Nonalcoholic Fatty Liver Disease: Focus on Lipoprotein and Lipid Deregulation

Abstract

Obesity with associated comorbidities is currently a worldwide epidemic and among the most challenging health conditions in the 21st century. A major metabolic consequence of obesity is insulin resistance which underlies the pathogenesis of the metabolic syndrome. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome. It comprises a disease spectrum ranging from simple steatosis (fatty liver), through nonalcoholic steatohepatitis (NASH) to fibrosis, and ultimately liver cirrhosis. Abnormality in lipid and lipoprotein metabolism accompanied by chronic inflammation is the central pathway for the development of metabolic syndrome-related diseases, such as atherosclerosis, cardiovascular disease (CVD), and NAFLD. This paper focuses on pathogenic aspect of lipid and lipoprotein metabolism in NAFLD and the relevant mouse models of this complex multifactorial disease.

Full text

Hindawi Publishing Corporation
Journal of Lipids
Volume 2011, Article ID 783976, 14 pages
doi:10.1155/2011/783976
Review Article
Nonalcoholic Fatty Liver Disease: Focus on Lipoprotein and
Lipid Deregulation
Klementina Fon Tacer and Damjana Rozman
Center for Functional Genomic and Biochips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Zaloˇ
ska 4,
1000 Ljubljana, Slovenia
Correspondence should be addressed to Damjana Rozman, damjana.rozman@mf.uni-lj.si
Received 4 January 2011; Revised 26 April 2011; Accepted 27 April 2011
Academic Editor: Shinichi Oikawa
Copyright © 2011 K. Fon Tacer and D. Rozman. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Obesity with associated comorbidities is currently a worldwide epidemic and among the most challenging health conditions in
the 21st century. A major metabolic consequence of obesity is insulin resistance which underlies the pathogenesis of the metabolic
syndrome. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome. It comprises
a disease spectrum ranging from simple steatosis (fatty liver), through nonalcoholic steatohepatitis (NASH) to fibrosis, and
ultimately liver cirrhosis. Abnormality in lipid and lipoprotein metabolism accompanied by chronic inflammation is the central
pathway for the development of metabolic syndrome-related diseases, such as atherosclerosis, cardiovascular disease (CVD), and
NAFLD. This paper focuses on pathogenic aspect of lipid and lipoprotein metabolism in NAFLD and the relevant mouse models
of this complex multifactorial disease.
1. Introduction
Nonalcoholic fatty liver disease (NAFLD) is progressively
diagnosed worldwide and is considered to be the most
common liver disorder in Western countries, estimated to
affect at least one-quarter of the general population [1,2].
NAFLD used to be almost exclusively a disease of adults
but is now becoming a significant health issue also in
obese children. The prevalence of childhood obesity has
significantly increased over the past three decades [3,4]and
boosted the prevalence of NAFLD in adolescents (reviewed
in [5]).
NAFLD covers a spectrum of hepatic pathologies, rang-
ing from simple steatosis to nonalcoholic steatohepatitis
(NASH). It strongly associates with obesity, insulin resis-
tance, hypertension, and dyslipidaemia and is now regarded
as the liver manifestation of metabolic syndrome [6].
Simple steatosis is largely benign and nonprogressive whereas
NASH is characterized by hepatocyte injury, inflammation,
and fibrosis and can lead to cirrhosis, liver failure, and
hepatocellular carcinoma [7].
Lipid accumulation in the liver is the major hallmark of
NAFLD. A comprehensive understanding of the mechanisms
leading to liver steatosis and further transition to nonalco-
holic steatohepatitis (NASH) still remains elusive. There is
no simple solution to understand the multi-factorial nature
of NAFLD appearance and progression, presumably due to
the nonlinear interactions of those factors. Abnormalities in
lipid and lipoprotein metabolism accompanied by chronic
inflammation are considered to be the central pathway for
the development of several obesity-related co-morbidities
such as NAFLD and cardio-vascular disease (CVD) [8,9].
NAFLD is not an innocent bystander in the metabolic
syndrome. Rather, it represents an important self-governing
risk for the development of CVD. In NAFLD patients, liver
overproduces several atherogenic factors, such as cytokines
and “bad” lipoproteins. In this manner, fatty liver is
associated with increased serum low-density lipoproteins
(LDL) and triglycerides, combined with decreased high-
density lipoproteins (HDL) that represent a threat for CVD
development (reviewed in [10]). There are many evidences
suggesting that NAFLD is linked to the increased incidence
of CVD, both in nondiabetic and type 2 diabetic patients
[11]. Furthermore, a prospective observational study of
Hamaguchi et al. implied that NAFLD may play a central
role in the cardiovascular risk of metabolic syndrome [12].

