the columns, compounds are separated due to their
affinity with the stationary phase. The affinity is affected by different factors such as
flow rate, mobile phase composition, and column temperature as well as the chemical
composition of the analytical mixture. The time required by a compound to elute from
a column is defined as its retention time (RT), and that is the parameter used to identify
the various compounds (Griffin et al, 2011). Based on the affinity between compounds
and the stationary phase, there are two major classes of chromatographic techniques:

 normal-phase liquid chromatography

 reverse-phase liquid chromatography.

Normal-phase LC uses a polar stationary phase and a non-polar mobile phase,

whereas the reverse-phase LC, uses a polar mobile phase and a non-polar stationary
phase. Besides the different approaches available, the extraordinary variety of
columns available permits the separation of a huge range of compounds, thus

44
rendering LC a very popular separation method in metabolomics. A key limitation of
LC is the overlapping RT of may compounds. To overcome this issue, the separated
metabolite can be further analysed by MS.
MS is a powerful analytical technique which enables direct identification of molecules
based on the mass-to-charge ratios (m/z) as well as fragmentation pattern (Urban,
2016). In its most basic form, a mass spectrometer is composed of an ionising source,
an analyser, and a detector. The ionisation of a metabolite is a mandatory step for its
separation and detection while one of the most common is the electrospray ionization
(ESI) or soft ionisation due to the small fragmentation generated during the ionising
process (2019). The ESI can be used in combination with a variety of analyser, which,
despite the different approaches used, ultimately rely on the use of the m/z as a
discriminant of separation (Kofeler et al, 2012). Two very common analysers include
the triple quadrupole (QqQ), and the orbitrap MS analyser. The former is used for
targeted assays, which refer to the measurement of a specific set of metabolites,
whereas the orbitrap is mostly used for open profile studies, which refer to the measure
of as many metabolites as possible without pre-defined mass transitions as used in
QqQ MS. Once a compound has been separated, it is finally directed towards the
detector, which generates an electrical signal that is proportional to the abundance of
the compound.
The QqQ MS employs two quadrupoles, acting as mass analysers, and one
quadrupole for fragmentation (collision cell), located between them. Quadrupoles
consists of four parallel charged metal rods arranged through a central axis. Static
potential (DC voltages) and alternative potentials (RF voltages) are applied to the
quadrupole roads with the two pairs of rods having opposite polarity. These voltages
generate an oscillating field that allows only specific ions with determined m/z to move
towards the detector, while other compounds will be deflected away. In the QqQ, the
first quadrupole filters the ion of interest (parent ion) according to its m/z and then
fragments it in the collision cell using collision-induced dissociation (CID). Specifically,
fragmentation is obtained through the collision of the ion with an inert gas (i.e helium,
nitrogen or argon), eventually generating a fragment ion. The latter is further separated
in the third analyser and then quantified by a detector. The use of tandem LC-MS/MS
is among the most common approaches in metabolomics, providing excellent
sensitivity but a relatively low mass accuracy as compared to other methods (Mann &
Kelleher, 2008).
45
A more recent analyser is the orbitrap MS. The latter is composed of two outer
electrodes and a central spindle electrode. Ions are injected into space between the
central and outer electrodes where are trapped by an electrostatic field. Here, ions
oscillate around the central electrode and along its axis. Ions with different m/z
oscillate at different frequencies, thus allowing their separation. The oscillating
frequencies generate an image current which is detected by the outer electrodes and
quantified according to its size, which represents the ion abundance. The image
current is then processed by a Fourier transformation. The orbitrap MS is
characterised by a high resolution and high mass accuracy (2-5ppm) (Hu et al, 2005),
which are of crucial relevance for untargeted approaches.

46
Chapter 2. Materials and methods

This chapter provides a general description of the materials and methods used
throughout this thesis. Methods specific for each study are reported in their respective
chapters.

2.1 Measurement of clinical biochemistry

Clinical biochemistry for the different studies, including full lipid profile, glucose, insulin,
circulating liver enzymes, and total free fatty acids, was performed by The Pathology
Partnership (Addenbrooke’s Hospital, Cambridge, UK). HOMA2 values were obtained
by the program HOMA Calculator v2.2.3 available at
https://www.dtu.ox.ac.uk/homacalculator/.

2.2 Measurement of lipid levels by mass spectrometry

2.2.1 Lipid extraction

Lipids were extracted from blood serum, previously stored at -80 °C, using an
adaptation of the Folch method (Folch et al, 1957). Briefly, 10 μL of blood serum was
mixed with chloroform/methanol (2:1, 1 mL) and then 10 uL of internal standard mix
including the following: N-palmitoyl-d31-D-erythro-sphingosine (16:0-d31 Ceramide,
12.2 µM), heptadecanoic-d33 acid (17:0-d33 FFA, 40.0 µM), , 1-palmitoyl(D31)-2-
oleyl-sn-glycero-3-phosphatidylcholine (16:0-d31-18:1 PC, 40.1 µM), 1-
palmitoyl(d31)-2-oleyl-sn-glycero-3-phosphoethanolamine (16:0-d31-18:1 PE, 38.0
µM), 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (16:0-d31-18:1
PG, 12.5 µM), N-palmitoyl(d31)-d-erythro-sphingosylphosphorylcholine (16:0-d31 SM,
47.4 µM), glyceryl tri(pentadecanoate-d29) (45:0-d87 TAG, 40.3 µM) (Avanti Polar
Lipids Inc, USA) was added. Samples were sonicated for 10 min and subsequently,
deionised water was added (400 μL). Samples were then centrifuged (17,000 × rpm,
10 min). 500 μL of the organic fraction was transferred into a new vial and dried under
a steam of nitrogen, whereas 500 L of the aqueous part was placed in a new vial
and dried in a CentriVap Centrifugal Concentrator with attached cold trap (78100

47
series, Labconco Co, Kansas City, USA). Dried samples were then stored into a - 80
C until analysis.

2.2.2 Lipid analysis by liquid chromatography-mass spectrometry

Before analysis, dried samples were reconstituted in 20 μL of 1:1

chloroform/methanol, sonicated for 10 minutes, and then diluted in isopropyl
alcohol/acetonitrile/water (2:1:1, 100 μL). The analysis of intact lipids was performed
through LC-MS using a Dionex Ultimate 3000 ultra-high performance liquid
chromatography system (UHPLC; Thermo Scientific) coupled to an LTQ Orbitrap Elite
Mass Spectrometer (Thermo Scientific, Hemel Hempstead, UK). 10 μL of sample was
injected onto a C18 CSH column, 1.7 μM pore size, 2.1 mm x 50 mm, (Waters Ltd,
Manchester, UK) maintained at 55 °C. A gradient of solvent A, 10 mM ammonium
formate in acetonitrile/water 6:4, and solvent B, 10 mM ammonium formate in
isopropanol/acetonitrile 9:1, was used for the positive mode acquisition. For the
negative mode, mobile phase remained the same except the use a 10 mM ammonium
acetate instead of ammonium formate. The HPLC was coupled to a heated
electrospray source optimised at 50:50 mobile phase A to mobile phase B for spray
stability (capillary temperature; 300 C, source heater temperature; 420 C, sheath gas
flow; 40 (arbitrary), auxiliary gas flow; 15 (arbitrary), spare gas; 3 (arbitrary), source
voltage; 4 kV. The mass spectrometer scan rate set at 4 Hz, giving a resolution of
25,000 (at 200 m/z) with a full-scan range of m/z 100 to 1800 m/z collected in positive
and negative ion mode.Two representative MS spectra (one positive and one
negative) highlighting the major lipid classes are reported in Figure 1. Peak
identification and mass spectra processing were carried out with Xcalibur Software
(Thermo Scientific). Peak areas were normalised to their class-specific internal
standard, leading to a semi-quantitative data, here referred to as normalised intensity.
Lipids with missing values higher than 40% were removed from the dataset, whereas
the others were imputed with the half minimum value method (Hinz et al, 2019). The
area ratios were then blank corrected (a blank was a sample that followed the entire
extraction procedure as done with the regular samples but without serum, thus blank,
and injected after each 10 regular samples). Specifically, normalised intensities with a
value less than three times the blank samples were set to zero and removed from the
dataset. As a quality control I used a cohort specific pooled sample (serum combined
from each participant and extracted as described above) injected every 10 regular
48
samples. Pooled samples were used to assess the coefficient of variation (CV) across
the LCMS run. Samples with a CV <25% were retained whilst higher CV led to the
exclusion of the specific lipid.

Figure 1. Representative mass spectrums (positive and negative modes) of total lipid extract of untargeted whole
serum lipidome. Cer, ceramide; DG, diacylglycerol; LPC, lysophosphatidylcholine; LPE,
lysophosphatidylethanolamine; CE, cholesteyl ester; TG, triacylglycerides; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; SM, sphingomyelin; PG phosphatidylglycerol; FFA, free fatty acids.

To assess the linearity and range covered within our lipidomic platform, by employing
calibration lines (lipid standards), we identified that our lipidomic data are in the range
between 5 nM and 25 uM with a linear response, while the calibration curve turns
quadratic after 25 uM. A representative calibration curve (duplicate) for a PC (PC 28:1)
is reported in Figure 2. This work, however, did not use calibration lines to obtain a

49
fully quantitative data but a semi-quantitative result was obtained by using a single
specific internal standard, as above detailed.

Figure 2. Representative calibration curve showing the linearity of a standard lipid (phosphatidylcholine 28:0, (PC
28:0)). In the y axes is reported the normalised intensity referred to as Area_ratio (peak intensity divided to a
labelled internal standard), while the x axes report the concentration expresses to as uM.

With regards to the reproducibility of our platform, below is reported the coefficient of
variation for the intraday variability and intraday variability (3 weeks) of several IS at 5
M concentrations (Table 1).

Table 1. Inter and intraday variability of the LCMS lipidomic platform.

LPC_(C14:0)- SM_(C16:0- PG_(C16:0- PC_(C16:0- C16-d31 PE_(C16:0- TG_(45:0-

Internal standard
d42 d31) d31/C18:1) d31/C18:1) Ceramide d31/C18:1) d29)

Internal standard
IS_LPC IS_SM IS_PG IS_PC IS_Cer IS_PE IS_TG
(shorthand notation)

Timing Duplicate at 3 weeks between the two analysis

Mean 48730606 320042416 198765580 509610817 146002237 170457773 396978106
AREA Inter-
Std Dev. 3817879 44769467 22146204 40744358 18335448 20393972 85852911
CV (%) 8 14 11 8 13 12 22
Timing Duplicate within the same day
Mean 50190859 329665328 213955398 527044342 138818410 176313186 412649285
AREA Intra-
Std Dev. 3527761 32840732 25850804 40151990 26724945 15694049 68439178
CV (%) 7 10 12 8 19 9 17

LPC, lysophosphatidylcholine; SM, sphingomyelin; PG phosphatidylglycerol; PC, phosphatidylcholine; Cer,

ceramide; PE, phosphatidylethanolamine; TG, triacylglycerides; IS, internal standard; CV, coefficient of variation.

50
2.2.3 Esterified cholesterol analysis – LC-MS/MS

The organic lipid-containing layer from the Folch extraction was analysed by targeted
LC-MS/MS using a UHPLC+ series coupled to a TSQ Quantiva mass spectrometer
(Thermos fisher scientific, Waltham, MA, US). Ten microlitres of sample containing
isotopically labelled cholesteryl-d7 pentadecanoate at the concentration of 42.5 µM
(Avanti Polar Lipids Inc, USA) was injected onto an Acquity C18 CSH column (Waters
Ltd., Warrington, UK; 100 × 2.1 mm, 1.7 μm) with a column temperature of 55 °C. A
gradient separation was used as described for the lipidomics experiments. A heated
electrospray ionisation source was operated in positive ion mode; desolvation
temperature and gas flow were 270 ˚C and 45 arbitrary units, respectively. Selected
reaction monitoring transitions used are included in Table 2. Xcalibur software
(Thermo Fisher Scientific) was used for peak integration. Peak areas were normalised
to the internal standard. Post-acquisition processing (blank correction, imputation and
QC analysis) was the same as above described.

Table 2. Targeted (LC MS/MS) parameters for the measurements of circulating cholesteryl esters (CE).

Metabolite HMDB ID Column Retention Mode Precursor Product Collision Lens

Time (RT) Energy
(CE)
(min) m/z m/z (V) (V)
CE15:0 d7 18 CSH 7.6 Positive 640.58 376.387 24.713 155
CE16:0 HMDB0000885 18 CSH 7.7 Positive 647.815 369.333 24.511 140
CE18:1 HMDB0005189 18 CSH 7.6 Positive 673.537 369.333 27.242 152
CE18:2 HMDB0000610 18 CSH 7.5 Positive 671.53 369.333 28.86 151
CE18:3 HMDB0010369 18 CSH 7.2 Positive 669.56 369.333 21 151
CE 20:4 HMDB0006726 18 CSH 7.4 Positive 695.567 369.333 30.124 148

CE, cholesteryl esters; HMDB, Human Metabolome Database; CSH, Charged Surface Hybrid.

2.3 Measurement of apolipoprotein levels by mass spectrometry

2.3.1 Apolipoprotein extraction

Apolipoprotein were extracted from blood serum as previously reported (Kay et al,
2007). As compared to the original method, we focused on a subset of apolipoprotein
found relevant to the circulating lipoprotein lipid remodelling based on a literature
search I performed at the beginning of my project. Briefly, 10 L of serum was diluted
by a factor of 10 with 50 mM ammonium bicarbonate in water. Diluted serum (10 μL)
then was transferred to 490 μL of 10 mM dithiothreitol in 50 mM ammonium

51
bicarbonate, with 0.5 mg/mL bovine serum albumin (BSA) as an internal standard.
Samples were incubated at 60°C for 1 hour, allowed to cool to room temperature and
100 μL of 100 mM iodoacetamide in 50 mM ammonium bicarbonate was added and
samples incubated at room temperature in the dark for 30 min. Trypsin (10 μL at 100
μg/ml) was added to a 100 μL aliquot of each sample and incubated for 16 hours at
37 °C. After incubation, 20 μL 1% formic acid (aq) was added to halt digestion prior to
analysis by LC-MS/MS.

2.3.2 Apolipoproteins analysis by liquid chromatography-mass

spectrometry
LC-MS/MS analysis was performed on an M-Class liquid chromatography system
coupled to a TQ-XS (Waters, Milford, MA, USA). A gradient of solvent A, 0.1% formic
acid in water, and solvent B, 0.1% formic acid in acetonitrile was used to separate
digested proteins. Sample (2 µL) was injected onto an HSS T3 50 x 1.0 mm column
(Waters) at 25 µL per minute at 15% B, rising to 50% B over 5 minutes. The column
was washed at 85% B for 2.5 minutes before returning to initial conditions for a total
run time of 10 minutes. Positive electrospray ionisation was performed with a needle
voltage of 3 kV, source and desolvation temperature of 450 ºC and 150 ºC
respectively, and a cone voltage of 30V. Selected reaction monitoring transitions used
are included in Table 3. Peptide peaks were integrated using TargetLynxXS (Waters)
and expressed a ratio of the average peak area to the mean peak area value of two
typically digested BSA peptides.

Table 3. Targeted (LC MS/MS) parameters for the measurements of circulating apolipoproteins.
Peptide sequence Q1 m/z Q3 m/z Collision
Protein
Energy (CE)
APOA-I DYVSQFEGSALGK 700.8 808.4 20
675.7 540.2 20
APOA-IV LGEVNTYAGDLQK
675.7 575.6 20
APOB-100 TEVIPPLIENR 640.8 838.4 20
516.8 620.3 20
APOC-I TPDVSSALDK
516.8 719.4 20
518.2 658.2 15
APOC-II TYLPAVDEK
518.2 771.2 15
598.7 854.2 20
APOC-III GWVTDGFSSLK
598.7 953.3 20
615.8 890.4 20
APOD NILTSNNIDVK
615.8 1003.5 20
484.7 399.7 15
APOE LGPLVEQGR
484.7 588.2 20
APOM EFPEVHLGQWYFIAGAAPTK 754.4 615.8 30
BSA 1 LVNELTEFAK 582.3 951.29 20
BSA 2 LGEYGFQNALIVR 740.5 1017.4 25

52
2.4 Lipoprotein separation by Fast protein liquid chromatography

The serum lipoprotein profile was performed by size exclusion chromatography (SEC)
using a previously described method (Innis-Whitehouse et al, 1998). Briefly, fractions
were determined in 50 L of serum samples diluted with 50 μl of a PBS solution via
SEC, using a Superose 6 increase column, (10/300GL, 10 × 300, 24 ml). SEC was
carried out using an ÄKTA purifier 10 (GE Healthcare; Uppsala, Sweden), equipped
with a fraction collector Frac-950. The system was controlled by a UNICORN control
system version 4.10 (GE Healthcare). SEC flow rate was set at 450 μl/min at 10°C. A
representative chromatogram of the total cholesterol FPLC eluate profiling in a healthy
and a NAFLD participant is reported in Figure 3. Eluting fractions were collected in 96
glass-coated well-plates (Eppendorf Protein Low- Bind; Hamburg, Germany). After
fractionation, samples were dried down under a gentle steam of nitrogen and stored
at - 80°C before further processing. Selected fractions obtained by Superose-6 SEC
were further investigated by MS as detailed above.

