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Nonalcoholic Fatty Liver Disease: Focus on Lipoprotein and Lipid Deregulation


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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.
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Hindawi Publishing Corporation
Journal of Lipids
Volume 2011, Article ID 783976, 14 pages
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,
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
aect 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, sucient 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
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 anity 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 dierence 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.
symbol Name Physiological role Insulin
Deregulation in
associated with
CETP Cholesteryl ester transfer protein Facilitates the transfer of TG from VLDL
to LDL and CE from LDL to VLDL Increased Increased + [1820]
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 +[2426]
SORT 1 Sortilin 1 Intracellular sorting receptors for
apolipoprotein B100 lipoproteins +[2729]
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 [3440]
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]
FADS2 Δ-5 and Δ-6 desaturase PUFA desaturation Decreased [47,48]
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
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.
symbol Name Physiological role Insulin
Deregulation in
associated with
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 [6264]
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
sdL LD
sdL LD
Fatty acids
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 dierent
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 eect. 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
FA synthesis
FA uptake
FA oxidation
FA accumulation
TG accumulation
TG synthesis
VLDL secretion
TG synthesis
VLDL secretion
FFA and cholesterol
Lipid peroxidation
Insulin resistance
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
eect 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 dierentiation
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 [3537].
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 eect: 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
suciently overcome the elevated rates of hepatic fatty acid
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 [4246]. 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 eects of atorvastatin [101].
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 oers 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 buering 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´
[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 sucient to cause
inflammatory response in the liver. As mentioned earlier,
increased VLDL and accompanied hypertriglyceridemia
underlie the synthesis of small, dense LDL with lower anity
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 [3840],
which are present on Kuper cells and prompt inflammatory
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 eux, 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
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
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 eective 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 aect 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 eect on lipid metabolism
[129]. Further, two dierent 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
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
oer a highly ecacious lipid-lowering strategy [150]and
were shown to be eective and safe also in NAFLD patients
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.
ACAT: Acyl-coenzyme A cholesterol acyltransferase
ACC: Acetyl-CoA carboxylase
CETP: Cholesteryl ester transfer protein
CD-36: Cluster dierentiation protein-36
ChREBP: Carbohydrate regulatory element binding
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.
This paper was supported by the Slovenian Research Agency
Program Grant P1-0104.
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... Hepatosteatosis, also known as fatty liver disease, is characterized by excessive fat accumulation in the liver. If unchecked, this condition can evolve into serious liver diseases, including, fibrosis, cirrhosis, and even cancer [16][17][18]. Given that these pathological conditions often manifest without noticeable symptoms over long periods, preventive strategies, including dietary intervention with effective nutraceutical agents, become particularly meaningful [15,19]. ...
Full-text available
Rice bran, a by-product of rice milling, is abundant in bioactive molecules and is highly recognized for its health-promoting properties, particularly in improving metabolic conditions. Building on this knowledge, we aimed to optimize the extraction conditions to maximize the functional efficacy of rice bran extract (RBE) and further validate its impact on lipid metabolism. We found that the optimized RBE (ORBE) significantly suppressed high-fat diet-induced weight gain, hyperlipidemia, and hepatosteatosis in mouse models. ORBE treatment not only suppressed lipid uptake in vivo, but also reduced lipid accumulation in HepG2 cells. Importantly, we discovered that ORBE administration resulted in activation of AMPK and inhibition of STAT3, which are both crucial players in lipid metabolism in the liver. Collectively, ORBE potentially offers promise as a dietary intervention strategy against hyperlipidemia and hepatosteatosis. This study underlines the value of optimized extraction conditions in enhancing the functional efficacy of rice bran.
... NASH is the inflammatory subtype of NAFLD and may lead to liver cirrhosis if not treated properly [57]. It is caused by complex interactions between metabolic and stress pathways in hepatocytes [58], triggered by chronically elevated lipid levels [59], and inflammatory processes mediated by multiple immune cell populations [60], collectively resulting in the histological appearance of active steatohepatitis. Despite being studied for decades, no treatment for NASH has been developed. ...
... Oxidative stress and inflammation are known to underlie the etiopathogenesis of NASH (40,41) and can be categorized as the second hit with regard to the two-hit hypothesis. pNaKtide decreased hepatic α1 carbonylation, p-Src, and downstream p-ERK expression to attenuate Na/K-ATPase-induced ROS amplification. ...