2Journal of Lipids
NAFLD is also very common in type 1 diabetes and
is strongly associated with increased prevalence of CVD
independent of other confounding factors [13]. In addition
to the liver-related causes, CVD represents the major survival
risk of patients with NASH [14]. However, the nature of the
relationship NAFLD/CVD is still under debate. McKimmie
and coauthors [15] did not find independent association
between hepatic steatosis and CVD in a subset of participants
in Diabetes Heart Study. They suggested that hepatic steatosis
is more a secondary phenomenon than a direct mediator
of CVD. Even so, sufficient evidence exists that CVD risk
assessment seems mandatory in NAFLD patients.
NAFLD pathogenesis as a two-hit model was initially
proposed by Day and James [16]. First, insulin resistance
causes lipid accumulation in hepatocytes; second, cellular
insults such as oxidative stress, lipid oxidation, and inflam-
mation result in NASH. Deregulation of fat metabolism in
the fatty liver is accompanied by overproduction of very-low-
density lipoproteins (VLDL), the characteristic lipoproteins
of the metabolic syndrome [17]. LDL has recently attracted
attention since small, dense LDL is the most atherogenic
subclass of LDL, and this subclass is elevated in metabolic
syndrome and fatty liver. However, elevated VLDL is likely
the key metabolic disturbance and correlates strongly with
obesity and metabolic syndrome. Fatty liver-associated dys-
lipidemic profile characterized by large VLDL, small dense
LDL, and decreased large HDL correlates with the intrahep-
atic lipid content. Herein, we review recent understanding
of lipid and lipoprotein homeostasis in the development of
NAFLD and the relevant polygenic mouse mode that help
in unraveling the pathogenesis of this disease. The studies
included in this review paper (31 clinical and 82 experi-
mental studies) were selected based on the involvement in
lipid and lipoprotein metabolism and reported association
with NAFLD and insulin resistance. Major pathways of lipid
and lipoprotein homeostasis relevant for the development of
NAFLD are depicted in Figure 1.Figure 2 summarizes the
association between insulin resistance-induced lipid abnor-
malities and pathogenesis of NAFLD. Tabl e 1 recapitulates
the physiologic role of all receptors and enzymes described
in the paper and the association with insulin resistance and
the pathogenesis of NAFLD. Finally, we briefly discuss how
reviewed pathways are involved in the therapeutic strategies
for NAFLD.
2. Lipoprotein Metabolism in NAFLD
Lipid transport in plasma utilizes highly specialized lipopro-
tein complexes. After a meal, dietary fat and cholesterol are
absorbed into intestinal cells and incorporated in nascent
chylomicrons. The liver is another important source of
lipoproteins. A central metabolic role of the liver is to
maintain plasma glucose within narrow physiological limits
regardless of the nutritional state of the animal. In energy
excess, glucose is converted to fatty acids, which are further
used to synthesize triglycerides. Triglycerides can be stored
as lipid droplets within hepatocytes or incorporated into
very-low-density lipoproteins (VLDL) and secreted into the
blood. Once in the blood, triglyceride content of these
particles is progressively reduced by the action of lipoprotein
lipase (LPL), eventually resulting in intermediate-density
lipoproteins (IDLs) and low-density lipoproteins (LDL) with
relatively high cholesterol content [69]. LDL circulates and
is absorbed by the liver by binding of LDL to LDL receptor
[70].
Patients with insulin resistance increase VLDL secretion
as they attempt to maintain hepatic lipid homeostasis.
Therefore, insulin resistance is associated with abnormal
concentration of lipoproteins [71,72], elevated VLDL pro-
duction, and increase in plasma LDL [73]. Elevated plasma
LDL was also found in patients with NAFLD [5,74].
Association of fatty liver and small dense LDL (sdLDL)
concentration is now well documented [75,76]. As the
triglyceride-rich VLDL is entering plasma at an accelerated
rate, small, dense LDL, the most atherogenic subclass of LDL,
develop after triglycerides are gradually removed from LDL.
Two enzymes are implicated in this process. First, cholesteryl
estertransferprotein(CETP)[18] facilitates the transfer
of triglycerides from VLDL to LDL (and cholesteryl esters
from LDL to VLDL), and, second, hepatic lipase increases
lipolysis of triglyceride-rich LDL resulting in the formation
of sdLDL [21].Thus,CETPremodelsVLDLincirculation,
enriches it in cholesterol, and also favors, together with
HL, the formation of sdLDL. CETP activity is increased
in hepatic steatosis patients [19]. LDL receptor shows a
lower affinity for smaller particles, therefore such particles
stay longer in the circulation [77]. Hyperlipidemia can be
further exacerbated by low activity of lipoprotein lipase, or
by high level of apolipoprotein C-3 (APOC-3), an inhibitor
of lipoprotein lipase. Indeed, APOC-3 polymorphisms have
been associated with fatty liver in humans [20]. However, this
association was not found in recent Dallas Heart Study [22].