Figure 3. Representative lipoprotein cholesterol profiles (colorimetric assay) of a fasting blood serum of a
healthy volunteer and a NAFLD patient. The fraction numbers, are represented on the x-axis and the absorbance
units in arbitrary units (ab), measured at 570 nm, are represented by the y-axis.

53
2.5 Statistical analyses
Data are shown as mean ± standard deviation unless otherwise specified. Normality
was visually assessed from plots of the data (skewness/kurtosis) obtained with the lm
function in R, and logarithmic transformations were applied to non-normally distributed
data. In Chapter 3, lipids are grouped into their major lipid classes by summing any
lipid species belonging to a specific class, while in Chapter 4 lipids are grouped as in
Chapter 3 (total lipids) in addition to a further classification based on their number of
double bonds within each lipid class as saturated, lipids with no double bonds,
monounsaturated, lipids with a maximum of 1 double bond per chain, polyunsaturated,
lipids with 2 or more double bonds per chain (further details are provided in the
respective chapters). Comparisons of clinical and omics data between healthy and
MetS/NAFLD were assessed using two-way or three-way ANOVA with “sex” and
“disease state” or “sex”, “T2DM” and disease state as covariates, respectively. Overall,
a P-value <0.05 was considered significant, yet multiple testing correction (Benjamini-
Hochberg procedure to control the False Discovery Rate (FDR)) were applied when
lipids were investigated as independent hits as specified in the method section of
Chapter 3 and 4. Correlation matrices between omics layers along with clinical
variables, were performed with Pearson correlation coefficient, with P-value <0.05
considered significant, or multiple regression (lm function, in R) with “sex” as a
covariate, when relevant; a P-value <0.05 was considered significant. Statistical
analysis was performed with R version 4.0.0 and Microsoft Excel 2016. Graphs were
obtained using R version 4.0.0 and Graph Pad (Graph Pad Prism 7.0).

54
Chapter 3. Lipidomic indices reveal reduced LCAT activity
in patients with metabolic syndrome

3.1 Abstract

The Metabolic Syndrome, a cluster of cardiovascular risk factors, has been associated
with changes in circulating lipidome, but underlying mechanisms remain unclear. The
present study aimed to define the fasting lipidomic differences between MetS (n=14)
and healthy people (n=11), and the contribution of circulating lipoprotein remodelling
enzymes in the observed changes. Compared to healthy people, MetS patients
showed increased: triglycerides, diglycerides, free fatty acids, and
phosphatidylcholines in their blood, whereas lysophosphatidylcholine alongside
cholesteryl ester to free cholesterol ratio were decreased. Employing lipidomic ratios,
used as an indirect proxy of enzymatic activity, along with direct measurements of the
circulating lipoprotein remodelling enzymes, we demonstrated that the lipidomic
changes seen in the MetS were attributable, in part, to reduced activity of the enzyme
lecithin cholesterol acyltransferase (LCAT). Both direct and indirect proxies of LCAT
activity showed a positive correlation with HDL-C and a negative correlation with
obesity and insulin resistance, thus suggesting a close link between LCAT activity and
the MetS. Taken together, these results provide, for the first time, a mechanistic
attempt to decipher the lipidomic profile associated with the MetS.

Simplified representation of the hypothesised mechanistic pathway involved in the lipidomic

remodelling occurring in MetS.

55
3.2 Introduction

The majority of lipidomic studies conducted to date have been performed in a fasting
status. When compared to healthy people, MetS patients display a plethora of lipid
changes that have only recently begun to be characterised. For instance, in line with
the hypertriglyceridemic nature of the MetS, lipidomic studies have shown that MetS
is characterised by elevated levels of circulating triglycerides (TG) and diglycerides
(DG) irrespective of their fatty acid composition (Capel et al, 2020; Surowiec et al,
2019). Interestingly, the same studies also reported discordant results for the
phosphatidylcholine (PC) levels. Specifically, when compared to healthy participants,
Capel et al., reported elevated levels of PC in MetS patients (Capel et al, 2020), while
Surowiec et al, reported reduced levels in MetS (Surowiec et al, 2019). In line with the
former study, Kulkarni et al, found that MetS patients showed a general increase in
plasma PC when compared to healthy subjects (Kulkarni et al, 2013b) whereas a
smaller study reported that only PC34:2, the most abundant circulating PC, was
increased in MetS when compared to healthy people, and is positively correlated with
the MetS defining criteria (Ramakrishanan et al, 2018). A lipidomic analysis of the
Framingham Heart Study, involving 658 participants, reported that PC and
lysophosphatidylcholine (LysoPC) were inversely associated, while sphingomyelin
(SM), TAG, and DAG were directly correlated with obesity (Yin et al, 2020). The same
study also reported that LysoPC were inversely associated, while specific SM were
positively associated with hyperglycaemia. Moreover, several TG and DG showed a
positive correlation with dyslipidaemia, while different PC and LysoPC were inversely
correlated with the latter (Yin et al, 2020). As pointed out in a recent systematic review
of the literature, the paucity of studies investigating the lipidomic signature of MetS,
along with differences in the study design, and lack of replication cohorts, except for
the Framingham Heart Study, make the interpretation of the different studies very
difficult (Monnerie et al, 2020). Most importantly, however, the lack of follow-up
experiments that would provide mechanistic insight into the observed changes is also
a major issue in the field.

This chapter aims to investigate the fasting lipidomic changes occurring in patients
with MetS and to provide a mechanistic explanation. For this purpose, I employed
state-of-the-art mass spectrometry lipidomics and targeted proteomics, along with
commercially available enzymatic assays which pointed out to an impaired

56
phospholipid remodelling in MetS, partly driven by a reduced activity of the enzyme
lecithin–cholesterol acyltransferase.

57
3.3 Methods

3.3.1 Ethics and the MetS study cohort

11 healthy volunteers and 14 patients at the first diagnosis of MetS were included in
this study. Briefly, the presence of MetS was defined when 3 out of 5 criteria for MetS
were present according to the NCEP: ATP III (Alberti et al, 2009). Exclusion criteria
were: the presence of diseases that could have influenced participants’ metabolism
(i.e. autoimmune disease, cancer, thyroid, and adrenal disorders, endocrine disorders,
and acute and chronic kidney failure), alcohol intake over 25 g/day, and
pharmacological treatment except for hypertension drugs in MetS patients. Moreover,
only patients with no evidence of organ damage (kidney function and interventricular
septum thickness within normal range) or atherosclerosis (carotid intima-media
thickness (CIMT) < 0.9 mm, no atherosclerotic plaques at the carotid doppler
ultrasound) were included in this study
The clinical characteristics of the study population are summarised in Supplementary
Table 1. The study protocol was approved by the Ethical Committee of the Azienda
Ospedaliero-Universitaria Policlinico di Bari, Italy, following the Declaration of Helsinki.
Participants were recruited at the University Hospital of Bari, Italy. All patients gave
written informed consent.

3.3.2 Sample collection and clinical biochemistry measurements

After overnight fasting, serum was collected in healthy and MetS participants for the
assessment of standard clinical biochemistry tests. Serum was separated by
centrifugation and stored at -80 °C. Details of the clinical measurements are described
in Chapter 2 Materials and Methods (section 2.1).

3.3.3 Measurement of lipid levels by mass spectrometry

Lipid extraction and their measurements through liquid chromatography coupled with
mass spectrometry are reported in Chapter 2 Materials and Methods (section 2.2).

58
3.3.4 Measurement of protein levels by mass spectrometry

Protein extraction and their measurements through liquid chromatography coupled

with mass spectrometry are reported in Chapter 2 Materials and Methods (section 2.3).

3.3.5 Circulating lipoprotein remodelling enzyme activity - fluorimetric

assay

3.3.5.1 Lp-PLA2
Lp-PLA2 activity of serum was measured in duplicates using a commercially available
kit (Cayman, Europe), following manufacturer’s instructions. Specifically, samples
were incubated for 30 minutes with Ellman’s reagent at room temperature with the
subsequent addition of 2-thio PAF, used as a substrate for Lp-PLA2 activity. The
reaction causes an increase in colorimetric absorbance measured once every minute
at 405-414 nm. Measurements were obtained using a plate reader Tecan Infinite 200
PRO (Tecan, Mannedorf, Switzerland).

3.3.5.2 LCAT activity

LCAT activity of serum was measured in duplicates using a commercially available kit
(Merck, St Louis, USA) following the manufacturer's instructions. Specifically, samples
were incubated with LCAT substrate for 4 h at 37 °C. The fluorescent substrate emits
fluorescence at 470 nm. When the substrate is hydrolysed by LCAT, a monomer is
released that emits fluorescence at 390 nm. The LCAT activity is expressed as a
change of 470/390-nm emission intensity and thus providing a semi-quantitative
analyses. Measurements were obtained using a plate reader Tecan Infinite 200 PRO
(Tecan, Mannedorf, Switzerland).

3.3.5.3 PLTP activity

PLTP activity of serum was measured in duplicates using a commercially available kit
(Merck, St Louis, USA) following the manufacturer's instructions. Specifically, the
assay uses a proprietary substrate to detect PLTP mediated transfer of the fluorescent
substrate. The transfer activity results in an increase in fluorescence intensity ((λex =
465 nm)/(λem = 535 nm)). Measurements were obtained using a plate reader Tecan
Infinite 200 PRO (Tecan, Mannedorf, Switzerland).

59
3.3.6 Statistics

Data are shown as mean ± standard deviation unless otherwise specified. Normality
was visually assessed from plots of the data (skewness/kurtosis) obtained with the lm
function in R, and logarithmic transformations were applied to non-normally distributed
data. Lipids are grouped into their major lipid classes by summing any lipid species
belonging to a specific class. Comparisons of clinical and omics data between healthy
and MetS were assessed using two-way ANOVA (with “sex” and “disease state” as
covariates), and P-value <0.05 considered significant. Correlation matrices between
omics layers along with clinical variables, were performed with Pearson correlation
coefficient, with P-value <0.05 considered significant, or multiple regression (lm
function, in R) with “sex” as a covariate, when relevant; a P-value <0.05 was
considered significant. Data of the whole serum lipidome, single lipid species, was
analysed by student two-sided T-Test reporting the unadjusted alongside the adjusted
P-value (Benjamini-Hochberg procedure to control the False Discovery Rate (FDR)).
Statistical analysis was performed with R version 4.0.0 and Microsoft Excel 2016.
Graphs were obtained using R version 4.0.0 and Graph Pad (Graph Pad Prism 7.0).

60
3.4 Results

3.4.1 Clinical characteristics, serum lipidomic and apoprotein profile of

healthy and MetS participants

Compared to controls, MetS patients were significantly older, with higher BMI and
abdominal obesity, and insulin resistant (Supplementary Table 1). They also displayed
the classical features of mixed dyslipidaemia: remarkably elevated TG and reduced
HDL-C, with an expected significant sex effect on these parameters, while they did not
show significant differences in total cholesterol, LDL-C, and blood pressure
(Supplementary Table 1).
The serum lipidome analyses included 8 major lipid species measured through ESI
LC-MS (TG, DG, FFA, PC, LysoPC, and SM), colorimetric assay (CE and FC) (Figures
1 a-h), and ESI LC-MS/MS (CE) (Supplementary Table 2). We also profiled the main
apoproteins involved in the serum lipidomic remodelling through ESI LC-MS/MS
(Figure 2).
In keeping with the definition of MetS and some studies pointing to AT-IR as a driver
of MetS (Azzu et al, 2020; Karpe et al, 2011), patients with MetS showed increased
levels of total TG, DG, and FFA (Figures 1a-c) compared to controls. Interestingly,
MetS patients displayed significantly increased levels of total PC along with reduced
LysoPC when compared to controls (Figures 1d-e), while total SM, CE, and FC
(Figures 1f-h) did not show significant changes.

61
Figure 1. Serum levels of major lipidomic classes from healthy and MetS participants. (a) TG, (b) DG, (c)
FFA, (d) PC were increased in MetS (n=14) whereas (e) LysoPC were reduced as compared to controls (n=11).
SM, FC, and CE (f-h) were not significantly changed. All lipid species were analysed by LC-MS except for FC and
CE analysed calorimetrically as reported in the method section. Statistical significance was assessed using two-
way ANOVA controlling for sex and interaction between MetS and sex, with a P-value <0.05 considered significant.
Data are mean ± standard deviation.

With regards to the apoprotein profile, compared to healthy participants, MetS patients
displayed a significant reduction in ApoA-I and ApoD (Figures 2a-b), this being in
agreement with the low levels of HDL-C in the MetS group. ApoA-IV, ApoM, ApoE,
ApoC-I, and ApoB-100, did not show a significant difference between the two groups
(Figures 2c-g), while Apo-CII and ApoC-III were remarkably increased in the MetS
group in comparison to controls (Figures 2h-i). Increased levels of ApoC-III contribute
to hypertriglyceridemia via the inhibition of a) the activity of lipoprotein lipases (LPL)
and b) the uptake of TG by the liver (Santos-Baez & Ginsberg, 2020), thus resulting in
an increased half-life of TG-rich lipoproteins. ApoC-II, however, is a cofactor of LPL

62
and promotes its activity (Wolska et al, 2017). Interestingly, elevated levels of ApoC-
II have been reported in patients with T2DM and obesity (Beliard et al, 2009; Ooi et al,
2016) and their levels were strongly associated with TG levels.

Figure 2. Serum levels of major apoproteins involved lipoprotein remodelling.

ApoA-I (a) and ApoD (b) were decreased in MetS (n=14), ApoA-IV, ApoM, ApoE, ApoC-I and ApoB-100 (c-g),
were not significantly different, whereas ApoC-II (h) and ApoC-III (i) were increased as compared with controls
(n=11). All apolipoproteins were analysed by LC-MS/MS as reported in the method section. Statistical significance
was assessed using two-way ANOVA using the disease state and Sex as covariates; a P-value <0.05 was
considered significant. Data are mean ± standard deviation.

63
To further assess the association between the lipidomic and targeted proteomic
changes, I correlated the significant features of both omics as shown in Figure 3. The
latter showed that apart from the expected positive correlations of TG, DG, and FFA
with ApoC-II and ApoC-III, PC and LysoPC reported the highest correlations with
ApoA-I and to a lesser extent to ApoD, thus suggesting a possible lipidomic
remodelling of the HDL fractions.

Pearson’s correlation coefficient

Figure 3. Heatmap of correlation matrix, Pearson’s correlation coefficient, among significantly changed
lipidomic and apoprotein species in MetS and healthy controls. Data are plotted based on whether they are
positively (red) or negatively (blue) correlated with red bold borders highlighting correlations with a p<0.01 and
black bold borders highlighting correlations with a p<0.05.

Apart from the anticipated lipidomic and apoprotein features of the MetS, these results
suggest an alteration of circulating lipoprotein remodelling enzymes. PC and LysoPC
are biochemically interconvertible species, and their metabolism can be modulated
both intracellularly (e.g. in the liver, intestine, adipose tissue, or macrophages), and
extracellularly (Law et al, 2019). In circulating blood, PC can be remodelled into
LysoPC by two enzymes: a) lecithin–cholesterol acyltransferase (LCAT), a key player
in the formation of large and mature HDL (Ossoli et al, 2016b) that transfers 18:1 or
18:2 fatty acids from PC to FC, thus generating CE and LysoPC (Rousset et al, 2009);
and b) lipoprotein-associated phospholipase A2 (Lp-PLA 2), an enzyme with pro- and
anti-inflammatory capabilities (Marathe et al, 2014) that catalyses the hydrolysis of
fatty acids at the sn-2 position of oxidised phospholipids ultimately generating a fatty
acid and a LysoPC.
I therefore decided to further investigate the changes in serum phospholipid
remodelling enzymes.