Full-text available
We have previously reported that the α1 subunit of sodium-potassium adenosine triphosphatase (Na/K-ATPase), acts as a receptor and an amplifier for reactive oxygen species, in addition to its distinct pumping function. On this background, we speculated that the blockade of Na/K-ATPase-induced ROS amplification with a specific peptide, pNaKtide, might attenuate the development of steatohepatitis. To test this hypothesis, pNaKtide was administered to a murine model of NASH: the C57Bl6 mouse fed a "western" diet containing high amounts of fat and fructose. The administration of pNaKtide reduced obesity as well as hepatic steatosis, inflammation and fibrosis. Of interest, we also noted a marked improvement in mitochondrial fatty acid oxidation, insulin sensitivity, dyslipidemia and aortic streaking in this mouse model. To further elucidate the effects of pNaKtide on atherosclerosis, similar studies were performed in ApoE knockout mice also exposed to the western diet. In these mice, pNaKtide not only improved steatohepatitis, dyslipidemia, and insulin sensitivity but also ameliorated significant aortic atherosclerosis. Collectively, this study demonstrates that the Na/K-ATPase/ROS amplification loop contributes significantly to the development and progression of steatohepatitis and atherosclerosis. Furthermore, this study presents a potential treatment, the pNaKtide, for the metabolic syndrome phenotype.
... The FA stored in the liver can be processed through several different pathways, including b-oxidation inside the mitochondria or peroxisomes, oxidation by the cytochrome P-450 enzymes in the smooth endoplasmic reticulum, or transformation into triglycerides (TG) that are either released as very low-density lipoproteins (VLDL) or stored in hepatocytes. The excessive FA accumulation leads to oxidative and endoplasmic reticulum stress, inflammation, inflammasome activation, and ultimately hepatocyte fibrosis and apoptosis [13]. ...
Full-text available
Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease, and is related to fatal and non-fatal liver, metabolic, and cardiovascular complications. Its non-invasive diagnosis and effective treatment remain an unmet clinical need. NAFLD is a heterogeneous disease that is most commonly present in the context of metabolic syndrome and obesity, but not uncommonly, may also be present without metabolic abnormalities and in subjects with normal body mass index. Therefore, a more specific pathophysiology-based subcategorization of fatty liver disease (FLD) is needed to better understand, diagnose, and treat patients with FLD. A precision medicine approach for FLD is expected to improve patient care, decrease long-term disease outcomes, and develop better-targeted, more effective treatments. We present herein a precision medicine approach for FLD based on our recently proposed subcategorization, which includes the metabolic-associated FLD (MAFLD) (i.e., obesity-associated FLD (OAFLD), sarcopenia-associated FLD (SAFLD, and lipodystrophy-associated FLD (LAFLD)), genetics-associated FLD (GAFLD), FLD of multiple/unknown causes (XAFLD), and combined causes of FLD (CAFLD) as well as advanced stage fibrotic FLD (FAFLD) and end-stage FLD (ESFLD) subcategories. These and other related advances, as a whole, are expected to enable not only improved patient care, quality of life, and long-term disease outcomes, but also a considerable reduction in healthcare system costs associated with FLD, along with more options for better-targeted, more effective treatments in the near future.
... More specifically, choline, a vitamin like water-soluble micronutrient and a lipotropic agent, plays a preventive role by enabling the fat utilization and transportation from the hepatic to the extrahepatic tissues (Biswas and Giri, 2015). It was reported that a diet with low choline content causes fatty liver syndrome due to low availability of carrier lipoproteins (Fon Tacer and Rozman, 2011), growth retardation and perosis in fast-growing broiler strains. To avoid the detrimental responses associated with choline deficiency, synthetic choline chloride (CCL) has been supplemented to the poultry diet to improve the growth performance and carcass quality (Gregg et al., 2022). ...
Full-text available
This study was conducted to evaluate the effect of polyherbal (phytogenic) formulation (PHF: containing Acacia nilotica and Curcuma longa) on performance parameters, liver histopathology and prevention of fatty liver in broilers. 700 day-old chicks were randomly distributed to seven groups (10 replicates / group; 10 birds each), namely positive control (T1) fed with basal diet + choline chloride (CCL) 60% (1000g), negative control (T2) fed with high energy (5% increment), low protein (24% reduction), high cholesterol (2% increment) diet, T3 (T2 + PHF; 1000g-full cycle), T4 (T2 + PHF; 2000g-full cycle), T5 (T2 + CCL 60% (1000g-full cycle)), T6 (T5 + PHF; 1000g-grower and finisher stage), T7 (T5 + PHF; 2000g-finisher stage). Average daily gain (ADG; g), average daily feed intake (ADFI; g) and feed conversion ratio (FCR) were calculated at 1-14 days, 15-28 days, 29-42 days, and 1-42 days. Serum triglycerides analysis, gross and histopathological observations of liver morphology were performed for the samples of control and experimental groups on day 42. The performance parameters; ADG, ADFI, FCR, and liveability were found to be improved in all the groups as compared to the negative control group. However, better performance was observed in PHF (2000g) top-up group (during the finisher stage) as compared to the negative control group. Serum triglyceride levels were increased non-significantly as compared to the negative control indicating that more fat is mobilized from liver to serum. In addition, PHF supplementation at 2000g during the finisher phase had restored the liver tissue architecture as well as improved the liver score when compared to the negative control group. It is concluded that PHF (2000g/ton) during the finisher stage can be used as a top-up to improve the performance parameters as well as to prevent the fatty liver condition in broiler chickens.