In contrast to simple steatosis, the steatohepatitis links
to dysfunctional VLDL synthesis and secretion [78]. Fujita
et al. [78] investigated the difference in lipid metabolite and
serum lipoprotein levels between patients with steatosis or
NASH. Hepatic lipid profiles in the two patient groups were
similar; however, VLDL synthesis and export were impaired
in NASH. This is in line with the two main experimental
animal models that are often used to study NAFLD [79]. A
high-fat/high-calorie diet is a generalized fatty liver model
[79]. A choline-deficient/1-amino acid-defined (CDAA) diet,
which disturbs VLDL secretion is an NASH model [80]. The
former involves only fatty liver whereas the latter includes
also steatohepatitis and liver cirrhosis [81].
The blockade of hepatic VLDL secretion results in accu-
mulation of triglycerides in the liver. Microsomal triglyceride
transfer protein (MTTP) is essential for the formation of
VLDL in the liver [24]. Mice that cannot secrete VLDL
due to the conditional knockout of Mttp in the liver
exhibit markedly reduced levels of triglycerides in the plasma
and develop hepatic steatosis [82,83], however, without
insulin resistance and inflammation [84]. In line with the
rodent data, human MTTP polymorphisms lead to decreased
MTTP activity and VLDL export and are associated with
greater intracellular triglyceride accumulation. Altogether,
this impacts NASH pathogenesis [25], possibly through

Journal of Lipids 3
Tab le 1: The physiologic role of all receptors and enzymes described in the paper and their association with insulin resistance and pathogenesis of NAFLD.
Gene
symbol Name Physiological role Insulin
resistance
Deregulation in
NAFLD
Loss-of-
function
models
and
NAFLD
Polymorphisms
associated with
NAFLD
Reference
CETP Cholesteryl ester transfer protein Facilitates the transfer of TG from VLDL
to LDL and CE from LDL to VLDL Increased Increased + [18–20]
HL Hepatic lipase Facilitates the clearance of TG from
VLDL, increases formation of sdLDL Increased [21]
LPL Lipoprotein lipase Decreased Decreased [20]
APOC-3 Apolipoprotein C-3 Inhibitor of lipoprotein lipase Increased Increased [20,22]
LDLR LDL receptor LDL intake Decreased [23]
MTTP Microsomal triglyceride transfer protein Formation of VLDL in the liver Decreased Develop
NAFLD +[24–26]
SORT 1 Sortilin 1 Intracellular sorting receptors for
apolipoprotein B100 lipoproteins +[27–29]
PCSK9 Proprotein convertase subtilisin/kexin type 9 Enhances LDLR degradation Resistance [30,31]
HSL Hormone-sensitive lipase Adipose tissue lipolysis Increased Increased Resistance [32,33]
CD-36 Fatty acid translocase; scavenger receptors FA uptake; oxidized LDL uptake Increased Increased Protective [34–40]
FASN Fatty acid synthase Catalyzes the last step in fatty acid
biosynthesis Increased Protective [41]
ACC Acetyl coenzyme A (acetyl-CoA) carboxylase Fatty acid biosynthesis Protective [42,43]
SCD1 Stearoyl-CoA desaturase Fatty acid desaturation Protective [44,45]
ELOVL6 Elongation of long-chain fatty acids Fatty acid elongation Protective [46]
FADS1
FADS2 Δ-5 and Δ-6 desaturase PUFA desaturation Decreased [47,48]
SREBP-
1c Sterol regulatory element binding protein 1c Increased Increased Protective [49,50]
ChREBP Carbohydrate regulatory element binding
protein Increased Increased Protective [51]
SIK2 Serine/threonine salt-inducible kinase 2 Increases ChREBP Protective [52]
CPT-1 Carnitine palmitoyl transferase-1 Shuttles fatty acids into mitochondria,
fatty acid oxidation Decreased [53,54]
DGAT2 Diacylglycerol acyltransferase 2 Triglyceride biosynthesis Increased Increased/decreased
in NASH
Improves
steatosis,
aggravates
NASH
[55–57]
ATGL Adipose triacylglycerol lipase Performs the first step in TAG lysis Decreased leads to
steatosis [58,59]
CGI-58 Gene identification-58 ATGL coactivator leads to
steatosis [60]

4Journal of Lipids
Tab le 1: Continued.