64
3.4.2 LCAT, but not Lp-PLA2, activity is reduced in MetS thus
justifying the changes in serum PC and LysoPC

To gain insight into the biology of lipoprotein remodelling, employing commercially

available kits, I determined the activity levels of circulating lipoprotein remodelling
enzymes. Specifically, I studied Lp-PLA 2, phospholipid transfer protein (PLTP), and
LCAT activity in serum. Lp-PLA2 showed no significant changes between the groups,
although there was a significant effect of sex, with the activity being increased in males
with MetS as compared to controls (Supplementary Figure 2a), while in agreement
with previous studies (Qin et al, 2014), PLTP was significantly increased in the MetS
group when compared with controls (Supplementary Figure 2b). Although PLTP
mediates the net transfer of PL from TG-rich lipoproteins into HDL particles, it is also
capable of transferring several lipids such as diacylglycerol, phosphatidic acid,
sphingomyelin, cerebroside and phosphatidylethanolamine, and α-tocopherol (Albers
et al, 2012), thus rendering this enzyme a non-specific transporter. The extent to which
the increased PLTP levels have impacted the PC and LysoPC in our study are of
challenging interpretation, but it is nonetheless an important enzyme to take into
account when characterising lipoprotein remodelling. LCAT activity was significantly
reduced in the MetS group when compared with controls (Figure 4a). I further
confirmed this observation assessing different product/substrate ratios that have been
proposed as indirect proxies of LCAT activity (Angelini et al, 2014; Gerl et al, 2018;
Ossoli et al, 2019); while LysoPC/PC ratio can be affected by both LCAT and Lp-PLA2
activity, the conversion of FC to CE in plasma can only be catalysed by LCAT (Ossoli
et al, 2016a). As shown in Figure 4b-c, both CE/FC and LysoPC/PC ratios were
significantly reduced in MetS patients. This was even more evident (Figure 4d) when
combining the information from cholesterol and phospholipids using a recently
proposed formula, called “non-equilibrium reaction quotient” (Q’; formula:
(CE*LysoPC)/(FC*PC)) (Gerl et al, 2018). Furthermore, in support of the notion that
these product/substrate ratios can be used as indirect proxies of LCAT activity, I found
significant correlations among these variables (Figure 5a-c). Taken together, these
data suggested that the changes in the whole serum lipidomics previously described
most likely reflect the suppressed LCAT activity seen in my cohort.

65
Figure 4. Direct and indirect measurements of LCAT activity. The direct measurement of LCAT activity (a) and
its lipidomic predictors (b-d) were significantly reduced in MetS (n=14) when compared with controls (n=11). All
lipid species were analysed by ESI LC-MS except for FC and CE analysed colorimetrically as reported in the
method section. LCAT activity was measured with a fluorometric assay. Statistical significance was assessed using
two-way ANOVA controlling for sex and interaction between MetS and sex, with a P-value <0.05 considered
significant. Data are mean ± standard deviation

Figure 5. Correlation between direct and indirect measurements of LCAT activity. Lipidomic predictors of
LCAT activity (a-c) positively correlated with LCAT activity, with LysoPC/PC and Q’ having the highest correlations.
All the measurements were performed on MetS (n=14) and controls (n=11). Lipid species were analysed by ESI
LC-MS except for FC and CE analysed calorimetrically as reported in the method section. The correlation between
variables was assessed with Pearson’s correlation coefficient. P-values < 0.05 were considered statistically
significant after adjusting for sex.

3.4.3 LCAT activity and its lipidomic proxies show an inverse

correlation with metabolic risk factors and positively correlate with
HDL-C

The role of LCAT in the pathophysiology of CVD is still debated, with conflicting results
coming from preclinical and clinical studies (Ossoli et al, 2016a). Contradictory findings
have also been reported in patients with metabolic risk factors (Dullaart et al, 2008;

66
Lucero et al, 2015; Magkos et al, 2009; Nakhjavani et al, 2011), further highlighting
the intricate nature of LCAT function. Because of the coherent reduction of LCAT
activity and its product to substrate ratios in MetS, I sought to understand the extent
to which these parameters correlated with characteristic metabolic risk factors linked
to the MetS, such as obesity (BMI), insulin resistance (HOMA2-IR), and HDL-C. LCAT
activity and all of the lipidomic indices investigated showed a remarkable and
significant negative correlation with BMI (figure 6a,d,g,j). The ratios were also
negatively correlated with HOMA2-IR (Figure 6b,e,h,k), but only the direct
measurement of LCAT (figure 5b) reached a significant correlation. Lastly, the LCAT
activity and relative ratios investigated showed a positive correlation with HDL-C
(figure 5c,f,i,l), with the Q` showing the highest association (R = 0.57) and significance
(p=0.0008). Taken together, these data suggest that LCAT and its proxies display a
strong relationship with metabolic risk factors, this being partially attributable to
reduced HDL-C levels in the MetS group compared to controls.

67
Figure 6. Correlation between indirect measurements of LCAT activity with metabolic risk factors. Lipidomic
predictors and direct measurement of LCAT activity (a-l) were negatively correlated with BMI and HOMA2-IR and
positively correlated with HDL. All the measurements were performed on MetS (n=14) and controls (n=11). Lipid
species were analysed by ESI LC-MS except for FC and CE analysed colorimetrically as reported in the method
section. The correlation between variables was assessed with Pearson's correlation coefficient. P-values < 0.05
were considered statistically significant after adjusting for sex.

68
3.4.4 Discussion

The MetS, a cluster of CVD risk factors, is characterised by mixed dyslipidaemia

(elevated VLDL-TG and reduced HDL-C), and its treatment is a cornerstone of CVD
primary and secondary prevention. Beyond the traditional lipid markers (i.e HDL-C and
TG), several studies have started to characterise the lipidomic signature of the MetS
(Monnerie et al, 2020), but underlying mechanisms are lacking. In this chapter,
besides the expected MetS-associated lipidomic changes (elevated TG, DG, and
FFA), I observed a phospholipid remodelling which was due, at least in part, to a
reduced LCAT activity. The latter, along with lipidomic ratios used as proxies of LCAT
activity, showed a strong correlation with some defining features of the MetS, thus
suggesting a close link between LCAT activity and metabolic risk factors. Changes in
circulating PC and LysoPC in MetS patients have been reported by several authors
(Monnerie et al, 2020), however, results are inconsistent and this has partially been
attributable to differences in study design. Elevated PC levels in MetS have been
reported by some authors (Capel et al, 2020; Kulkarni et al, 2013b; Ramakrishanan et
al, 2018) but not from others (Surowiec et al, 2019; Yin et al, 2020). Because PC, in
healthy people, are predominantly associated with HDL particles, and reduced HDL-
C is one of the inclusion criteria of the MetS, increased PC in MetS is somehow
unexpected. However, different studies have found elevated levels of PC in MetS and
this suggests a probable enrichment of these lipids in fractions other than HDL.
Indeed, PC constitute about 8% of the VLDL lipidome (Christinat & Masoodi, 2017),
therefore, it is tempting to speculate that the increased VLDL-TG fraction observed in
the MetS, can be paralleled by an increase in PC, thus leading to an overall PC level
in the whole plasma. The use of lipidomic ratios, as opposed to single lipid
investigation, might be a more informative approach in describing biological
processes. In this chapter, I used the Q’ ratio as a proxy of LCAT activity. Q’ is defined
as the products to substrates of the LCAT reaction: (CE*LysoPC)/(FC*PC), and was
recently proposed by Gerl and colleagues (Gerl et al, 2018). Q’ was used as a proxy
of the LCAT activity in a cohort of patients with CVD, where they showed a reduction
of this index in patients when compared to controls. However, the authors did not
report a direct validation of their proxy against the LCAT activity or the enzyme mass
(Gerl et al, 2018). My findings are in line with their work suggesting that Q' is also
altered in patients at high CVD risk but without organ damage. Furthermore, my study

69
confirmed, for the first time, the positive correlation between Q' with a direct
measurement of the LCAT activity. Although it was beyond the scope of this work to
characterise the mechanisms by which LCAT was reduced in MetS, the targeted
proteomics data further supported the notion of a reduced LCAT activity in MetS.
Indeed, as compared to healthy people, MetS participants were characterised by lower
levels of ApoA-I, which is the main activator of LCAT activity although the mechanism
has not been fully characterised. Furthermore, MetS participants also displayed
elevated ApoC-III levels, which have been shown to dose-dependently inhibit the
LCAT activity in reconstituted HDL (Cho, 2009). Taken together, these data coherently
point towards a reduced LCAT activity in the MetS. It is, however, important to
underline that despite the crucial role of LCAT in the maturation of HDL, and its
intensive investigation over the last 50 years, the role of LCAT in cardiovascular health
is still debated with studies showing conflicting results in both pre-clinical and clinical
settings (Norum et al, 2020). Despite the controversies in the field, my data support
the notion of LCAT being suppressed in a cohort of patients at high of CVD risk.

70
3.4.5 Limitations

The findings in this chapter are subject to at least two limitations. First, the present
study involved a secondary analysis of a previously published study, so I utilised data
from the highest number of available participants and no formal sample size
calculation was performed. This implies that results must be interpreted with caution,
although two layers of evidence, enzyme activity, and lipidomic ratios, support my
findings. Second, The MetS group was significantly older than the healthy controls,
and after adjustment for ageing, the significant differences in the lipidomic ratios
between the two groups were no longer present. Linear regression indicated the
increased age in the MetS group explained 22% of the variance in the Q' (p=0.02)
while for the LCAT activity it only explained 11% of the variance (p=0.1). This further
weakens my results as I cannot rule out the role of ageing as a driver for the observed
lipidomic changes. However, because the direct measurement of LCAT is not
influenced by age, along with a previous study that reported no association between
Q’ and age (Gerl et al, 2018), I still find relevant the suppression of LCAT in MetS. To
address this issue it is important to further investigate these indices in age-matched
studies.

71
3.4.6 Conclusion

The purpose of the current study was to characterise the circulating lipidomic changes
associated with the MetS and to provide a mechanistic explanation. The lipidomic data
showed that compared to healthy controls, MetS patients were characterised by
elevated PC and reduced LysoPC levels, along with the expected increased TG and
DG. Correlation between targeted proteomics and lipidomics data suggested a
remodelling of the HDL fractions in MetS. Lipidomic ratios, along with fluorimetric
enzymatic assays, pointed to a reduction of LCAT activity while ruling out the
involvement of Lp-PLPA2 in MetS when compared to healthy people. Direct
measurements and indirect proxies of LCAT activity were also positively correlated
with HDL-C and inversely correlated with BMI and HOMA2-IR. Taken together, these
data suggest that LCAT activity might be a key modulator of the circulating lipidome in
patients with MetS and that LCAT is strongly related with the metabolic derangement
observed in this condition. These results warrant confirmation in larger study
populations.

72
Supplementary materials
Supplementary Table 1. Clinical characteristics of the study cohort.

P-value
Healthy MetS
Sex MetS
N (M/F) 6/5 9/5 0.6
Age (years) 29 ± 2 42 ± 10 0.4 0.0004
Body mass index (Kg/m2) 22.8 ± 1.9 32.7 ± 3.4 0.1 0.00000003
Waist circumference (cm) 86 ± 8 111 ± 7 0.08 0.00000004
Systolic blood pressure (mm/Hg) 116 ± 8 122 ± 12 0.02 0.2
Diastolic blood pressure (mmHg) 78 ± 6 80 ± 7 0.3 0.6
Creatinine (mg/dl) 0.7 ± 0.17 0.9 ± 0.18 0.00003 0.008
Microalbuminuria (mg/l) 11.7 ± 1.6 15 ± 3.2 0.9 0.7
Total cholesterol (mmol/L) 5.5 ± 1 5 ± 1.1 0.2 0.4
HDL (mmol/L) 1.6 ± 0.4 0.9 ± 0.2 0.001 0.000002
LDL (mmol/L) 3.5 ± 0.9 3.4 ± 1.1 0.4 0.8
Triglycerides (mmol/L) 0.8 ± 0.3 1.5 ± 0.3 0.003 0.00006
Glucose (mmol/L) 5 ± 0.4 5.7 ± 0.7 0.7 0.006
Insulin (pmol/L) 53 ± 17 101 ± 61 0.4 0.002
HOMA2-IR 1 ± 0.3 1.9 ± 1.1 0.5 0.01

Data are mean ± SD. Differences between the two groups were analysed with 2-way ANOVA, and significance
accepted at p < 0.05. M = Male, F = Female, BMI = Body Mass Index, TC = total cholesterol, HDL-C = high-density
lipoprotein cholesterol, LDL-C = low-density lipoprotein cholesterol, TG = triglycerides, HOMA2-IR = Homeostasis
Model Assessment 2 of Insulin Resistance.

73
Supplementary Table 2. Average value for lipid species in the whole serum of Healthy and MetS participants.

Whole Average normalised Average normalised P-value FDR

serum peak intensity - Healthy peak intensity - MetS
lipidome
CE 16 0 0.411 0.402 0.884534653 0.930604166
CE 18 0 0.325 0.373 0.380575892 0.463110422
CE 18 1 39.352 39.596 0.832339241 0.884908035
CE 18 2 51.843 35.758 0.0196652 0.043010482
CE 20 4 24.528 19.485 0.115112903 0.189336767
DG 32 0 0.159321086 0.092046459 0.243699636 0.33261707
DG 34 1 0.108778231 0.102718975 0.012856743 0.031837802
DG 37 0 0.084824089 0.069177777 0.11622653 0.189336767
DG 38 8 0.349389706 0.940988321 0.020014779 0.043010482
DG 41 5 0.082156665 0.177741432 2.46519E-06 4.81E-05
LysoPC 15 0 0.010360738 0.007563991 0.319470554 0.403331574
LysoPC 16 0 0.692126414 0.693633619 0.909608664 0.937363362
LysoPC 16 1 0.014687905 0.015281642 0.918801711 0.937363362
LysoPC 17 0 0.012790905 0.011126363 0.580806068 0.690134268
LysoPC 18 0 0.325172269 0.252231062 0.170994451 0.257767755
LysoPC 18 1 0.269313457 0.156068212 0.002245602 0.00986112
LysoPC 18 2 0.268527176 0.154704396 0.003621153 0.013592192
LysoPC 18 3 0.140838234 0.149435702 0.720441127 0.823681686
LysoPC 20 3 0.063191507 0.046477716 0.104020014 0.178068159
LysoPC 20 4 0.043007377 0.026013381 0.000801808 0.005061411
LysoPC 20 5 0.052824818 0.024530703 0.000186948 0.001452442
PC 30 0 0.080767222 0.085586673 0.898560647 0.935614694
PC 32 0 0.281899667 0.313913895 0.242038273 0.33261707
PC 32 1 0.228358318 0.323204525 0.035343376 0.071393619
PC 32 2 0.062821669 0.083520648 0.068165627 0.122941577
PC 34 1 3.535585472 4.929156768 0.331956372 0.413920908
PC 34 2 6.568506053 9.593782425 0.067592892 0.122941577
PC 34 4 0.025174981 0.026685511 0.737229718 0.823681686
PC 35 2 0.138002281 0.142590461 0.590082186 0.693003497
PC 36 4 3.98809162 4.884301395 0.261416843 0.352041349
PC 36 5 0.308982022 0.388173434 0.314094329 0.401563635
PC 36 6 0.039237277 0.082340383 0.06977506 0.12363651
PC 38 7 0.57729321 0.774130529 0.241262138 0.33261707
PC 38 8 0.059159191 0.07102255 0.740415753 0.823681686
PC 40 0 0.08085624 0.155468724 0.124382921 0.196291797
PC 40 5 0.093837589 0.160207878 0.094168001 0.163982209
PC 40 9 0.271965242 0.409207094 0.013470016 0.032392181
PC 42 8 0.026458309 0.032343396 0.239925001 0.33261707
PC 42 9a 0.064336461 0.108317521 0.006892067 0.019888537
PC 42 9b 0.064336461 0.108317521 0.006892067 0.019888537
SM 26 1 0.038403762 0.07439313 0.138833026 0.215725163
SM 32 1 0.093972905 0.092958474 0.816508278 0.884908035
SM 33 1 0.31081373 0.040715321 8.61905E-05 0.000818946
SM 34 0 0.044982104 0.03273963 0.00446445 0.015030316