... Upon lipid uptake by hepatocytes, fatty acids undergo β-oxidation in mitochondria, peroxisomes, and microsomes, still with existing controversy regarding the rate of lipid oxidation (whether increased or decreased) in patients with NAFLD [51,53], and accumulation of triglyceride-rich lipoproteins in hepatocytes is increased [51,54]. Likewise, in patients with NAFLD increased production of small dense LDL-C was demonstrated [55]. ...
Full-text available
Increasing evidence implicates the enzyme Heparanase in the development and progression of liver steatosis and fibrosis, where high heparanase expression was demonstrated. Morever, inhibition of heparanase activity significantly attenuated the development of fatty liver in animal models. Non-alcoholic fatty liver disease is the most common liver disease in the western world, with the natural course of a chronic progressive condition that is expected to worsen with time. Potential complications of the disease are steatohepatitis, liver fibrosis, liver cirrhosis and even liver malignancies, such as hepato-cellular carcinoma. As such, non-alcoholic fatty liver disease is considered a leading etiology for liver transplantation in the western world. No effective treatment for fatty liver is available so far, and seeking effective treatment strategies is of great importance. The aim of this chapter is to shed light on the knowledge regarding the involvement of Heparanase in the development and progression of fatty liver, opening the opportunity for future research of potential therapeutic options for treating this common liver pathology.
... Generally, fatty acids entering the liver are either assembled into TGs or oxidized by mitochondria in hepatocytes. It is thought that aP2, Cebpα, Lep and LPL play key regulatory roles in fatty acid uptake, transport, and metabolism process (Fon Tacer and Rozman, 2011;Maradonna and Carnevali, 2018). The current study showed that TCC/TCS enhanced the expression of aP2, Lep and LPL, which might contribute to the increased fat deposit in hepatocytes induced by TCC/TCS co-exposure with fatty acids. ...
Triclosan (TCS) and triclocarban (TCC) have become ubiquitous pollutants detected in human body with concentrations up to hundreds of nanomolar levels. Previous studies about the hepatic lipid accumulation induced by TCS and TCC were focused on pollutant itself, which showed weak or no effects. High-fat diet (HFD), as a known environmental factor contributing to lipid metabolism-related disorders, its synergistic action with environmental pollutants deserves concern. The present study aimed to demonstrate the combined effects and potential molecular mechanisms of TCS and TCC with HFD at cellular and animal levels. The in vitro studies showed that TCC and TCS alone had negligible impact on lipid accumulation in HepG2 cells but induced lipid deposition at nanomolar levels when co-exposure with fatty acid. TCC exhibited much higher induction effects than TCS, which was related to their differential regulatory roles in adipogenic-related genes expression. The in vivo studies showed that TCC had little influence on hepatic lipid accumulation in mice fed with normal diet (ND) but could exacerbate the lipid accumulation in mice fed with HFD. Meanwhile, TCC-induced dyslipidemia in mice fed with HFD was more significant than that fed with ND. Therefore, we speculated that TCC might increase the risk of nonalcoholic fatty liver disease (NAFLD) and atherosclerosis in HFD humans. Molecular mechanism studies showed that TCC and TCS could bind to and activate estrogen-related receptor α (ERRα) and ERRγ as well as regulate their expression. TCC had higher activity on ERRα and ERRγ than TCS, which explained partly the differential regulatory roles of two receptors in the lipid accumulation induced by TCC and TCS. This work revealed synergistic effects and molecular mechanisms of TCC and TCS with excessive fatty acid on the hepatic lipid metabolism, which provided a novel insight into the toxic mechanism of pollutants from the perspective of dietary habits.