Gene
symbol Name Physiological role Insulin
resistance
Deregulation in
NAFLD
Loss-of-
function
models
and
NAFLD
Polymorphisms
associated with
NAFLD
Reference
SREBP-2 Sterol regulatory element binding protein 1c Induces cholesterol synthesis Decreased [23,61]
LXR Liver X receptor Induces cholesterol secretion and FA
synthesis, induces CD-36 and Idol Increased Increased [62–64]
Idol Inducible degrader of the LDLR Idol catalyzes the ubiquitination of LDLR
and targets it for degradation Increased Increased [65]
ACAT2 Acyl-Coenzyme A: cholesterol acyltransferase
Cholesterol esterification; provides
esterified cholesterol for incorporation
into VLDL and storage
Protection [66]
NPC1L1 Niemann-Pick C1-like 1 Cholesterol absorption Protection [67,68]

Journal of Lipids 5
Acetyl-CoA
Acetyl-CoA
Cholesterol
Bile
acids
Glucose
Pyruvate
Insulin
SREBP-1c SREBP-2
Glucose
ChREBP
LXR
FFA
FFA
CE
VLDL
VLDL
VLDL
ACC
FASN
SCD1
ELOVL6
DGAT2
sdL LD
sdL LD
sdLDL
MTTP
oxLDL
oxLDL
LPL IDL IDL
APOC-3
Acetyl-CoA
∗
L-PK
ACAT
CD-36
Kreb,s
cycle∗
Fatty acids
Triglycerides
CETP
CETP
CE
TG
LDL
LDL
Blood
Liver
IDOL
PCSK9
LDLR
HL
Figure 1: Lipid and lipoprotein pathways in the pathogenesis of NAFLD. NAFLD is considered to be liver manifestation of obesity and
metabolic syndrome. In response to insulin and glucose, transcription factors SREBPs and ChREBP are activated and induce the expression
of genes involved in the synthesis of fatty acids and cholesterol in the liver. Enhanced lipogenesis leads to enhanced VLDL production, a
major metabolic perturbation in NAFLD. Increased VLDL secretion in plasma results in increase in LDL through CETP-mediated exchange
of cholesteryl esters and triglycerides between LDL and VLDL, followed by triglyceride removal from LDL by hepatic lipase (HL). Liver
removes LDL from circulation by LDLR-mediated endocytosis. Oxidized LDL and FFA are transported to the liver by CD-36, FA translocase,
and scavenger receptor. Italics: metabolic genes, black lines: metabolic pathways, black dash lines: coming out or in the liver, white lines:
transcriptional regulation, ∗process in the mitochondria.
modulating postprandial lipid profile [26]. On the other
hand, a high-fat diet was shown to induce the methylation
of MTTP and consequently reduce its mRNA level [85]. The
postprandial phase has been linked to increased oxidative
stress [86], and increased lipid peroxidation is implicated
in NASH pathogenesis. Oxidized LDL can activate hepatic
stellate cells that are crucial in NASH pathogenesis [87].
Sortilins, intracellular sorting receptors for apolipopro-
tein B 100 (APO-B 100), are new players in lipoprotein
metabolism. Genome-wide association studies (GWAS) of
common genetic variations associated sortilin 1 (Sort 1)gene
with LDL metabolism. Several findings provide evidences
that sortilin 1 is involved in the hepatic metabolism of
lipoproteins containing APO-B, although the precise mech-
anism waits for further elucidation. Kjolby et al. showed that
Sort 1 interacts with APO-B 100 in the Golgi and facilitates
the formation and hepatic export of VLDL lipoproteins,
thereby regulating plasma LDL concentration. They showed
that Sort 1 overexpression stimulated hepatic release of
lipoproteins and increased plasma LDL [27]. Using different
mouse models, Musunuru et al. [28] showed an inverse
relationship between Sort1 1 expression and circulating LDL
cholesterol. Namely, liver-specific overexpression of Sort 1 in
mice decreased serum LDL cholesterol whereas knockdown
had opposite effect. Moreover, Linsel-Nitschke showed that
Sort 1 enhanced LDL endocytosis [29]. Further studies are
needed to understand the mechanistic link between sortilin
1 and lipoprotein metabolism.
Plasma levels of LDL, the major cholesterol carrying
lipoprotein in humans, are determined by the relative
rates of production and clearance by LDL receptor. The
groundbreaking work of Goldstein and Brown defined
the pathway of LDL receptor-mediated endocytosis and
its regulation by cholesterol-dependent negative feedback

6Journal of Lipids
Cholesterol
synthesis
FA synthesis
FA uptake
FA oxidation
lipolysis
ADIPOSE
FA accumulation
TG accumulation
TG synthesis
VLDL secretion
Cholesterol
accumulation
Inflammation
NASH
TG synthesis
VLDL secretion
FFA and cholesterol
accumulation
Lipid peroxidation
Insulin resistance
Liver
Fatty liver
Figure 2: Summary of insulin resistance-induced lipid abnor-
malities and consequent pathogenesis of NAFLD. In an insulin
resistant setting, insulin is unable to inhibit lipolysis in adipose
tissue leading to overflow of free FA into the bloodstream and
in the liver. In the liver, hyperinsulinemia and hyperglycemia
induce the synthesis of fatty acid and cholesterol which results in
increased triglyceride synthesis and VLDL assembly and secretion.