74
SM 34 2 0.128144362 0.114845383 0.304990085 0.397366428
SM 34 4 0.060446233 0.026742823 0.217432725 0.318271091
SM 35 3 0.02886031 0.015415948 0.000113006 0.000951138
SM 35 4 0.054145167 0.018189352 0.065268148 0.122075611
SM 36 2 0.080053791 0.080049914 0.742129044 0.823681686
SM 36 4 0.359171212 0.36641058 0.947735205 0.957212557
SM 39 1 0.046499889 0.042180325 0.343569225 0.423176728
SM 40 1 0.45293963 0.630525155 0.057244268 0.111185981
SM 41 1 0.162961689 0.144313469 0.302269104 0.397366428
SM 41 2 0.076223142 0.080969042 0.976977997 0.976977997
SM 42 2 0.702713625 0.615523888 0.185853935 0.276047757
SM 42 4 0.157507504 0.143491559 0.111666428 0.18797182
SM 43 5 0.034128588 0.025486603 0.032657117 0.06731365
SM 44 4 0.114987985 0.099041189 0.119949413 0.192299852
SM 44 5 0.367182916 0.235831394 0.019932648 0.043010482
TG 39 1 0.278069549 1.300649016 0.005025656 0.016373912
TG 46 1 0.119341106 0.13000335 0.234485553 0.33261707
TG 48 1 0.394532932 0.610449585 0.017553444 0.040293133
TG 48 2 0.286131151 0.369823345 0.047246376 0.093566353
TG 50 1 0.836468903 1.815968634 6.45962E-05 0.000724912
TG 50 2 1.248247296 1.905620078 0.003902709 0.013592192
TG 50 3 0.495514687 0.794676077 0.005346223 0.016874017
TG 51 2 0.129254553 0.207446742 0.001014893 0.006029658
TG 51 3 0.076587605 0.123505963 0.003224022 0.01356776
TG 52 1 0.61358433 1.421168547 3.04712E-08 3.08E-06
TG 52 2 2.600571787 6.08536918 1.82628E-06 4.61E-05
TG 52 3 2.075300893 5.046089209 8.26253E-06 0.000119217
TG 52 4 0.950946332 1.782204882 0.003397666 0.013592192
TG 53 2 a 0.075540129 0.155602421 1.46336E-07 7.39E-06
TG 53 2 b 0.075760345 0.137174887 0.001333268 0.007481114
TG 53 3 0.081434785 0.508221769 8.91922E-05 0.000818946
TG 54 1 0.114106124 0.244500483 2.85585E-06 4.81E-05
TG 54 2 0.346611382 0.69036496 1.31754E-06 4.44E-05
TG 54 3 1.105323274 1.805550841 0.001989238 0.009132412
TG 54 4 0.898778359 1.602380526 0.00193844 0.009132412
TG 54 5 0.398020176 0.678646158 0.012924256 0.031837802
TG 54 6 0.143875205 0.217944487 0.011898269 0.030813465
TG 55 2 0.17100972 0.412848174 1.19937E-05 0.000151421
TG 56 6 0.219177678 0.286022492 0.014695336 0.034516952
TG 56 7 0.135282204 0.212061272 0.010284861 0.027336077
TG 56 9 0.261915077 1.481717308 0.00385904 0.013592192
TG 57 3 0.054153722 0.106533008 0.000447025 0.003009969
TG 57 4 0.048980741 0.091410204 0.001743356 0.009110976
TG 57 9 0.034622606 0.054390902 0.010198682 0.027336077
FFA 14 0 0.302 0.389 0.000351032 0.002532448
FFA 15 0 0.035 0.040 0.064928655 0.122075611
FFA 16 0 7.142106257 8.600472092 0.00797862 0.022384463
FFA 17 0 0.074683697 0.095778543 0.003683136 0.013592192

75
 FFA 17 1 0.035861929 0.035933378 0.795237938 0.873032954
FFA 18 0 6.853645383 8.433201254 0.006469691 0.019801175
FFA 18 1 1.06005879 1.331500654 0.159480253 0.244053115
FFA 18 2 0.270751664 0.30841671 0.306877044 0.397366428
FFA 18 3 0.07722938 0.059335179 0.529180317 0.636276333
FFA 20 4 0.092097111 0.086052748 0.827656182 0.884908035
FFA 21 0 0.012456402 0.018018219 0.001804154 0.009110976
FFA 22 0 0.012527612 0.018430634 0.027877688 0.058659302
FFA 22 6 0.026136606 0.025782511 0.691882355 0.803219745

Statistical significance was calculated using two-tailed Student-T test on log2-transformed lipids; adjusted P-value
< 0.05 was considered significant; the adjusted P-value was calculated using the false discovery rate (FDR) as
described in the methods.

Supplementary Table 3. Unadjusted and adjusted for multiple testing, false discovery rate (FDR), P-values
related to Figure 1.

Supplementary Figure 2. Lipoprotein remodelling enzymes. While LP-PLA2 (a) did not show significant
changes between MetS and the control group, PLTP activity (b) was increased in the MetS group. LP-PLA2 and
PLTP assays (a-b) were performed on serum from healthy (n=11) and MetS (n=14) participants. Enzyme activities
were measured with a fluorometric assay. Statistical significance was assessed using two-way ANOVA controlling
for sex and interaction between MetS and sex, with a P-value <0.05 considered significant. Data are mean ±
standard deviation.

76
Chapter 4. NAFLD is characterised by a reduced reverse
polyunsaturated fatty acid transport from peripheral tissues
to the liver

4.1 Abstract

Non-alcoholic fatty liver disease (NAFLD) is a cluster of liver diseases, ranging from
simple steatosis to more aggressive forms, which can ultimately lead to liver failure,
hepatocellular carcinoma, and death. While hepatic lipidomic studies have coherently
reported a “signature” of lipid changes characterising the NAFLD spectrum, studies
using whole serum have reported conflicting results. The latter is in part due to the fact
that circulating lipidome is influenced by the nutritional status/habits of the subjects but
also due to the fact that the whole serum lipidome is a mixture of multiple
pathophysiological events that can be better studied looking into the composition of
specific lipoprotein fractions. Here, I aimed to define the whole serum lipidomic profile,
alongside its HDL and VLDL fractions, to study the inter-organ cross-talk between liver
and peripheral tissues (and vice versa) in NAFLD. In the whole serum the most
important changes occurring in NAFLD were related to a depletion in the absolute
values of phospholipids (PL) and free fatty acids (FFA), particularly the
polyunsaturated fatty acids (PUFA) components of these lipid classes. A deeper look
into the composition of the HDL fraction, confirmed that they were the main
contributors of the observed serum PUFA/PL depletion. On the contrary, the lipidome
of the VLDL fraction demonstrated a generalised increase in PL, which was driven by
their saturated and monounsaturated fatty acid fraction, but unchanged PUFA, likely
reflecting the well described enhanced hepatic de novo lipogenesis (DNL) occurring
in NAFLD. Taken together, these data show that NAFLD is characterised by a reduced
absolute content of PUFA and PL within HDL, and allows us to speculate that an
impaired PUFA transport from peripheral tissues to the liver (via FFA and HDL) might
probably be a contributing factor in the pathophysiology of NAFLD.

77
Simplified representation of the hypothesised mechanistic pathway by which reduced PUFA
HDL might contribute to the established depletion of hepatic PUFA observed in the NAFLD
spectrum.

78
4.2 Introduction

NAFLD is a continuum of diseases ranging from simple steatosis (intrahepatic fat

deposition), to steatohepatitis (steatosis in the presence of inflammation) (NASH),
fibrosis, and cirrhosis, which can ultimately evolve to hepatocellular carcinoma (HCC)
(Vacca et al, 2015). NAFLD has reached pandemic proportions with a global
prevalence of 24%, thus being a public health priority (Younossi et al, 2016). Of note,
the leading cause of death in these patients is CVD, with a mechanism not entirely
understood (Targher et al, 2020). NAFLD is often associated with mixed dyslipidaemia
(low HDL-C, and increased VLDL-TG), which partly explain the elevated CVD risk
(Targher et al, 2020). A deeper understanding of the lipid derangements observed in
this condition is being revealed by lipidomic studies (Musso et al, 2018). Specifically,
hepatic lipidomic studies have coherently reported a lipid remodelling alongside the
NAFLD spectrum, which is characterised by elevated levels of saturated fatty acids
(SFA) and reduced levels of polyunsaturated fatty acids (PUFA). For example, Araya
et al. first reported a depletion in PUFA within the liver (total lipids alongside the TG
fraction) in NAFLD compared to non-NAFLD subjects (Araya et al, 2004). Chiappini et
al. also reported reduced total hepatic PUFA in NASH patients when compared to
controls. Additionally, they reported a decrease in phospholipids (PL) [(i.e
phosphatidylcholines (PC) and phosphatidylethanolamines (PE)], sphingomyelins
(SM) but not ceramides (Cer) in NASH compared to controls (Chiappini et al, 2017).
Moreover, Allard et al. found a depletion of total hepatic PUFA in NASH patients
compared to controls, despite a similar dietary intake among the groups (Allard et al,
2008). The latter study suggested that the PUFA depletion observed in NASH patients
was attributable to a dysfunctional FA metabolism rather than a nutritional deficiency
per se. AT, the major provider of FA to the liver (Donnelly et al, 2005), might be partly
responsible for these changes. However, AT FA pool is mostly derived from the FA
released by the lipolysis of VLDL and chylomicron TG. These events render thus
difficult to trace the flux of lipids among organs and the extent to which AT influences
the liver and vice versa. Moreover, in humans, DNL in AT is quantitatively less
responsive than liver DNL after glucose infusion (Diraison et al, 2003). Overall, in
human AT, DNL contributes for a minimal part of the total AT TG pool (Morigny et al,
2021). It should be noted that the in vivo study of AT in humans has been hampered

79
by the complexity of directly labelling pathway precursors in AT coupled with the slow
AT turnover (years) (Arner et al, 2011).

A mechanism by which hepatic PUFA are depleted in NAFLD has been partly
attributed to a decreased activity of the enzyme fatty acid desaturase 1 (Araya et al,
2004; Chiappini et al, 2017). However, others have found no changes (Allard et al,
2008) or increased activity of FADS1 in NAFLD (Lopez-Vicario et al, 2014). This
pathway is thus still debated.

Because of the risks and cost associated with liver biopsy procedures, the search for
non-invasive alternatives such as blood samples (serum/plasma) to study lipid
metabolism in NAFLD has been increasingly investigated. Compared to healthy
controls, NAFLD patients display increased level of SFA and MUFA and reduced
PUFA in the TG fraction of the whole plasma (Mayo et al, 2018; Sanders et al, 2018;
Yang et al, 2017; Zhou et al, 2016), which has been explained by the enhanced DNL
characterising this condition. However, apart from these findings, whole plasma
lipidomic studies have reported conflicting results regarding the other lipid classes
(Capel et al, 2020; Tiwari-Heckler et al, 2018; Zhou et al, 2016). Because these lipids
are found in HDL and LDL, they are consequently less directly related to the hepatic
metabolism and thus more challenging to interpret (if not studied as isolated fractions).

Several factors might contribute to the discrepancies observed in the circulating

lipidome of NAFLD patients. Some of them include the differences in inclusion criteria
such as presence of dyslipidaemia, elevated blood pressure, sex, different ethnicity,
and lack of studies investigating isolated lipoproteins (lipoprotein lipidomics). Among
these factors, I believe that the information obtained by the study of lipoprotein
lipidomics could be more informative as compared to whole serum lipidomics, and thus
helping us to better understand the lipid remodelling which occurs in NAFLD.

This chapter aims to investigate the quantitative and qualitative lipidomic changes
occurring in patients across the NAFLD spectrum, and whether these changes can be
explained by changes in lipoprotein abundance and composition. Specifically, the aim
of this chapter is to understand whether lipidomics of specific lipoprotein fractions can
provide a better biological matrix to investigate the lipid remodelling occurring in the
liver of NAFLD subjects. For this purpose, I employed state-of-the-art mass
spectrometry lipidomics to profile whole serum and the lipoprotein fractions (HDL and

80
VLDL) obtained through fast protein liquid chromatography in healthy and biopsy-
confirmed NAFLD participants.

81
4.3 Methods

4.3.1 Ethics and the BioNASH study cohort

89 patients with a biopsy-proven NAFLD (patients with alternate diagnoses and

aetiologies and kidney dysfunction were excluded), and 20 healthy volunteers were
involved in this study. Patients were recruited at the NASH Service at the Cambridge
University Hospital, whereas healthy volunteers were recruited amongst members of
the NIHR Cambridge BioResource (http://www.cambridgebioresource.org.uk), at the
NHS Blood and Transplant Unit, Cambridge, UK. The recruitment of NAFLD patients
was approved by the local Ethics Committee, whereas healthy volunteers’ recruitment
was approved by the NHS Research Ethics Committees (REC 12/EE/0040). The
biopsy was scored by an experienced liver pathologist for steatosis (0-3), ballooning
(0-2), inflammation (0-2), and fibrosis (0-4) and were classified according to the SAF
system (Bedossa et al, 2012), whereas the healthy controls were selected according
to the non-invasive score proposed by Kotronen et al. (Kotronen et al, 2009a) since
liver biopsy can only be performed when clinically required. The study protocols
followed the principles of the Declaration of Helsinki and all participants gave written
informed consent.

4.3.2 Sample collection and clinical biochemistry measurements

Serum was collected in healthy and NAFLD participants for the assessment of
standard clinical biochemistry tests. Serum was separated by centrifugation and
stored at -80 °C. Details of the clinical measurements are described in Chapter 2
Materials and Methods (section 2.1).

4.3.3 Measurement of lipid levels by mass spectrometry

Lipid extraction and their measurements through liquid chromatography coupled with
mass spectrometry are reported in Chapter 2 Materials and Methods (section 2.2).

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4.3.4 Measurement of ApoA1

ApoA1 of HDL fractions was measured by liquid chromatography coupled with mass
spectrometry and details are reported in Chapter 2 Materials and Methods (section
2.3). In addition to the use of a specific internal standard per lipid class, ApoA1 was
used to normalise the lipidomic data of HDL.

4.3.5 Measurement of ApoB

ApoB of VLDL fractions was measured in duplicates using a commercially available

ELISA kit (Abcam, Cambridge, UK) following the manufacturer's instructions.
Measurements were obtained using a plate reader Tecan Infinite 200 PRO (Tecan,
Mannedorf, Switzerland). In addition to the use of a specific internal standard per lipid
class, ApoB was used to normalise the lipidomic date of VLDL.

4.3.6 Statistics

Data are shown as mean ± standard deviation unless otherwise specified. Normality
was visually assessed from plots of the data (skewness/kurtosis) obtained with the lm
function in R, and logarithmic transformations were applied to non-normally distributed
data. Comparisons of clinical data between healthy and NAFLD patients were
assessed using ANOVA. Regarding categorical variables, a chi-square test was
adopted. Whole serum lipidomic data were analysed using three-way ANOVA
controlling for sex, presence of diabetes mellitus (T2DM) and interaction between sex,
T2DM and disease state, with a P-value <0.05 considered significant. However, when
lipids were investigated as independent hits, multiple testing correction (Benjamini-
Hochberg procedure to control the False Discovery Rate (FDR)) was applied as
specified in the legend to tables. Lipoprotein lipidomics data, where participants were
only males, were analysed using two-way ANOVA controlling for presence of T2DM
and the interaction between disease state and T2DM, with a P-value <0.05 considered
significant. As with whole serum, when lipids were considered as an independent unit,
FDR was reported along with the raw P-value. To assess the power of the of this study,
whole serum and lipoprotein lipidomics, I performed a PostHoc power analyses by
assessing first the effect size with the following freely available tool:
https://webpower.psychstat.org/models/means03/effectsize.php, while the power was

83
assessed with G*power software
(https://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-
arbeitspsychologie/gpower). Variables with an effect size (f) below 0.3 (whole serum)
and 0.5 (lipoproteins) fell below an acceptable power level of 0.7, therefore being
potentially exposed to type 2 error. All the significant variables had a power above the
optimal power of 0.8. Univariate correlations were carried out using the Pearson
correlation coefficient, with P-value <0.01 considered significant. Statistical analysis
was performed with R version 4.0.0 and Microsoft Excel 2016. Graphs were obtained
using R version 4.0.0 and Graph Pad (Graph Pad Prism 7.0).

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4.4 Results

4.4.1 Clinical characteristics and whole serum lipidomic profile of healthy

and NAFLD patients

This study involved 109 participants, including 20 healthy volunteers (age and sex-
matched), 36 patients with NAFL, 31 patients with mild NASH, and 22 patients with
NASH with moderate/advanced fibrosis (Supplementary Table 1). As expected, the
patients in the NAFLD spectrum displayed a significantly higher BMI, impaired glucose
metabolism (HOMA2-IR, insulin, and glucose), and mixed dyslipidaemia (elevated TG
and decreased HDL-C), along with elevated liver enzymes (ALT, AST), while LDL and
total cholesterol were not significantly different across the groups (Supplementary
Table 1).
Lipids were analysed as total lipid class (sum of each lipid measured), and according
to their acyl chain saturation levels (saturated, monounsaturated and
polyunsaturated), as imbalances in the FA saturation levels have been involved in the
pathophysiology of NAFLD.
The whole serum lipidomic analysis revealed a quantitative and qualitative depletion
of several phospholipid classes [phosphatidylcholines (PC),
phosphatidylethanolamines (PE), phosphatidylglycerols (PG)] in the NAFLD group as
compared to the controls (Figure 1a-e). The total reduction of PC and PE were mainly
driven by the depletion of their PUFA component, while PG were markedly reduced
irrespectively of their acyl chain saturation levels (Figure 1b,d,f). Furthermore, I
observed a significant sex and T2DM effect on most of the PL measured (Figure 1),
this being in line with the differences in the lipoprotein metabolism among men and
women alongside the presence of diabetes. Within the lysophosphatidylcholines
(LPC), the only significant change observed was in the saturated fraction, which
increased in NAFLD compared to controls (Figure 1 g,h), whereas within the
lysophosphatidylethanolamines (LPE), only the MUFA content was significantly
changed, which was reduced in NAFLD compared to controls (Figure 1i,j). The
sphingolipids sphingomyelins (SM) and ceramides (Cer) did not show significant
differences between NAFLD and controls (Figure 1 k-n). Of note both lipids were
characterised by a significant sex effect. Several observational studies have reported
sex differences in circulating ceramides, with females showing higher ceramide levels

85
than males (Bui et al, 2012; Hammad et al, 2010; Mielke et al, 2015). Difference in sex
but lack of interaction is in line with a previous human study assessing the mid- to late-
life trajectories of plasma ceramide levels (Mielke et al, 2015). While the mechanism
of these differences have not been fully elucidated, differences in sex hormones have
been suggested as putative mediators of sphingolipid metabolism in rodent studies
(Muralidharan et al, 2021; Norheim et al, 2018).
While between NAFLD and controls total free fatty acids (FFA) and LC-MS measured
total triglycerides (TG) were not significantly different, when compared to controls,
NAFLD subjects showed a marked increase in saturated FFA and TG, coupled with a
depletion in polyunsaturated FFA and TG (Figure 1 o,p, q,r). Lastly, to better
understand the extent to which lipidomic data related to metabolic parameters, I
correlated the significantly changed lipids with key clinical data (Figure 2). Pearson
correlation analysis showed that PG (total, SFA, MUFA, PUFA) had the strongest
inverse correlation with obesity and insulin resistance, whereas SFA from LPC and
FFA were positively correlated with HOMA-IR (Figure 2). Moreover, most of the lipids
significantly reduced in the NAFLD groups were also positively correlated with HLD-C
(Figure 2).
Taken together, these data show that in NAFLD, the whole serum lipidome is depleted
of PL, predominantly PUFA containing PL, in keeping with previous hepatic lipidomic
studies, and suggest that there might be a close relationship between IR, HDL-C, and
the circulating lipidome. However, this type of whole serum lipidomic studies leave
unresolved which is the main lipoprotein acting as a driver of the observed changes:
these results can be justified either by a reduced liver export of PUFA-PL through
VLDL, or by a reduced PUFA-PL transport from peripheral tissues to the liver via HDL.
I therefore investigated further this paradigm by separating the lipoprotein fractions
HDL and VLDL and then performing lipidomic analyses.