Since triglyceride synthesis prevails over VLDL secretion, excess
triglycerides accumulate and lead to fatty liver development. In
NASH, triglyceride synthesis and VLDL assembly is impaired and
free FA and cholesterol accumulate. Increased lipid accumulation
leads to lipid peroxidation and inflammation which exacerbates
liver damage.
transcriptional regulation (reviewed in [88]). In addition
to transcriptional regulation, LDLR is also regulated on
the protein level. PCSK9 (proprotein convertase subtilisin/
kexin type 9) enhances LDLR degradation, resulting in
low-density lipoprotein accumulation in plasma. PCSK9
binds the EGF-A domain of the low-density lipoprotein
receptor (LDLR) and favors the targeting of the LDLR
to endosomes/lysosomes and its degradation (reviewed in
[30]). Individuals with loss-of-function mutations in PCSK9
have reduced plasma levels of LDL cholesterol and are
protected from CHD [89]. Inhibiting the action of PCSK9
on the LDLR has emerged as a novel therapeutic target
for hypercholesterolemia. It was shown recently that PCSK9
deficiency confers resistance to liver steatosis; however, this
effect seemed to be LDLR independent [31].
3. Cholesterol and Triglyceride
Homeostasis in NAFLD
Abnormal lipoprotein concentration in plasma reflects dis-
turbances in homeostasis of major lipid components of
lipoproteins, triglycerides, cholesterol, and cholesterol esters.
Excessive accumulation of triglycerides in the liver is the hall-
mark of NAFLD. The potential sources of fat contributing
to hepatic steatosis are dietary fatty acids through uptake
of intestine-borne chylomicron remnants, increased lipolysis
of peripheral fat store, and de novo synthesis. Tracer studies
in obese humans with NASH demonstrated that 60% of
triglycerides in the liver arose from free fatty acids, 25% from
de novo lipogenesis, and 15% from the diet [90].
3.1. Fatty Acids and Triglycerides. Insulin resistance is asso-
ciated with deregulation of adipose-derived fatty acid flux
in the fasting state [91]. In NAFLD patients, insulin does
not suppress adipose tissue lipolysis to the same extent that
it does in healthy individuals [32]. Studies on mice have
revealed that hormone-sensitive lipase- (HSL-) knockout
mice show increased insulin sensitivity with decreased
hepatic triglyceride content [33]. Fatty acid uptake was
believed to be predominantly passive; however, this concept
has been challenged by the discovery of cluster differentiation
protein-36 (CD-36), fatty acid translocase [34], and its
association with NAFLD. CD-36 is regulated by insulin and
can induce hepatosis [92]. In NASH, development of disease
is associated with increased expression of CD-36 [35–37].
Among major causes of fat accumulation in NAFLD is
the inability of the liver to regulate the changes in lipogenesis
in the transition from fasted to fed state. Several studies
suggested increased hepatic lipogenesis in hepatic steatosis.
Increased lipogenesis may have dual effect: increased triglyc-
eride synthesis and decreased fatty acid oxidation through
production of malonyl-CoA [93], both leading to increased
triglycerides content in fatty liver. Sanyal et al. reported
that β-oxidation of fatty acids in the liver was increased in
patients with NASH [94]. However, this increase might not
sufficiently overcome the elevated rates of hepatic fatty acid
synthesis.
De novo fatty acid synthesis is the metabolic pathway
converting excess carbohydrates into fatty acids, which are
ultimately esterified to form triglycerides. Several indications
show an increase in fatty acid synthesis in NAFLD. Humans

Journal of Lipids 7
[95]andmice[96] with fatty liver accumulate oleic acid, the
end product of fatty acid synthesis, indicating increase in this
pathway. Fatty acid synthase (FASN) catalyzes the last step in
fatty acid biosynthesis, and thus, it is believed to be a major
determinant of the maximal hepatic capacity to generate
fatty acids by de novo lipogenesis. The expression of FASN
mRNA in human liver is higher in hepatic steatosis [41].
Further on, knocking down several genes of the fatty acid
synthesis, desaturation, and elongation, such as Acc,Scd1,
and Elovl6, reversed several metabolic defects associated with
hepatic steatosis in experimental animals [42–46]. NAFLD is
also associated with depletion of n-3 polyunsaturated fatty
acids (PUFA), a consequence of decreased liver Δ-6 and Δ-5
desaturase in obese NAFLD patients ([47], reviewed in [48]).
The mouse strain C57BL/6 is one of the most frequent
laboratory strains for studies of high lipid-related metabolic
disorders. The strain has a long life period and a high pre-
disposition for development of induced hyperlipidemias.
Hepatic lipid metabolic processes are circadian in this strain
[97]. An 8-week hyperlipidemic diet (1.25% cholesterol,
0.5% cholic acid, 15% fat) leads to fatty liver [98,99], and
a 5-week atherogenic diet to NASH [100]. This mouse strain
has been successfully applied also in studies of cholesterol-
lowering effects of atorvastatin [101].