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87
88
Figure 1. Whole serum lipidomic signature of NAFLD. (a,b) PC, (c,d) PE, (e,f) PG, were reduced across the
NAFLD spectrum. (g,h) LPC were qualitatively increased, and (i,j) LPE qualitatively reduced in NAFLD compared
to controls. (k-n) SM and Cer were unchanged and (o,p,q,r) FFA and TG showed increased SFA, MUFA, and
reduced PUFA as compared to controls. All lipid species were analysed by LC-MS. Statistical significance was
assessed using three-way ANOVA controlling for sex, presence of T2DM and interaction between sex, T2DM and
disease state, with a P-value <0.05 considered significant. Data are mean ± standard deviation. In Supplementary
Table 3 are reported the specific lipid species analysed highlighting whether they were considered saturated,
monounsaturated and polyunsaturated.

Pearson’s correlation coefficient

Figure 2. Heatmap of a correlation matrix, Pearson’s correlation coefficient, among significantly changed
lipid species and clinical data in NAFLD and healthy controls. Data are plotted based on whether they are
positively (red) or negatively (blue) correlated, with a red circle highlighting the significant correlation with a p-value
<0.01.

4.4.2 HDL lipidomics of NAFLD patients suggest a reduced reverse PUFA

transport from peripheral tissues to the liver

The strong correlation observed between whole serum lipidomics and HDL-C
suggested a potential key role of HDL particles in carrying less PL and PUFA from

89
peripheral tissues to the liver. To confirm this observation, I isolated HDL and studied
their lipidome from a subpopulation of our cohort, which included 40 age-matched
males distributed as follows: 9 healthy, 11 NAFL, 11 mild NASH, and 9 NASH with
moderate/advanced fibrosis (Supplementary Table 2). The lipoprotein lipidomics was
performed on a smaller subgroup because of the reduced time available in the lab due
to the lockdown related with the COVID-19 pandemic. I particularly focused on male
participants to reduce the variability attributable to sex differences. Because NAFLD
patients were characterised by low HDL-C levels as compared to controls, it was
reasonable to assume that HDL lipoproteins were reduced in the NAFLD group and
so would be the other lipids since cholesterol account for ~40% of total lipids within
HDL. To account for this difference, the lipidomic data of the HDL fractions, after the
internal standard lipid normalisation, were further normalised for the ApoA-I
abundance (Supplementary Figure 1), since the latter is the most abundant protein
found within HDL particles, accounting for ~70% of its protein content (Dominiczak &
Caslake, 2011). In light of these considerations, the further normalisation for ApoA-I
was considered a reasonable way to overcome the issue related to different levels of
HDL-C. Of note, it has been shown that after a meal, HDL temporarily increases its
ApoA-I levels (Averill et al, 2020; Burnap et al, 2021) without changing its total mass.
In this scenario, a normalisation for total protein content, providing albumin is depleted
from the HDL fraction, would provide a much better normalisation matrix.
Compared to controls, the HDL lipidomic profile of NAFLD patients was characterised
by decreased PC levels, mainly driven by their PUFA content (Figure 3 a,b). These
results suggested, after lipid normalisation (absolute values) to Apo1, that the HDL
fractions showed reduced PUFA-PC. Reduced PC and, especially their PUFA content,
have been reported in peripheral tissues of IR obese patients: peripheral tissues
including AT provide lipids such as PL to HDL (Borkman et al, 1993; Ferrara et al,
2021; Pietilainen et al, 2011). LPC and PE did not show significant changes between
the groups, while total PG were dramatically reduced in NAFLD patients (Figure 3 c-
f). PG represent a minor component of the HDL lipidome, however, it can influence
the HDL cholesterol efflux capacity (Camont et al, 2013). Furthermore, when
compared with controls, the NAFLD group showed a reduced total SM (mostly MUFA
and PUFA), increased levels of SFA Cer, and no changes in the TG fraction (Figure 3
g-l). Within the HDL-TG, it is worth noticing how the NASH F2-3 behave differently
from the previous stages (being closer to control). This has been previously shown by
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others (in whole serum) and attributable to lower functionality of the liver in advanced
stages of NASH (Guerra et al, 2021). Moreover, this is also supported at gene
expression levels (i.e. gene related with lipid synthesis) performed on liver biopsies of
a cohort of NASH patients partially overlapping with this one (Azzu et al, 2021).
Reduced SM levels have also been associated with a decreased HDL efflux capacity
(Sano et al, 2007), while increased HDL Cer have been less investigated. The liver is
the main site of synthesis of Cer, and they have been found increased in in liver of
patients with NAFLD (Samuel & Shulman, 2018), my data suggest that HDL might
also be contributing to this increase. Surprisingly, T2DM did not show a significant
effect in any of the lipids studied within the HDL fraction, this suggesting that the
changes observed were mainly driven by the NAFLD state. To further assess the
extent to which the HDL lipidome related with metabolic parameters, I correlated the
significantly changed lipids between NAFLD and healthy controls with key clinical
metabolic data (Figure 4). Pearson correlation analysis showed that the lipid species
significantly reduced in the NAFLD group were negatively correlated with insulin
resistance and, within these species, (Total, MUFA, and PUFA) SM having the
strongest correlation; on the other hand, SFA Cer had an opposite trend (Figure 4).
Furthermore, the latter was also positively correlated with liver enzymes, with AST
showing the strongest correlation (Figure 4). In summary, these data show that
independently of the low HDL-C levels, in NAFLD, HDL is characterised by reduced
PUFA PL and increased SFA Cer levels and that IR is intimately related to these
changes. The fact that the liver largely reuses HDL-derived PL either incorporating
them into membranes or converting them into different lipid classes has been
previously suggested with tracer experiments (van der Veen et al, 2012): my
observational results show in real-life data from HDL from patients with NAFLD, that
the HDL lipidome carries the hepatic lipidomic signatures previously described in
NAFLD patients, thus pointing at HDL composition as a partial contributor in the
pathogenesis of NAFLD. Together with the more studied FFA, HDL might therefore be
a largely understudied but equally important player in the “reverse (phospho)lipid
transport” from peripheral organs able to influence liver function and pathophysiology
in hepatic metabolic diseases.

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Figure 3. HDL lipidomic signature of NAFLD. (a,b) PC were reduced in NAFLD as compared to controls while
(c,d,e) LPC and PE did not show any changes. (f,g,h) PG and SM were reduced across the NAFLD spectrum,
whereas (i,j) Cer increased qualitatively and no changes were observed among the (k,l) TG. All lipid species were
analysed by LC-MS and normalised to its internal standard as with whole serum, in addition to the ApoA-I
concentration (Supplementary Figure 1). Statistical significance was assessed using two-way ANOVA controlling
for presence of T2DM and interaction between T2DM and disease state, with a P-value <0.05 considered
significant. Data are mean ± standard deviation. In Supplementary Table 4 are reported the specific lipid species
analysed within the HDL fraction.

93
 Pearson’s correlation coefficient
Figure 4. HDL heatmap correlation matrix, Pearson’s correlation coefficient, among significantly changed
lipid species and clinical data in NAFLD and healthy controls. Data are plotted based on whether they are
positively (red) or negatively (blue) correlated, with a red circle highlighting the significant correlation with a p-value
<0.01.

4.4.3 VLDL lipidomics of NAFLD patients shows an enhanced SFA export

from the liver to peripheral tissues

VLDL fractions are an important biological matrix for the study of liver diseases
because of their intimate connection with this organ. While the remodelling of the
VLDL-TG has been widely investigated, less is known about other major lipid classes.
In an attempt to fill this gap, I analysed the lipidomic profile of separated VLDL fractions
belonging to the sub-cohort described above (Supplementary Table 2). As compared
to controls, NAFLD patients showed a generalised increase in PC, which reached
significance only for the SFA fraction (Figure 5a,b), whereas total LPC were markedly
increased, mostly driven by their SFA content (Figure 5c,d). Moreover, the NAFLD
group showed increased levels of total SM, driven by their MUFA content, and no
significant differences were observed among the Cer (Figure 5e-h). With regards to
TG, the most striking results were observed in their PUFA content which were depleted
in the NAFLD as compared to controls (Figure 5i-j). The presence of T2DM had a
significant effect only on the LPC fraction, whereas none of the analyses performed
showed a significant interaction between disease state and T2DM (Figure 5). To
further study the association between clinical data and significantly changed VLDL
lipids (NAFLD vs controls), I performed a correlation analysis. Specifically, Pearson

94
correlation showed that among all the selected lipids, the strongest association was
with circulating liver enzymes, particularly AST, which was positive for all lipids except
for the PUFA TG (inversely correlated) (Figure 6). These data indicate that the VLDL
lipidome was strongly related to hepatocyte cytotoxicity more than with markers of
metabolic impairment; however, the lack of fibrosis-associated changes in the data
tempts me to speculate that the reshaping of hepatocytes metatabolic programs in
NAFLD might not be a sole and sufficient driver of liver disease progression.
Also, most of the changes observed in VLDL were due to SFA-containing lipids, which
is in line with the increased arrival to the liver of saturated FFA, alongside the
enhanced DNL characterising NAFLD (Donnelly et al, 2005). Taken together these
data show that, in NAFLD, VLDL are crucial contributors of the SFA and MUFA plasma
pool within several lipids classes while having a minimal role, except for the TG, in the
observed PUFA depletion that cannot be only interpreted as a sole consequence of
the relative increase of DNL-like products. My data therefore point to the fact that the
reduced circulating lipidome described in the whole serum lipidomics of NAFLD
patients results from a combination of events driven by both HDL (PUFA-PL) and
hepatic secretion of VLDL (PUFA-TG).

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Figure 5. VLDL lipidomic signature of NAFLD. (a,b,c,d,e,f) PC, LPC and SM showed both a quantitative and
qualitative generalised increase in NAFLD as compared to controls. (g,h) Cer were not significantly changed
whereas (i,j) TG were qualitatively depleted in the NAFLD as compared to controls. All lipid species were analysed
by LC-MS and normalised to its internal standard as with whole serum, in addition to the ApoB-100 concentration
(Supplementary Figure 2). Statistical significance was assessed using two-way ANOVA controlling for presence of
T2DM and interaction between T2DM and disease state, with a P-value <0.05 considered significant. Data are
mean ± standard deviation. In Supplementary Table 5 are reported the specific lipid species analysed within the
VLDL fraction.

Pearson’s correlation coefficient

Figure 6. VLDL heatmap correlation matrix, Pearson’s correlation coefficient, among significantly changed
lipid species and clinical data in NAFLD and healthy controls. Data are plotted based on whether they are
positively (red) or negatively (blue) correlated, with a red circle highlighting the significant correlation with a p-value
<0.01.

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4.4.4 Discussion

The liver is a key player in regulating lipoprotein metabolism. The latter is due to the
liver's role in VLDL, LDL, and HDL synthesis and clearance. An imbalance between
the influx and efflux of these particles is considered crucial in the pathogenesis of
NAFLD (Donnelly et al, 2005).
Different studies have coherently reported a hepatic lipidomic signature of NAFLD
(Araya et al, 2004; Chiappini et al, 2017; Musso et al, 2018). On the other hand, whole
serum lipidomic studies have produced conflicting results, often discordant with the
liver findings (Mannisto et al, 2019; Puri et al, 2009). Discrepant findings have indeed
been reported even among major lipidomic classes: For instance, in morbidly obese
women with NASH, Anjani et al. found elevated levels of PC, PE, and PG when
compared to healthy females (Anjani et al, 2015). Likewise, increased levels of PC
and SM in NAFL and NASH compared to healthy subjects were reported by Tiwari-
Hecklerw et al. (Tiwari-Heckler et al, 2018). On the other hand, Zhou et al. reported
reduced levels of PC and LPC in NALF and NASH compared to controls (Zhou et al,
2016). In line with the previous findings, decreased levels of LPC in NAFLD compared
to controls were also reported by Oresic et al. (Oresic et al, 2013). Increased levels of
SM in NAFL and NASH compared to controls have been reported by some authors
(Tiwari-Heckler et al, 2018), while others found no significant changes (Anjani et al,
2015) or reduced levels (Zhou et al, 2016). It is therefore clear that the current
approaches to investigate the circulating lipidome requires different strategies to find
a unifying signature of plasma NAFLD.
Mass-spectrometry-based lipoprotein lipidomics of NAFLD has mainly focused on
VLDL (Boon et al, 2013; Carlier et al, 2020; Kotronen et al, 2009b), with limited
information regarding the other lipoprotein fractions. In this chapter, I first described
the whole serum lipidomic features of NAFLD compared to healthy controls. The latter
showed that NAFLD is characterised by a depletion in PL (PC, PE, PG) and specifically
in their PUFA fraction. Moreover, NAFLD patients had increased saturated and
decreased polyunsaturated FFA. Circulating FFA are released by the AT, and elevated
levels of FFA have been attributed to AT-IR-driven enhanced lipolysis (Azzu et al,
2020). Elevated FFA play a major role in the onset and development of NAFLD
(Bevilacqua et al, 1987; Lomonaco et al, 2012). The FFA changes observed in serum
are broadly in line with the results previously observed in liver biopsies (Chiappini et

98
al, 2017; Puri et al, 2007) and previous literature in whole serum lipidomics (de
Almeida et al, 2002; Feng et al, 2017; Gambino et al, 2016) despite the reduction of
polyunsaturated FFA is a debated finding (de Almeida et al, 2002; Feng et al, 2017;
Gambino et al, 2016). It is worth mentioning that the view of obesity as a driver for
elevated FFA due to increased lipolysis in AT has however been challenged by recent
re-evaluation of the literature (Hodson & Karpe, 2019; Karpe et al, 2011). A closer look
at the matter suggests that obese people have reduced lipolysis per unit of fat mass,
so when normalising FFA release per body fat, obese people show reduced lipolysis.
In this view, the conflicting literature on FFA changes might be due to the different
inclusion criteria among studies.
While the mechanism by which our NAFLD cohort displayed reduced polyunsaturated
FFA levels is not inferable from our data, the reduced PUFA TG in VLDL suggests a
strong cross-talk between liver and AT. I think that the discrepant results also
previously reported in whole lipidome studies for complex lipids are due, at least in
part, to the fact that the whole serum lipidome provides averaged information of
lipoproteins concentration and composition varying according to disease state, dietary
habits, fasting/fed states, sex and many other factors (Ding & Rexrode, 2020; Kontush
et al, 2013). I therefore wonder, if as it happens for cholesterol, where the different
biological significance of its concentration in VLDL/LDL and HDL is differentially
studied, this approach should also be used for other lipid classes.
Since NAFLD liver biopsies are depleted in PUFA in multiple lipid classes (Kartsoli et
al, 2020), and I observed a generalised PUFA reduction in the whole serum, I sought
to understand whether whole serum levels were reflecting “hepatic input”, i.e. lipids
deriving from peripheral tissues via HDL to the liver, or “output”, i.e. liver VLDL export.
To better understand these changes, I employed FPLC to isolate HDL and VLDL
fractions, where I then performed LC-MS lipidomic analyses. HDL lipidome showed a
generalised reduction in PC and PUFA across different lipid classes. These changes
have shown to affect HDL’s functionality as decreased PC levels in reconstituted HDL
(in vitro experiments) have been associated with reduced cholesterol efflux capacity,
thus rendering these particles potentially less atheroprotective (Jebari-Benslaiman et
al, 2020; Schwendeman et al, 2015). Moreover, the lipid profile of these HDL are in
line with reduced PL and PUFA content of peripheral tissues, such as skeletal muscles
and AT (both providing lipids to HDL), of obese and IR patients (Borkman et al, 1993;
Ferrara et al, 2021; Pietilainen et al, 2011). These findings are particularly relevant as
99
they highlight an often overlooked role of HDL in NAFLD, which is its transport of lipids
from peripheral tissues to the liver beyond its cholesterol content (van der Veen et al,
2012). Indeed, much attention has been given to reverse cholesterol transport but not
to HDL PL content and function, despite the elevated content of PL within HDL
(Kontush et al, 2013). This chapter therefore introduces the new paradigm that HDL,
whose PL content has been mainly studied with regards to consequences in HDL
physical properties, might be also acting as carriers of a “reverse phospholipid
transport” from periphery to the liver.
This concept emphasises the impaired lipid inter-organ cross-talk occurring in IR and
NAFLD and might be of help to better understand the pathophysiology of NAFLD.
Lastly, to investigate the role of the liver to the circulating lipidome, I studied the
isolated VLDL lipidome. In this regard, I observed, a different trend in the different lipid
classes. Indeed, as compared to control, NAFLD’s VLDL were enriched in PC and SM
(mostly driven by their SFA and MUFA content while their PUFA levels were
unchanged). Of note, while the contribution of the DNL, FFA, and dietary fat to VLDL
TG has been investigated, their contribution to the synthesis of other lipids (i.e PL and
sphingolipids) is not currently established. Although some studies have investigated
the VLDL lipidome of obese and IR subjects, there is very little information on the
biological role of these changes in the pathophysiology of IR, NAFLD and on
lipoprotein functions (Boon et al, 2013; Carlier et al, 2020; Kotronen et al, 2009b).
These results align with the increased SFA arrival to the liver from FFA and the already
described enhanced DNL in NAFLD (de Almeida et al, 2002; Donnelly et al, 2005; Puri
et al, 2007; Sanders et al, 2018). However, the extent to which de novo vs. peripheral
lipid recycling contribute to the PL and other lipid classes remodelling in NAFLD has
not been systematically investigated yet. Previous studies suggested that HDL PL can
be used to build up membranes and/or disassembled to use FA which were esterified
into TG (van der Veen et al, 2012). This hypothesis is really fascinating and I am
tempted to speculate that the reduced PUFA-TG pool could be, at least in part,
influenced by reduced transport of polyunsaturated FFA and HDL PUFA PL to the
liver. However, this hypothesis requires further validation with stable isotopes
approaches.

In conclusion, by using mass-spectrometry-based lipidomics, this chapter suggests

that NAFLD is characterised by an impaired reverse PL/PUFA transport (via FFA and
100
HDL) from peripheral tissues to the liver that, in turn exports mainly SFA and MUFA
enriched lipids through VLDL. More studies are needed to understand the metabolic
basis of this observation in terms of hepatic lipid fluxes, which of the two events is
cause or consequence, and the pathophysiological consequences of these altered
lipid fluxes in NAFLD progression.

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4.4.5 Limitations

The findings in this chapter are subject to different limitations. First, as an exploratory
analysis, I used data from the highest number of available participants and no formal
sample size calculation was performed. This implies that results must be interpreted
with caution. Second, the observational nature of the study does not allow to draw
causality among the changes observed. Third, I could only see differences among
healthy and diseased participants without being able to distinguish patients according
to disease severity. Fourth, variables not significantly different because of low effect
size should be interpreted with caution because of the risk of false negatives due to
the low power. Lastly, our study described a real-life cohort where medications such
as statins and metformin were not used as exclusion criteria. The latter medications
were present in the NAFLD spectrum but not in the controls, thus potentially further
confounding our results of circulating lipidome (data on medication were only present
for a subset of patients, thus rendering a further sub-analysis more complex due to
smaller numbers). These results thus warrant further confirmations in cohorts where
patients are not medically treated.

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Supplementary materials

Supplementary Table 1. Clinical characteristics of the BIONASH cohort. Data are mean ± SD and n (%).
Statistical tests are ANOVA-test, followed by Bonferroni correction and Pearson χ2 test, as appropriate. M = Male,
F = Female, BMI = Body Mass Index, HOMA2-IR = Homeostasis Model Assessment 2 of Insulin Resistance, TG
= Triglycerides, TC = Total Cholesterol, HDL-C = High-density lipoprotein cholesterol, LDL-C = Low-density
lipoprotein cholesterol, AST = Aspartate aminotransaminase, ALT = Alanine aminotransaminase. “a” means
different from “healthy controls”, “b” means different from “NAFL”.

Supplementary Table 2. Clinical characteristics of the BIONASH sub-cohort. Data are mean ± SD and n (%).
Statistical tests are ANOVA-test, followed by Bonferroni correction and Pearson χ2 test, as appropriate. BMI =
Body Mass Index, HOMA2-IR = Homeostasis Model Assessment 2 of Insulin Resistance, TG = Triglycerides, TC
= Total Cholesterol, HDL-C = High-density lipoprotein cholesterol, LDL-C = Low-density lipoprotein cholesterol,
AST = Aspartate aminotransaminase, ALT = Alanine aminotransaminase. “a” means different from “healthy
controls”.

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Supplementary Table 3. Average value for lipid species in the whole serum of Healthy and NAFLD
participants (all stages grouped together). Lipids classified as saturated are not highlighted, lipids
classified as monounsaturated are highlighted in yellow while lipids classified as polyunsaturated are
highlighted in green.

Whole Average normalised Average normalised

serum peak intensity - peak intensity - p value FDR
lipidome Healthy NAFLD
PG 36 0 0.014748201 0.005179818 4.22158E-10 6.54345E-08
LPC 18 0 0.171692782 0.30997664 8.07312E-10 6.25667E-08
FA 18 0 379.2858021 682.6838731 1.46962E-09 7.59305E-08
PC 40 3 0.014297539 0.0067915 5.75396E-09 2.22966E-07
PC 40 4 0.056544391 0.031717683 1.02056E-08 3.16372E-07
SM 36 0 0.011812975 0.020500518 3.8519E-08 9.95073E-07
LPC 20 3 0.053765111 0.0806669 4.30925E-08 9.54191E-07
PG 34 0 0.034846243 0.012994663 6.72028E-08 1.30205E-06
PC 36 3 2.137494094 1.415853174 2.14785E-07 3.69907E-06
FA 16 0 942.9547552 1394.123296 2.5921E-07 4.01776E-06
Cer 36 0 0.008456599 0.019749949 3.75514E-07 5.29134E-06
PG 36 1 0.02765252 0.010054597 3.78198E-07 4.88506E-06
PG 36 2 0.016776836 0.004994688 9.7554E-07 1.16314E-05
LPE 18 0 0.000359174 0.000570023 1.38961E-06 1.53849E-05
PC 40 5 0.118727491 0.071905907 1.82108E-06 1.88178E-05
LPC 20 5 0.046482622 0.028819538 2.26199E-06 2.1913E-05
LPC 16 0 0.4996007 0.672453364 2.43387E-06 2.21912E-05
PC 37 4 0.032959614 0.015358542 2.51935E-06 2.16944E-05
PC 38 4 1.295007127 0.916974658 5.09316E-06 4.15495E-05
PC 38 6 1.359269863 0.591195805 7.29985E-06 5.65738E-05
PC 36 4 2.12967252 1.375102926 1.00297E-05 7.4029E-05
PC 34 2 6.811594223 4.581828431 1.48476E-05 0.000104608
PC 40 7 0.003904402 0.002694416 2.79448E-05 0.000188323
PC 37 2 0.016597678 0.008354148 2.89966E-05 0.00018727
LPC 18 2 0.143743901 0.08905674 3.88665E-05 0.000240973
TG 56 7 0.384708987 0.146362584 5.55915E-05 0.000331411
TG 56 6 0.329124615 0.176450535 6.51753E-05 0.000374154
PC 40 6 0.352494449 0.185670388 6.63943E-05 0.00036754
PC 40 2 0.001762744 0.000958538 7.12518E-05 0.000380828
PC 35 2 0.179878633 0.098529668 8.17349E-05 0.000422297
TG 58 7 0.048293371 0.015221884 9.4929E-05 0.000474645
TG 54 2 0.471921768 0.673406027 0.000121986 0.000590872
LPC 18 3 0.130961449 0.159659379 0.000126492 0.000594129
SM 34 1 1.701129358 1.316848081 0.000135461 0.000617541
PC 34 0 0.047653084 0.03458482 0.000136753 0.000605619
SM 33 5 1.829952174 1.414013775 0.000139817 0.000601989
TG 58 9 0.058068907 0.01495279 0.000155005 0.000649346
PC 36 2 3.678560323 2.610111306 0.0001606 0.000655079
PC 34 3 0.356582789 0.233711842 0.000190129 0.00075564
TG 58 8 0.08240187 0.018608729 0.000252734 0.000979344
SM 40 2 0.298696337 0.223489711 0.00026105 0.000986895

104
PC 38 5 0.352628075 0.166031886 0.000300813 0.001110142
SM 34 0 0.075556586 0.055711834 0.000370318 0.001334866
PC 35 1 0.082458649 0.055512795 0.000392795 0.001383711
FA 18 2 329.6861672 215.5474681 0.000548402 0.00188894
TG 54 5 1.059217064 0.715614653 0.000652886 0.002199942
PC 33 2 0.05846998 0.033434261 0.000706675 0.002330525
PC 38 2 0.055368122 0.039913992 0.000800529 0.002585042
PE 36 2 0.000375027 0.006689366 0.000925772 0.002928462
PE 38 6 0.042240911 0.021048568 0.000958848 0.00297243
TG 54 6 0.507911836 0.295730154 0.000973446 0.002958513
TG 52 1 0.487064832 0.763458754 0.001213994 0.003618635
SM 39 1 0.062874069 0.043825517 0.002229345 0.006519781
TG 50 1 1.621117601 2.442823402 0.002311435 0.006634675
TG 55 4 0.010164041 0.007456292 0.002504056 0.007056885
SM 38 4 1.014174888 0.8327084 0.002672551 0.00739724
PE 36 4 0.029040167 0.017546235 0.002993897 0.0081413
PC 32 0 0.258742029 0.210212061 0.003120848 0.008340198
PC 33 0 0.018504835 0.012530957 0.003549836 0.009325841
SM 35 1 0.042782193 0.033584709 0.004581632 0.011835882
SM 38 2 0.077144951 0.064668715 0.005029367 0.012779538
PC 35 0 0.001093739 0.000703513 0.005422184 0.013555461
TG 53 4 0.078399038 0.058923079 0.006028379 0.014831726
Cer 42 1 0.368421738 0.295529379 0.006035074 0.014616195
TG 52 2 4.924785129 6.378896703 0.006708683 0.015997628
SM 35 2 0.00905709 0.006859068 0.007603464 0.017856619
TG 55 1 0.011231395 0.01594091 0.007886216 0.018244231
TG 50 2 2.162033725 3.037472134 0.010370271 0.023638119
SM 40 1 0.365680519 0.311965229 0.010397079 0.023355758
PE 38 4 0.068963139 0.048708654 0.012544773 0.027777712
SM 36 3 0.002015663 0.001565165 0.012751561 0.027837916
LPE 18 3 0.000100289 0.000142343 0.01570424 0.033807739
PC 32 2 0.047240408 0.033790582 0.01791477 0.03803821
PC 33 1 0.06973977 0.051307991 0.019239685 0.04029934
FA 20 3 23.16625311 28.38401948 0.020478851 0.042322959
PC 31 0 0.018639458 0.012848567 0.021474323 0.043796316
TG 48 1 0.712691074 1.066147329 0.025374494 0.051078528
TG 54 4 1.586045512 1.293319848 0.025820765 0.051310494
SM 40 0 0.006597577 0.008343683 0.026108764 0.051226057
PC 34 1 3.875880386 3.420680493 0.02656267 0.051465174
SM 38 0 0.005011409 0.006210849 0.029640868 0.056720179
TG 46 1 0.197976916 0.299050733 0.030136271 0.056964902
PC 38 3 0.511548876 0.424514255 0.030853664 0.057618288
TG 53 2 0.123605181 0.156476199 0.03157715 0.05826736
TG 51 4 0.043387531 0.033037759 0.033839288 0.061706938
SM 38 0 0.278119378 0.242951991 0.039494924 0.071182711
Cer 38 0 0.019795919 0.025148434 0.04410776 0.078582791
PE 36 1 0.006039451 0.008090838 0.046778831 0.082394532
LPC 20 0 0.000883901 0.001037751 0.050977364 0.088780802

105
TG 51 1 0.097711733 0.135934903 0.051003672 0.087839658
FA 17 0 18.75606812 23.21320572 0.051186727 0.087186184
LPC 17 1 0.001519162 0.001165227 0.053344317 0.089873578
SM 38 3 0.00037632 0.00055999 0.054060133 0.090100221
TG 52 3 4.16136592 4.890910516 0.057620101 0.095011869
LPC 19 0 0.000736451 0.000880904 0.060349524 0.098465013
LPE 18 1 0.00016746 0.000137501 0.061458795 0.099230346
SM 38 1 0.195680778 0.171537605 0.068750023 0.109858284
SM 33 0 0.001426946 0.001010083 0.112862627 0.178507216
TG 48 2 0.583440119 0.751996778 0.114072625 0.178598555
Cer 40 1 0.076138956 0.066939959 0.115000602 0.178250933
SM 36 2 0.168274169 0.152689324 0.119172875 0.182889065
PC 36 1 0.645693803 0.574379417 0.128152073 0.194740895
TG 51 2 0.257213028 0.319187716 0.130720519 0.196715344
LPC 17 0 0.010530699 0.012240134 0.147281183 0.219505609
TG 55 6 0.001591285 0.001247413 0.150801893 0.222612318
TG 44 1 0.055972231 0.074357854 0.171576459 0.250890105
TG 46 2 0.106103849 0.13505879 0.187510301 0.271627071
TG 49 1 0.136225244 0.171783481 0.224032474 0.321528088
PC 34 4 0.013789747 0.015578987 0.243746934 0.346612612
LPC 20 4 0.053524631 0.050064085 0.248855344 0.350659804
TG 36 0 0.002210239 0.00283418 0.251518342 0.351219307
LPC 22 5 0.002424331 0.002656169 0.268273739 0.371271692
TG 45 2 0.004564572 0.005761857 0.271011196 0.371741021
FA 16 1 23.50909592 25.79354486 0.280760255 0.381735435
FA 20 5 9.716240896 8.380809181 0.304505575 0.410420557
PC 30 0 0.031726939 0.027817631 0.305906705 0.408754649
TG 55 3 0.012260371 0.013568166 0.313309919 0.415068697
FA 20 0 2.023399641 1.7895456 0.319994999 0.420332414
TG 50 3 0.994422187 1.124475927 0.332915581 0.433629539
FA 22 6 21.49378451 20.01904109 0.437775157 0.565459578
SM 36 0 0.466111983 0.448381576 0.485122122 0.621437429
FFA 18 1 265.0450125 279.6138833 0.488633653 0.620805051
SM 36 1 0.284009176 0.273626569 0.51036215 0.643139294
TG 53 3 0.142474067 0.150757901 0.521631262 0.652039078
TG 51 0 0.015856844 0.01483812 0.526995528 0.653474455
TG 45 1 0.008921788 0.010128522 0.555892662 0.683836211
LPE 20 3 0.001036273 0.000976271 0.571096811 0.697007919
LPE 17 0 0.004698619 0.004498117 0.58426526 0.707508713
TG 55 5 0.004808685 0.004519492 0.584745523 0.70260121
LPC 14 0 0.004697222 0.004498589 0.587587657 0.700585283
TG 52 4 1.708569832 1.611895384 0.599481036 0.709309623
TG 47 1 0.039755673 0.044500191 0.600716923 0.705387296
TG 51 3 0.147955213 0.139405562 0.615217102 0.716982337
PG 40 8 7.41952E-05 6.38839E-05 0.6179688 0.714814657
TG 48 3 0.17420052 0.188341371 0.635775514 0.729964479
TG 47 2 0.016519234 0.018034179 0.653110791 0.744354211
TG 48 0 0.083973661 0.089417022 0.671094178 0.759267136

106
TG 47 0 0.012234878 0.013336855 0.680200471 0.763993283
FA 15 0 7.286185265 7.579770547 0.688512616 0.767765867
Cer 40 0 0.121337589 0.126324167 0.690230699 0.764183988
LPC 18 4 0.003775193 0.003632668 0.707727787 0.777998631
TG 49 2 0.102614755 0.109312505 0.712023805 0.777209083
SM 41 0 0.002207009 0.002132269 0.714596223 0.77456234
LPC 22 6 0.009051539 0.008824695 0.748542917 0.805723279
FA 20 4 67.0786296 68.40998139 0.762806895 0.815414267
LPC 16 1 0.011428513 0.011679651 0.798254515 0.847461985
PC 30 1 0.004854268 0.004649218 0.801534555 0.845155483
TG 49 0 0.010699581 0.01029592 0.822109425 0.860992979
TG 49 3 0.028759009 0.027870656 0.844841365 0.878861823
LPC 18 1 0.131916 0.130171544 0.848305029 0.876581864
Cer 42 0 0.194352039 0.198117391 0.850721456 0.873257123
TG 45 0 0.005677316 0.005998039 0.853520013 0.870365803
LPC 15 0 0.003861245 0.003816067 0.901685994 0.91347274
TG 53 0 0.005240329 0.005176851 0.902781942 0.908644162
PC 32 1 0.328226925 0.329184533 0.981004724 0.981004724

107
Supplementary Table 4. Average value for lipid species within the HDL fractions of Healthy and NAFLD
participants (all stages grouped together).