B¨
ubger et al. developed a unique polygenic model of
NAFLD—so-called “fat” mice [102]. Genetic studies have
confirmed a typical polygenic basis of this obesity [103–
105]. In contrast to many other animal models of obesity
and NAFLD, this line develops NAFLD already on a low-
to-moderate food intake, and development of later stages of
NAFLD is accelerated on high-fat diet [106,107]. Plasma
and liver lipid profiles are perturbed similarly as in NAFLD
patients. Liver transcriptome exhibits dramatic changes with
perturbations in genes of de novo cholesterol synthesis,
bile and glucose metabolism, liver receptors, and immune
response [107]. This polygenic model is unique since it
allows studies of the genetic and molecular mechanisms of
both early hepatic fat accumulation and advanced forms of
NAFLD. Other mouse models with defects in cholesterol
synthesis might also represent useful tools for understanding
NAFLD [108].
Insulin and glucose both drive lipogenesis [109]by
respective transcription factors, SREBP-1c (sterol regulatory
element binding protein 1c), and ChREBP (carbohydrate
regulatory element binding protein). Hyperinsulinemia
despite insulin resistance induces the expression of SREBP-
1c, a transcriptional activator of all lipogenic enzymes,
resulting in increased rate of fatty acid synthesis [49,50].
Overexpression of Srebp-1c in transgenic mouse livers leads
to classic fatty liver due to increased lipogenesis [110].
In contrast, inactivation of Srebp-1 gene in ob/ob mice
results in 50% reduction of triglycerides in these mice [111].
SREBP-1c also activates acetyl-coA carboxylase 2 (Acc2)
[53] that produces malonyl-CoA. Increase in malonyl-CoA
results in decreased fatty acid oxidation due to inhibition
of carnitine palmitoyl transferase-1 (Cpt-1), which shuttles
fatty acids into mitochondria [54]. Indeed, Acc2 knockout
mice are resistant to obesity with increased Cpt-1 activity and
consequent fatty acid oxidation [112].
In addition to insulin, glucose activates lipogenesis
through transcriptional factor ChREBP. ChREBP simulta-
neously activates liver-type pyruvate kinase (L-PK), key
regulator of glycolysis and all lipogenic genes [113,114],
including acetyl-CoA carboxylase and fatty acid synthase
[115]. ChREBP gene expression and nuclear protein content
are significantly increased in liver of ob/ob mice. Liver-
specific inhibition of ChREBP improves hepatic steatosis
and insulin resistance in ob/ob mice [51]. Bricambert
et al. [52] went on and showed upstream regulation of
ChREBP. Serine/threonine salt-inducible kinase 2 (SIK2)
directly regulates hepatic lipogenesis through the inhibition
of p300 acetylation of ChREBP, which in turn increases
ChREBP-induced transcription. Inhibition of hepatic p300
thus offers a novel target for treating hepatic steatosis.
Triglyceride accumulation in hepatocytes was considered
to be the major pathogenic trigger in the development of
NAFLD. Diacylglycerol acyltransferase 2 (DGAT2) catalyzes
the final step in hepatocyte triglyceride biosynthesis. Sup-
pression of DGAT2 reversed diet-induced hepatic steatosis
and insulin resistance [55] as well as attenuates hyperlipi-
demia [56]. However, recent findings suggest that triglyceride
synthesis per se may not be harmful to hepatocytes. Rather,
it provides a useful mechanism for buffering free fatty
acid accumulation [57]. Yamaguchi et al. [57] show that
inhibiting triglyceride synthesis by inhibiting DAGT2 does
improve hepatic steatosis, yet it exacerbates liver damage
and fibrosis in obese mice with nonalcoholic steatohepatitis.
Lipotoxicity arises when hepatic triglyceride synthesis is
unable to accommodate increased free fatty acid accumula-
tion. Thus, rather than being hepato-toxic, liver triglyceride
accumulation is actually hepatoprotective in obese, insulin-
resistant individuals.
Hepatic fatty acids are derived from several sources,
including adipose tissue lipolysis, chylomicron-TAG lipol-
ysis, and de novo lipogenesis, and can be stored as TAG
in lipid droplets located within the cytosol [116]. Hepatic
TAG stores are mobilized by several hepatic lipases. Adipose
triacylglycerol lipase (Atgl) that selectively performs the
first step in TAG in the liver is reduced in several rodent
models of obesity, and Atgl ablation leads to steatosis,
although increased TAG content in the hepatocytes from
Atgl-deficient mice does not enhance insulin sensitivity [58,
59]. Similarly, inhibiting expression of ATGL coactivator
(gene identification-58 (Cgi-58)) resulted in a large increase
in hepatic TAG content, yet in a decrease in insulin resistance
[60]. Further, overexpressing ATGL specifically in the liver
of obese mice did decrease liver steatosis, but it only mildly
enhanced liver insulin sensitivity [58], suggesting that ATGL
might be a pharmacological therapeutic target for NAFLD
but not type 2 diabetes.
3.2. Cholesterol and Cholesteryl Esters. Cholesterol is either
synthesized de novo in the liver or delivered to the liver
by lipoproteins (reviewed in [117]). The metabolism of
cholesterol in NAFLD remains poorly explored. Insulin
resistance is associated with increased cholesterol synthesis
[118,119]. Recent metabolomic analysis implicated that
cholesterol synthesis in NAFLD patients is increased, in

8Journal of Lipids
contrast to diminished absorption of cholesterol [120]. The
expression of cholestrogenic genes was also found elevated
in NAFLD patients, accompanied by decreased SREBP-2
and LDLR expression [23]. In rodents, excess cholesterol
intake contributes to the development of NAFLD even in the
absence of obesity [121,122].
Cholesterol rich-atherogenic diet induces oxidative stress
and provokes inflammation. The transition towards hepatic
inflammation is the key factor in NASH pathogenesis and
promotes progression to liver damage. Currently, NASH is
thought to develop via the “two hit” model [16]. According
to this hypothesis, hepatic steatosis represents “first hit”
and is still reversible. The “second hit” includes NASH
progression beyond hepatic steatosis that promotes oxidative
stress, inflammation, cell death, and fibrosis [93]. Mar´
ıetal.
[123] provided evidence that mitochondrial loading of free
cholesterol, but not free fatty acids or triglycerides, sensitizes
the liver toTNF-α-induced steatohepatitis. In line with these
results, Wouters et al. [124] found that dietary cholesterol
rather than lipid accumulation is an important risk factor for
the progression to hepatic inflammation. High-cholesterol
diet led to increased VLDL and was sufficient to cause
inflammatory response in the liver. As mentioned earlier,
increased VLDL and accompanied hypertriglyceridemia
underlie the synthesis of small, dense LDL with lower affinity
for LDL receptor. Therefore, these particles stay in circulation
for longer period and are prone to oxidation [77]. Oxidized
LDL can bind scavenger receptors, such as CD-36 [38–40],
which are present on Kupffer cells and prompt inflammatory
response.
Inflammatory cytokine TNF-αis overexpressed in the
liver of obese mice and mediates insulin resistance [125,126].
Furthermore, TNF-αis required for the development of fatty
liver and subsequent liver damage by alcohol [127,128].
We [129,130] and others have showed that TNF-αactivates
cholesterol synthesis and inhibits cholesterol elimination
through bile acids, which together contribute to increase in
LDL-cholesterol and reduction of HDL-cholesterol.
Cholesterol is indispensible, however, toxic in excess.
Intracellular level of cholesterol is tightly regulated by
a number of mechanisms that govern uptake, synthesis,
catabolism, and export. Two master regulators of these
pathways are the transcription factors SREBP-2 and LXR
(liver X receptor). When intracellular cholesterol levels drop,
SREBP-2 induces cholesterol biosynthesis and uptake [61].
In contrast, excess intracellular cholesterol inhibits SREBP-2
and activates LXR, which in turn promotes cholesterol export
and elimination [62].
LXRs are nuclear receptors that control lipid metabolism.
Nuclear receptors may have a crucial role in lipid-related
genesis of NAFLD [131,132]. LXRs were discovered as sterol
sensors that regulate cholesterol homeostasis [63,64]. In
rodents, LXR promote cellular cholesterol efflux, transport,
and excretion [133]. LXRs have emerged as promising drug
targets for antiatherosclerotic therapies. However, pharma-
cological LXR activation also induces hepatic steatosis and
promotes the secretion of VLDL particles by the liver, com-
plicating the clinical application of LXR agonists. Namely,
LXR activates SREBP-1c, a master transcriptional regulator
of fatty acid synthesis [134,135]. Moreover, LXR has a central
role in insulin-mediated activation of SREBP-1c-induced
fatty acid synthesis in liver [136]. In addition, LXR can also
promote lipogenesis in an SREBP-1c-independent manner
[137,138] and activate other lipogenic transcriptional
factor ChREBP [139]. Further on, LXR was also shown to
induce the expression of CD-36, fatty acid transporter, and
scavenger receptor, suggesting another mechanism by which
LXR can promote fatty liver [92]. LXR also exerts negative
control of LDLR-mediated cholesterol uptake by inducing
the expression of Idol (inducible degrader of the LDLR).
Idol catalyzes the ubiquitination of LDLR and targets it for
degradation [65].