Average Average
normalised peak normalised peak
HDL P-value FDR
intensity - intensity -
Healthy NAFLD
PC 32 0 0.05085952 0.02769058 0.000410406 0.053763125
SM 38 2 0.014903302 0.00661459 0.001090314 0.071415553
PC 34 0 0.006525886 0.002699913 0.001602173 0.069961541
SM 34 1 0.374719877 0.185130709 0.00253388 0.082984554
SM 42 2 0.064525964 0.035238397 0.002684714 0.070339512
TG 47 2 0.006736223 0.008350617 0.003530756 0.077088181
PC 40 7 0.008322957 0.004108538 0.005373129 0.100554272
PC 37 6 0.00112323 0.000529108 0.006219044 0.10183685
PC 35 2 0.029028764 0.016692424 0.006981667 0.101622041
PC 35 4 0.004018736 0.002291535 0.007575312 0.099236581
PG 34 0 0.001571234 0.000579927 0.008777752 0.104535042
PC 35 3 0.002811492 0.001317773 0.009766183 0.106614163
PC 33 2 0.013372644 0.007879047 0.009988851 0.100656879
PC 38 5 0.176164556 0.091061586 0.011621184 0.108741074
Cer 40 0 0.0002682 0.000438229 0.011913793 0.104047122
PG 40 3 0.000403659 0.000149143 0.016245732 0.133011929
TG 58 7 0.006086781 0.008566875 0.018006103 0.138752908
Cer 48 0 0.00091826 0.001523649 0.020050576 0.145923635
PC 33 0 0.001051196 0.000461825 0.020780006 0.143272674
TG 57 0 0.000697469 0.000898659 0.020825665 0.136408106
PC 38 6 0.209794713 0.123996832 0.020831395 0.129948225
TG 55 4 0.000243245 0.000567363 0.022260658 0.132552099
SM 33 1 0.01057071 0.006482438 0.023960053 0.136468128
TG 55 0 0.001140398 0.001469393 0.024081906 0.131447071
PC 37 3 0.002124625 0.001123846 0.024335873 0.127519973
SM 36 2 0.017858075 0.010028779 0.024436482 0.123122276
TG 56 1 0.001422822 0.001866669 0.02554961 0.123962921
TG 52 1 0.052041468 0.066044327 0.026708773 0.124958903
PC 31 0 0.002873701 0.000988602 0.026921938 0.121612894
PC 37 4 0.004772476 0.002259412 0.030959684 0.13519062
Cer 34 0 0.002496829 0.00375933 0.034072717 0.143984706
TG 55 1 0.000811525 0.001064585 0.03445897 0.14106641
Cer 46 0 0.000869744 0.001592473 0.035023552 0.139032887
SM 35 2 0.000692432 0.000416835 0.03507376 0.135137132
Cer 48 0 0.000462525 0.000753895 0.03751466 0.140412012
PC 36 3 0.468446937 0.272827832 0.037615925 0.13688017
TG 56 0 0.001329916 0.001673076 0.039046567 0.138245954
PC 35 1 0.014485887 0.006535817 0.039158132 0.134992509
PG 40 4 0.000210055 0.000109974 0.039575149 0.132931911
PC 40 4 0.005265963 0.002682494 0.041418015 0.135643998
PE 34 2 0.068024704 0.086362548 0.041747403 0.133388042

108
 PC 34 2 1.848612215 1.095762999 0.043856345 0.136790028
TG 54 1 0.010248092 0.013082273 0.044493144 0.135548881
Cer 38 0 0.002177946 0.002815909 0.044822177 0.133447844
PC 34 1 1.066324225 0.806822323 0.053682771 0.156276513
Cer 47 0 0.000352289 0.000580778 0.053944319 0.153624038
PE 38 6 0.002188674 0.001170495 0.054967785 0.153208081
SM 34 1 0.00030193 0.000143185 0.055103002 0.150385276
TG 45 1 0.005946912 0.00902467 0.057175417 0.152856728
TG 47 1 0.012083555 0.014750736 0.058417672 0.153054301
PC 37 5 0.001463517 0.000451129 0.058682049 0.150732322
SM 36 3 0.00085933 0.000452718 0.059683234 0.150355838
Cer 47 0 0.000742792 0.001127729 0.059761853 0.14771326
PG 40 2 0.001060883 0.000854005 0.061722016 0.149733038
PC 40 6 0.058079051 0.040437512 0.061819915 0.147243798
Cer 45 0 0.000636864 0.001328443 0.062689476 0.146648596
Cer 46 0 0.001560305 0.002571366 0.067665067 0.155510944
PC 33 1 0.010670792 0.00550487 0.072598892 0.163973359
SM 40 1 0.038154012 0.030172651 0.072981884 0.162044521
PC 40 5 0.021481642 0.013801563 0.074462304 0.162576031
PC 36 1 0.123948226 0.076838214 0.082241525 0.176617045
SM 34 2 0.033069178 0.023532713 0.09425912 0.1991604
LPC 20 0 0.002650322 0.002058234 0.094617216 0.196743735
PC 34 3 0.063140735 0.045552233 0.095505434 0.195487685
PC 36 0 0.00203329 0.001086177 0.095990602 0.193457982
Cer 45 0 0.000556893 0.000987656 0.096686469 0.191907992
PC 36 4 0.427986686 0.309426209 0.097197927 0.190043708
SM 30 1 0.001156955 0.001486407 0.097258226 0.187365112
PC 38 3 0.097072817 0.067185527 0.102889526 0.195340984
TG 53 1 0.002658631 0.00318575 0.118204448 0.221211182
TG 45 0 0.00747789 0.009436071 0.122803768 0.2265816
PC 36 2 0.66462548 0.472585073 0.122956756 0.223712987
Cer 44 0 0.000942936 0.001937748 0.126453874 0.226924075
SM 37 2 0.000157081 0.00020828 0.13375016 0.236773932
Cer 46 2 0.000415948 0.000820583 0.159514812 0.278619206
Cer 50 0 0.000331418 0.00053406 0.16019855 0.276131711
PC 39 6 0.000583222 0.000265312 0.174623413 0.297086586
Cer 44 2 0.000614002 0.002040343 0.175799761 0.295253445
Cer 44 1 0.00058102 0.001856446 0.175998605 0.291845788
LPC 16 0 0.271583998 0.340535084 0.176833964 0.289565616
Cer 37 0 0.003350846 0.003906992 0.177119012 0.286451736
Cer 48 1 0.000338548 0.000673484 0.177371262 0.283361406
Cer 47 1 0.000337619 0.000593188 0.180902078 0.285520147
Cer 45 1 0.000424347 0.000936705 0.181339496 0.282803262
Cer 46 1 0.000683785 0.001595733 0.184293277 0.284028462
Cer 42 0 0.000573395 0.001524667 0.187367337 0.285408386
Cer 44 1 0.000907713 0.00251857 0.188233859 0.283432592
PC 38 4 0.307278372 0.251030266 0.194427491 0.289431833
LPC 18 1 0.068863495 0.082299971 0.199065126 0.293005971

109
Cer 50 2 0.00011764 0.000214472 0.205499604 0.299116091
Cer 38 0 0.00011096 0.000352217 0.218922529 0.315152212
SM 40 0 0.000867028 0.000995811 0.221038585 0.314739724
Cer 38 1 0.000231363 0.000594209 0.233190991 0.328473332
LPC 18 2 0.025584271 0.020252743 0.240767767 0.335538058
Cer 45 1 0.000229777 0.000600876 0.246756046 0.3402636
TG 58 10 0.00158694 0.001808095 0.247698548 0.338005311
PC 30 0 0.005665716 0.0044129 0.24989518 0.337487305
Cer 43 1 0.000199657 0.000769718 0.251326995 0.335957514
Cer 45 2 0.000230362 0.000599635 0.25417787 0.336336374
PC 34 4 0.002902288 0.00219471 0.277265979 0.363218433
Cer 34 1 9.66E-05 0.00060191 0.283405777 0.367585711
Cer 41 2 5.25E-05 0.000267954 0.301482808 0.387198509
TG 47 0 0.015521254 0.017713545 0.317365541 0.403639669
Cer 43 1 0.000816992 0.001430037 0.324614575 0.408889513
Cer 37 1 0.003357093 0.003757293 0.333692808 0.416321504
LPC 18 3 0.058635942 0.06837232 0.333697267 0.412399452
Cer 43 0 0.000272674 0.000647105 0.334489313 0.409514953
Cer 43 2 0.000366229 0.001095554 0.335696666 0.407187623
LPC 17 0 0.008115858 0.009252024 0.337868757 0.406062451
SM 42 0 0.000865978 0.000966307 0.368553471 0.438913679
TG 52 0 0.011744753 0.010090233 0.380963317 0.449605357
PE 34 1 0.000420276 0.000503506 0.391600389 0.458032598
Cer 50 1 0.000232988 0.000350105 0.409336678 0.47454075
PC 30 1 0.00113586 0.001453306 0.429073963 0.493058676
PC 38 2 0.009704364 0.007720001 0.436390973 0.497106239
SM 40 0 0.000599772 0.000531182 0.441948236 0.499096715
LPC 20 4 0.018976849 0.020816203 0.445179159 0.49844846
TG 49 0 0.007797999 0.008680456 0.487472758 0.541177384
LPC 20 3 0.06199402 0.057138466 0.504842927 0.555751458
Cer 39 1 8.42E-05 0.000150757 0.507115275 0.553600842
TG 48 0 0.028411831 0.025552231 0.630091534 0.682165214
Cer 41 1 0.001123668 0.001381184 0.632048507 0.678675036
Cer 42 1 0.003408553 0.003934894 0.689872192 0.734741928
PC 28 0 0.000656556 0.000594263 0.756596046 0.799307114
PC 32 1 0.048536816 0.045397828 0.791463516 0.829453765
TG 53 0 0.001399954 0.001440666 0.795537843 0.827106805
TG 51 0 0.003078209 0.003176127 0.810334644 0.835856995
SM 33 0 0.001930055 0.001961189 0.918122958 0.939641465
LPC 18 0 0.302046828 0.304767406 0.939637594 0.954205619
Cer 40 1 0.000477375 0.000484648 0.961649446 0.969046749
PC 32 2 0.010488942 0.010425302 0.968044067 0.968044067

110
Supplementary Table 5. Average value for lipid species within the VLDL fractions of Healthy and NAFLD
participants (all stages grouped together).

Average Average
normalised peak normalised peak
VLDL P-value FDR
intensity - intensity -
Healthy NAFLD
SM 38 0 1.64E-05 5.90E-05 0.000725908 0.103078998
SM 36 0 0.000118311 0.000296425 0.002462228 0.174818164
PC 36 0 0.000120779 0.000299941 0.003337048 0.157953596
PC 32 0 0.002282174 0.00409094 0.005229781 0.185657229
SM 32 0 1.85E-05 3.64E-05 0.007697312 0.218603656
SM 30 1 1.52E-05 3.91E-05 0.009326217 0.220720474
SM 32 1 0.001231918 0.002055163 0.009742023 0.19762389
SM 42 1 0.002200887 0.003664717 0.010568205 0.187585639
PC 34 0 0.000317408 0.000590306 0.011338917 0.178902917
SM 36 1 0.003014769 0.005194076 0.01229138 0.174537595
SM 38 1 0.001865159 0.003201536 0.013756776 0.177587478
TG 54 6 0.007253429 0.001557512 0.015267723 0.180668059
Cer 42 0 3.11E-05 5.46E-05 0.01994314 0.217840451
SM 40 1 0.003280978 0.004872498 0.021782806 0.220939886
TG 54 5 0.010891758 0.003286594 0.022404919 0.212099897
SM 34 1 0.017287935 0.02599398 0.027295636 0.242248767
SM 33 0 0.001355826 0.000339181 0.027486423 0.229592473
TG 56 8 0.0021035 0.000307393 0.028550824 0.225234275
TG 54 4 0.014081464 0.00533033 0.029703566 0.22199507
TG 58 7 0.000711575 0.000195432 0.029835832 0.211834409
SM 35 1 0.000313305 0.00053147 0.036530803 0.24701781
SM 38 2 0.000716539 0.001124055 0.045110114 0.291165282
TG 51 4 0.000518805 0.000173617 0.046419712 0.286591268
SM 44 1 0.000286624 6.04E-05 0.055187822 0.326527948
TG 53 4 0.000656809 0.000261498 0.061595775 0.349864002
Cer 36 0 4.32E-05 5.23E-05 0.062780787 0.342879685
TG 56 3 0.000247314 0.000115154 0.064418784 0.338795086
TG 52 4 0.02094862 0.009924854 0.066712281 0.338326567
TG 58 9 0.000414199 6.12E-05 0.066989418 0.328017149
LPC 16 0 0.000822757 0.001539891 0.074331903 0.351837675
SM 36 3 1.39E-05 2.60E-05 0.074428726 0.340931584
TG 56 6 0.003713296 0.00079967 0.080371001 0.356646319
SM 34 0 0.000563524 0.00082278 0.082301229 0.354144681
Cer 36 1 1.53E-05 2.63E-05 0.08285178 0.346028021
TG 55 5 0.000129901 2.80E-05 0.09244864 0.375077338
Cer 40 0 7.75E-06 1.62E-05 0.09414458 0.371348065
TG 58 10 0.00022681 2.49E-05 0.108574809 0.416692511
TG 51 3 0.001508399 0.000682267 0.110133616 0.411551935
PC 33 0 5.87E-05 9.98E-05 0.112947492 0.411244715
TG 53 3 0.001332957 0.000598287 0.116176495 0.412426557
TG 54 3 0.011789519 0.007093287 0.119602708 0.414233769
TG 52 3 0.04727754 0.025458108 0.123194471 0.41651464

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TG 49 3 0.000476086 0.000179315 0.127520985 0.421115811
PC 36 1 0.005654631 0.008234784 0.13276145 0.428457407
TG 47 2 0.000399637 0.00019667 0.146511756 0.462325984
SM 34 2 0.001288182 0.001770359 0.15113638 0.466551434
PC 38 2 0.000370736 0.000528636 0.164290046 0.496365672
TG 55 3 7.32E-05 2.74E-05 0.169947128 0.502760253
TG 51 2 0.002711071 0.001185441 0.173602076 0.503091732
TG 45 1 0.000253797 0.000141791 0.176613851 0.501583338
Cer 46 1 1.47E-05 7.97E-06 0.178000419 0.49560901
LPC 18 1 0.000204534 0.000291911 0.181958627 0.496887021
TG 56 7 0.002701672 0.000607087 0.186193723 0.498858655
TG 49 2 0.001219126 0.000571831 0.186975833 0.491677191
TG 55 4 8.46E-05 2.20E-05 0.188444226 0.48652873
PC 31 0 0.000124441 0.000189325 0.188866165 0.478910633
TG 48 3 0.002606029 0.001093743 0.189321376 0.471642727
TG 46 4 9.36E-05 2.75E-05 0.193701662 0.474235103
Cer 45 1 8.37E-06 5.44E-06 0.194050447 0.467036669
TG 50 3 0.012366626 0.00621292 0.198459254 0.469686901
TG 49 1 0.001577092 0.00066469 0.202455819 0.471290596
SM 36 2 0.000710086 0.001020051 0.210232542 0.481500338
PC 36 2 0.022462112 0.029938416 0.222651821 0.501850137
SM 40 0 0.002141875 0.002697262 0.228044857 0.505974526
PC 30 0 0.000163443 0.000244546 0.229218712 0.500754725
TG 53 2 0.00096699 0.000440526 0.237360521 0.510684758
Cer 47 0 8.20E-06 6.33E-06 0.23850698 0.505492405
TG 47 1 0.000623068 0.000329739 0.243801517 0.509114932
PC 34 1 0.043380747 0.058753576 0.248249666 0.510890617
SM 34 0 1.29E-05 1.80E-05 0.253433724 0.514108412
SM 40 2 0.003211464 0.003926275 0.254049782 0.508099564
PC 38 3 0.003417012 0.004806974 0.264511169 0.521674806
TG 52 2 0.045966498 0.029146265 0.266586926 0.518566349
TG 48 2 0.008406837 0.004188476 0.270296832 0.518677705
TG 53 1 0.00010632 5.48E-05 0.276945135 0.524349456
TG 51 1 0.000888865 0.000376012 0.285535452 0.53350045
TG 46 2 0.001926717 0.000940073 0.288628023 0.532275056
TG 45 2 0.000142335 8.59E-05 0.292791843 0.533031304
Cer 46 0 1.48E-05 1.06E-05 0.292966201 0.526597475
TG 50 0 0.001294721 0.000748263 0.297469409 0.5280082
Cer 48 0 1.54E-05 1.08E-05 0.305400444 0.535393371
Cer 47 1 4.76E-06 2.94E-06 0.30955713 0.536062348
TG 50 2 0.025864674 0.014963728 0.310040655 0.530431
PC 30 1 3.62E-05 5.28E-05 0.318600618 0.538586759
PC 32 2 0.000262703 0.000344008 0.329245018 0.550032853
TG 56 2 8.23E-05 5.62E-05 0.338395283 0.5587457
SM 40 0 0.000137659 7.67E-05 0.34051691 0.555786221
TG 44 1 0.00110792 0.000582715 0.34755793 0.560832114
TG 46 1 0.003223549 0.001700473 0.348255493 0.555643596
TG 48 0 0.003183064 0.00208503 0.34861262 0.550033245

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Cer 42 1 0.000408799 0.000521489 0.352211975 0.5496055
LPC 18 2 3.48E-05 4.67E-05 0.382033288 0.589660075
TG 48 1 0.009331883 0.005341443 0.38292265 0.584677594
Cer 44 0 1.72E-05 1.30E-05 0.383658506 0.579569232
Cer 38 1 1.46E-05 1.94E-05 0.404152063 0.604100978
SM 35 2 3.14E-05 3.87E-05 0.406492636 0.601270358
TG 54 2 0.002740124 0.002026569 0.411315337 0.602131731
Cer 42 0 5.46E-05 7.15E-05 0.422114427 0.61163519
TG 50 1 0.016634978 0.011168324 0.445998603 0.639715168
SM 37 2 1.29E-05 1.68E-05 0.456267902 0.647900421
LPC 17 0 2.20E-05 3.27E-05 0.478494018 0.672734164
TG 52 1 0.003693083 0.002434799 0.479390481 0.667386748
Cer 40 1 0.000102664 0.000130243 0.480834756 0.662898402
PC 38 6 0.004243898 0.003277305 0.482727466 0.659108655
PC 38 5 0.001515105 0.001098437 0.484330202 0.65499894
LPC 18 0 0.001939338 0.002520599 0.497906314 0.667006571
PC 32 1 0.002266018 0.002929076 0.501122582 0.665041184
LPC 18 3 0.000217798 0.000300851 0.562226283 0.739223446
Cer 45 0 5.72E-06 4.70E-06 0.566192448 0.73760851
PC 37 3 8.77E-05 7.24E-05 0.58449671 0.754532116
TG 54 1 0.000228347 0.000182819 0.589935329 0.754692042
PC 40 5 0.000634337 0.00050541 0.592426974 0.751112771
Cer 41 1 0.000118245 0.000138361 0.596826607 0.749994497
PC 35 1 0.000589558 0.000693357 0.602699645 0.750731137
Cer 37 0 0.000122097 0.00011145 0.606448142 0.748831619
Cer 43 1 6.68E-05 7.45E-05 0.611576216 0.748653643
Cer 37 1 0.000121854 0.000111452 0.614018663 0.745219232
Cer 39 1 7.88E-06 9.53E-06 0.615933145 0.741207682
PC 34 2 0.052485566 0.059834824 0.630243515 0.752055287
Cer 44 1 3.31E-05 2.94E-05 0.642375872 0.760144782
PC 33 2 0.000267724 0.000297702 0.691534383 0.811552747
LPC 20 4 7.55E-05 8.45E-05 0.749745875 0.872655035
PC 37 2 4.13E-05 3.60E-05 0.764644898 0.882760777
PC 40 4 0.000216256 0.000181847 0.767755297 0.879203646
Cer 47 0 2.59E-06 2.26E-06 0.793408132 0.901311638
PC 36 4 0.011704723 0.010650792 0.794283663 0.895145081
Cer 38 0 5.59E-05 5.79E-05 0.802063052 0.896794909
SM 33 1 0.000920576 0.000960887 0.808104388 0.896490806
Cer 35 1 0.000338189 0.000325308 0.812418315 0.894289928
PC 40 6 0.001801183 0.00167113 0.823975417 0.900034686
TG 61 12 2.53E-05 2.71E-05 0.834277463 0.904331295
LPC 20 3 0.000356414 0.000319264 0.869336661 0.935195499
PC 36 3 0.012801294 0.012351956 0.903062197 0.964171669
PC 33 1 0.000415532 0.000433378 0.923070797 0.978179501
Cer 41 0 8.83E-06 9.11E-06 0.92870562 0.976860726
PC 34 3 0.001637708 0.001590517 0.939706352 0.981163985
Cer 43 0 8.75E-06 8.60E-06 0.956174461 0.991071339
Cer 48 0 4.15E-06 4.08E-06 0.968987409 0.997074

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 PC 38 4 0.00976357 0.009819216 0.98540401 1.006671722
PC 35 2 0.001074877 0.001077542 0.991660455 1.005827033
PC 40 7 0.000493895 0.000492985 0.996335056 1.003401262
PC 34 4 0.000127041 0.000127335 0.997301532 0.997301532

Supplementary figure 1. HDL ApoA-I levels in healthy and NAFLD participants. ApoA-I levels were
significantly reduced within isolated HDL fractions of NAFLD patients compared to controls independently from the
presence of type 2 diabetes mellitus (T2DM). ApoA-I was analysed by LC-MS and normalised to bovine serum
albumin as detailed in the method section. Statistical significance was assessed using two-way ANOVA controlling
for presence of T2DM and interaction between T2DM and disease state, with a P-value <0.05 considered
significant. Data are mean ± standard deviation.

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Supplementary figure 2. VLDL ApoB-100 levels in healthy and NAFLD participants. ApoB-100 levels were
not significantly different within isolated VLDL fractions of NAFLD patients compared to controls. ApoB-100 was
analysed by an ELISA kit as detailed in the method section. Statistical significance was assessed using two-way
ANOVA controlling for presence of T2DM and interaction between T2DM and disease state, with a P-value <0.05
considered significant. Data are mean ± standard deviation.

115
Chapter 5. Conclusion

5.1 Summary and conclusion

The epidemic spread of obesity has rendered this condition a public health priority.
Obesity and AT dysfunction promote IR, and together they increase the risk for
NAFLD, T2DM, and CVD. Lipoprotein dysmetabolism is believed to be a key mediator
in this process. However, most of the studies conducted thus far have focused on their
LDL/HDL cholesterol levels or triglycerides in VLDL, thus leaving largely unexplored
multiple other lipid classes and their changes in health and disease.
In this thesis, I sought to understand the lipoprotein remodelling occurring in obesity-
related metabolic disorders, the MetS and NAFLD, since they are well known to be
associated to altered lipoprotein metabolism and dyslipidaemia.

Studies investigating the whole serum/plasma lipidome of MetS and NAFLD have
reported conflicting findings (Kartsoli et al, 2020; Monnerie et al, 2020) and there
currently lacks an accepted mechanistic explanation as to why certain lipoprotein
changes are associated with MetS and NAFLD. Part of these discrepancies are due
to the fact that whole serum/plasma lipidome is an average of lipid signals from
different lipoprotein fractions thus being highly influenced by the recruitment criteria
and characteristics of the study population.

In chapter 3, I first profiled the major lipidomic classes in the whole serum of healthy
and MetS patients. In addition to the expected increased levels of TG and DG in MetS
compared with controls, the most intriguing results were attributable to increased PC
and reduced LPC levels, alongside a decreased CE/FC ratio in MetS compared to
controls. By using a lipidomic index as an indirect measure of enzyme activity, termed
the Q index (Gerl et al, 2018), my data suggested that LCAT, the only circulating
enzyme capable of esterifying CE from FC, could be responsible for the changes
observed. The use of enzyme activity assays confirmed a reduced activity of LCAT in
MetS compared with controls while ruling out the contribution of the other major
enzyme involved in plasma PC metabolism, namely PLPA2. The whole serum
targeted proteomic data further supported the hypothesis of reduced activity of LCAT
in MetS compared with controls since ApoA-I, the predominant activator of LCAT, was
significantly reduced, and ApoC-III, a potent in vitro inhibitor of LCAT, was markedly

116
increased in MetS (Cho, 2009; Sorci-Thomas et al, 2009). Gerl et al. demonstrated
that the Q index was reduced in patients with CVD (Gerl et al, 2018). In line with their
study, I also showed that in patients at high CVD risk, the Q index is also reduced,
thus suggesting that it could possibly serve as an early marker of CVD risk also in
primary prevention. Of note, I also validated the utility of the Q index as a surrogate
LCAT activity marker correlating it with the direct measurement of LCAT. Whether
MetS patients experience elevated CVD risk via LCAT is yet to be established:
although the role of LCAT in CVD has been already investigated, conflicting results
directly linking LCAT activity to CVD have been reported in both clinical and pre-clinical
studies (Norum et al, 2020). While our small cohort does not address the clinical
implication of LCAT in CVD it strongly suggests that LCAT is an important player in
regulating the plasma lipidome. This chapter demonstrates how combined omics
approaches along with fluorometric assays can provide a clearer picture of the
lipoprotein remodelling in metabolic diseases. Although promising, these results
require further validations in larger cohorts designed to confirm the clinical implications
of our findings.

In chapter 4, I investigated the lipoprotein remodelling occurring in NAFLD. Several

studies have coherently reported a reduction of PUFA in the liver of NAFLD patients.
However, the circulating lipidomic studies found conflicting results which do not always
match the liver findings (Kartsoli et al, 2020). In this chapter, I showed that the whole
serum lipidome of NAFLD patients was indeed characterised by a reduction of PUFA
lipids and specifically in the PL species. Furthermore, increased levels of saturated
and reduced polyunsaturated FFA in NAFLD compared with controls, suggested that
AT is an important player in the hepatic lipid composition of NAFLD, this being in line
with previous research (Donnelly et al, 2005). To understand whether the PUFA
reduction in circulation was led by a reduced hepatic “input” (HDL and FFA) or a
consequence of reduced PUFA export from the liver (VLDL), I isolated the lipoprotein
fractions to study in more detail their lipidomic profile. These data showed that
NAFLD’s HDL were depleted in PL and specifically in PUFA lipids, suggesting that the
peripheral tissues provide less PL and PUFA to the liver also via HDL. These findings
go alongside previous literature showing that AT and skeletal muscle biopsies of
obese and IR subjects are depleted in PL and PUFA (Borkman et al, 1993; Ferrara et
al, 2021; Pietilainen et al, 2011). Intriguingly, with tracer experiments, previous

117
literature suggested that the liver largely re-uses HDL-derived PL either incorporating
them into membranes or converting them into TG (van der Veen et al, 2012); this
seems to be confirmed in my VLDL data where the VLDL lipidome of NAFLD patients
appears depleted in PUFA-TG compared to controls. Besides TG, the remaining lipids
had unchanged PUFA levels and increased SFA and MUFA lipids in several PL
classes probably reflecting a well-known upregulation of DNL and lipid remodeling
occurring in NAFLD. These results indicated that the plasma PL depletion observed in
NAFLD could be driven, at least in part, by the HDL lipid composition; however, it
cannot be excluded that peripheral organs become PUFA-depleted because they
receive less PUFA-TG from the liver (VLDL). Another intriguing hypothesis is that
PUFA-PL might also be depleted as a consequence of their utilisation for the
production of bioactive lipid species, such as eicosanoids, which exert signalling
functions (Musso et al, 2018) either in the liver or in peripheral tissues. However, it
should be said that these are only speculations and that the extent to which AT
influences the liver lipidome and vice versa is not inferable from our data, thus
warranting further investigation with stable isotope approaches to measure fluxes.
The strong correlation between VLDL lipidome and circulating liver enzymes confirms
the role of the hepatic lipidome in driving hepatocyte lipotoxicity; however, the lack of
stepwise changes with the severity of the disorder suggest that other factors might be
also influencing disease progression.

The hypothesis of HDL as a nexus of peripheral and hepatic lipid disorders has been
largely unexplored except for its cholesterol content or in mouse studies (van der Veen
et al, 2012). This chapter therefore introduces a new paradigm where HDL, whose PL
content has been mainly studied with regards to consequences in HDL physical
properties, might be also acting as carriers of a “reverse phospholipid transport” from
the periphery to the liver.

The reader might have noticed some differences in the findings between the MetS
(Chapter 4) and NAFLD (Chapter 5) cohorts, despite these disorders being both
obesity-driven. This could be attributable to the different criteria of recruitment in the
two studies leading to pretty different metabolic profiles of the NAFLD/MetS groups.
The MetS patients inclusion criteria was the presence of 3 ATPIII criteria for MetS, in
the case of NAFLD the diagnosis is clinically made irrespectively of the metabolic

118
background. Also, the nutritional background between the 2 cohorts was completely
different as the MetS patients were recruited in Southern Italy (in a region
characterised by a high dietary consumption of extra-virgin olive oil and fish) while the
NAFLD cohort was recruited in the UK therefore possibly being less exposed to
nutritional PUFA and potentially more prone to depletion. In this work, I could not rule
out the influence of the dietary habits of these two cohorts. It is thus essential for future
lipidomic studies to also account for dietary habits.

In conclusion, the work done in this thesis highlighted a novel role for the circulating
lipoprotein remodelling enzyme LCAT in shaping the plasma lipidome in MetS patients
alongside providing a new role for HDL in the pathogenesis of NAFLD. If confirmed,
both findings could lead to novel therapeutic strategies.

119
5.2 Future directions

The work presented in this thesis has highlighted future areas of research that would
increase the understanding of lipoprotein metabolism in obesity-related metabolic
disorders and potentially lead to new drug targets.

In chapter 3, I identified LCAT as a putative modulator of the PL and cholesterol

dysmetabolism observed. A follow-up experiment would be to profile the serum
lipidome of patients with genetic LCAT deficiency. The latter would provide evidence
of the plasma remodelling occurring as a consequence of LCAT deficiency while
excluding the influence of other factors characterising the MetS, which could also be
involved in the lipid changes observed. In parallel, a further question to answer would
be to assess if the use of recombinant LCAT, which has proven to restore the HDL
cholesterol levels in LCAT deficient patients (Simonelli et al, 2013), would also rescue
the changes in the lipidomic phenotype described in this chapter. In terms of
functionality, it would also be relevant to characterise how the changes in the Q index
affects the cholesterol efflux capacity (with in vitro experiments), thus providing a
functional relevance of these lipids for HDL function and CVD. Lastly, larger
prospective cohorts would also be necessary to confirm the role of the Q index as a
hallmark of cardiometabolic risk. While these findings are still preliminary, my work
also supports the increasingly established role of shorter and more saturated TG as
hallmark of IR and MetS. Because increased levels of these TG are already present
at early stages of IR, my work also suggests that the use of a specific set of TG,
measured through liquid chromatography mass-spectrometry, might prove a better
identifying criteria for the MetS as compared to total TG.

In chapter 4, I found that HDL lipid composition matches the profile described in the
liver of NAFLD patients. Interestingly, the contribution of de novo vs. dietary FA in
promoting lipotoxic species such as ceramides in different tissues is still poorly
described, and a better understanding of these pathways in clinical and pre-clinical
models would improve our understanding of the NAFLD pathophysiology.
The use of stable isotopes in mice with humanised livers (mouse models which better
recapitulate the human lipoprotein metabolism) placed on western-style diets (high-
fat, high-cholesterol diets enriched in refined carbohydrates either in the form of

120
sucrose or fructose) with different ratios between PUFA and MUFA/SFA, would allow
to study if the defective reverse PUFA/PL transport observed in NASH patients and
likely due to a reduced accumulation of PUFA in peripheral tissues (AT, skeletal
muscle) is secondary to reduced PUFA intake, increased PUFA consumption in
different organs, or rather an event secondary to the enhanced de novo hepatic FA
synthesis. These settings would also allow to characterise in parallel gene/protein
expression of enzymes involved in FA metabolism in different tissues to inform
whether the elongation and desaturation of preformed essential PUFA is a primary
liver event or rather a peripheral event leading to the PL depletion. These findings
could also be translated clinically via the administration of stable isotopes to patients
scheduled for liver biopsy to study hepatic fluxes of different lipid classes, and to
investigate if lipotoxic species (i.e. ceramides) are products of intra-hepatic de novo
synthesis. Lastly, profiling of eicosanoids in tissues and plasma would shed light on
the contribution of these pathways to the PL and its PUFA remodelling across the
NAFLD spectrum. Despite the preliminary nature of the results provided here, our data
highlights that whole serum lipidomics is highly influenced by lipoprotein remodelling
and that can provide useful insights into these processes; however, the use of
lipoprotein lipidomics will ultimately be necessary to allow a better understanding of
the biology underlying lipoprotein metabolism and remodelling.

121
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