Intracellular free cholesterol is converted into choles-
teryl ester by acyl-Coenzyme A: cholesterol acyltransferase
(ACAT). The function of ACAT2 in the hepatocyte is to
provide esterified cholesterol for incorporation into very-
low-density lipoprotein (VLDL), as well as to provide
cholesteryl ester for cytoplasmic lipid droplets, a means for
storage when liver cholesterol is abundant. Increased VLDL
cholesteryl ester secretion occurs in livers of monkeys fed
dietary cholesterol (reviewed in [140]). Mice, genetically
engineered to lack Acat2 in both the intestine and the liver,
were dramatically protected against hepatic neutral lipid
(TG and cholesteryl ester) accumulation, in particular with
elevated cholesterol diet. Inhibition of hepatic Acat2 can
prevent dietary cholesterol-driven hepatic steatosis in mice
[66].
Recently, Niemann-Pick C1-like 1 (NPC1L1) has been
shown to play a pivotal role in cholesterol absorption
[67]. Unlike mouse NPC1L1 protein that is predominantly
expressed in the intestines, human and rat NPC1L1 is also
abundantly expressed in the liver. Loss of NPC1L1 expression
has been shown to protect against diet-induced fatty liver
[68].
4. Conclusion
Nonalcoholic fatty liver disease (NAFLD) is a multi-factorial
disorder with contribution of a variety of genetic and
environmental factors that up till now no effective treatments
exist for them. By poorly defined mechanisms, including
free fatty acid and cholesterol accumulation accompanied
by oxidative stress and inflammation, NAFLD may progress
to the irreversible steatohepatitis (NASH) and further to
cirrhosis or hepatocellular carcinoma. Herein, we review
the major advances in the understanding of the pathogenic
aspect of lipid and lipoprotein metabolism in NAFLD that
could affect future therapeutic strategies. Currently, the only
established treatment is weight loss since obesity underlies
insulin resistance and NAFLD (reviewed in [141]). Caloric
restriction reverses hepatic insulin resistance and steatosis
in rats [142]. Fasting inhibits cholesterol and fatty acid
synthesis and has protective effect on lipid metabolism
[129]. Further, two different obesity-treatment drugs are
currently on the market: orlistat, which reduces intestinal
fat absorption, and sibutramine, an appetite suppressant
(reviewed in [141]).

Journal of Lipids 9
NAFLD and cardiovascular disease share common risk
factors, in particular disturbed lipid homeostasis accom-
panied by dyslipidemia. In patients with NAFLD, inhibi-
tion of cholesterol synthesis by statins alone [143]orin
combination with antioxidants was shown beneficial [144].
However, the statin therapy shares common risks of drug
failure in some individuals that can develop hepatoxicity or
drug interactions [145]. Further on, inhibition of SREBP
pathway represents another potential treatment for NAFLD.
Inhibition of SREBP by a small molecule, betulin, decreased
the biosynthesis of cholesterol and fatty acid [146]. Recent
reports suggest a potential benefit of inhibiting intestinal
cholesterol absorption by ezetimibe NPC1L1-inhibitor [147,
148]. Fenofibrates were also shown to decrease cholesterol
absorption at the level of intestinal NPC1L1 expression
[149].
Due to the complex multi-factorial nature of the disease,
combined treatment may be needed to achieve better
results. Indeed, dual inhibition of cholesterol absorption
and synthesis and coadministration of ezetimibe/simvastatin
offer a highly efficacious lipid-lowering strategy [150]and
were shown to be effective and safe also in NAFLD patients
[151].
In the complex pathology of NAFLD/NASH, the inte-
grative approaches focusing on networks rather than on
individual molecules, and applying environmental perturba-
tions (diet, drugs, rhythm, etc.) in suitable animal models,
can represent new venues towards predictive markers and
successful therapies.
Abbreviations
ACAT: Acyl-coenzyme A cholesterol acyltransferase
ACC: Acetyl-CoA carboxylase
CETP: Cholesteryl ester transfer protein
CD-36: Cluster differentiation protein-36
ChREBP: Carbohydrate regulatory element binding
protein
DGAT2: Diacylglycerol acyltransferase 2
ELOVL6: Family member 6, elongation of long-chain
fatty acids
FASN: Fatty acid synthase
HL: Hepatic lipase
HSL: Hormone-sensitive lipase
IDL: Intermediate-density lipoprotein
Idol: Inducible degrader of the LDLR
LPL: Lipoprotein lipase
LDL: Low-density lipoprotein
LDLR: LDL receptor
L-PK: Liver-type pyruvate kinase
LXR: Liver X receptor
MTTP: Microsomal triglyceride transfer protein
NAFLD: Non-alcoholic fatty liver disease
NASH: Non-alcoholic steatohepatitis
PCSK9: Proprotein convertase subtilisin/kexin type 9
SCD1: Stearoyl-CoA desaturase
SREBP: Sterol regulatory element binding protein
TNF-α: Tumor necrosis factor α
VLDL: Very-low-density lipoprotein.
Acknowledgment
This paper was supported by the Slovenian Research Agency
Program Grant P1-0104.
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