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The most frequent cause of chronic liver disease globally is non-alcoholic fatty liver disease (NAFLD). Potential risk factors for NAFLD have not received enough attention in Pakistan even though NAFLD has received substantial research. Objective: To assess risk factors for Non-alcoholic fatty liver disease. This study was conducted at the Department of Medicine Liaquat University, Hospital Jamshoro from 7th September 2020 to 6th March 2021. Methods: This research was cross-sectional. The study recruited a total of 195 patients via nonprobability sequential sampling. The ultrasound of all the patients was done by a sonologist for detecting NAFLD. Results: The stratification of NAFLD according to smoking, hypertension, obesity, hyperlipidaemia, uncontrolled diabetes mellitus, vitamin D deficiency was done. The statistical significance was observed for NAFLD in accordance with smoking, (p=0.00), hypertension (p=0.04), obesity (p=0.04), hyperlipidaemia (p=0.03), uncontrolled diabetes mellitus (p=0.04), vitamin D deficiency (p=0.04). Conclusions: This study has shown that the percentage of NAFLD was highest in age group (40-49 years). Males were more affected as compared to females. Smokers, hypertensives, hyperlipidemics and diabetics were more prone to develop NAFLD.
Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this anti-tumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. While the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.
Discovery efforts leading to the identification of ervogastat (PF-06865571), a systemically acting diacylglycerol acyltransferase (DGAT2) inhibitor that has advanced into clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) with liver fibrosis, are described herein. Ervogastat is a first-in-class DGAT2 inhibitor that addressed potential development risks of the prototype liver-targeted DGAT2 inhibitor PF-06427878. Key design elements that culminated in the discovery of ervogastat are (1) replacement of the metabolically labile motif with a 3,5-disubstituted pyridine system, which addressed potential safety risks arising from a cytochrome P450-mediated O-dearylation of PF-06427878 to a reactive quinone metabolite precursor, and (2) modifications of the amide group to a 3-THF group, guided by metabolite identification studies coupled with property-based drug design.
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The oxidation of low density lipoprotein (LDL) in the arterial wall is thought to contribute to human atherosclerotic lesion formation, in part by the high affinity uptake of oxidized LDL (OxLDL) by macrophages, resulting in foam cell formation. We have utilized cloning by expression to identify CD36 as a macrophage receptor for OxLDL. Transfection of a CD36 clone into 293 cells results in the specific and high affinity binding of OxLDL, followed by its internalization and degradation. An anti-CD36 antibody blocks 50% of the binding of OxLDL to platelets and to human macrophage-like THP cells. Furthermore, like mouse macrophages, 293 cells expressing CD36 recognize LDL which has been oxidized only 4 h, whereas more extensive oxidation of the LDL is required for recognition by the other known OxLDL receptors, the acetylated LDL (AcLDL) receptor and FcgammaRII-B2. CD36 may play a role in scavenging LDL modified by oxidation and may mediate effects of OxLDL on monocytes and platelets in atherosclerotic lesions.
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Previous studies have shown that the rate of fatty acid synthesis is elevated by more than 20-fold in livers of transgenic mice that express truncated nuclear forms of sterol regulatory element-binding proteins (SREBPs). This was explained in part by an increase in the levels of mRNA for the two major enzymes of fatty acid synthesis, acetyl-CoA carboxylase and fatty acid synthase, whose transcription is stimulated by SREBPs. Fatty acid synthesis also requires a source of acetyl-CoA and NADPH. In the current studies we show that the levels of mRNA for ATP citrate lyase, the enzyme that produces acetyl-CoA, are also elevated in the transgenic livers. In addition, we found marked elevations in the mRNAs for malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase, all of which produce NADPH. Finally, we found that overexpressing two of the SREBPs (1a and 2) led to elevated mRNAs for stearoyl-CoA desaturase 1 (SCD1), an isoform that is detectable in nontransgenic livers, and SCD2, an isoform that is not detected in nontransgenic livers. This stimulation led to an increase in total SCD activity in liver microsomes. Together, all of these changes would be expected to lead to a marked increase in the concentration of monounsaturated fatty acids in the transgenic livers, and this was confirmed chromatographically. We conclude that expression of nuclear SREBPs is capable of activating the entire coordinated program of unsaturated fatty acid biosynthesis in mouse liver.
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The discovery of oxysterols as the endogenous liver X receptor (LXR) ligands and subsequent gene targeting studies in mice provided strong evidence that LXR plays a central role in cholesterol metabolism. The identification here of a synthetic, nonsteroidal LXR-selective agonist series represented by T0314407 and T0901317 revealed a novel physiological role of LXR. Oral administration of T0901317 to mice and hamsters showed that LXR activated the coordinate expression of major fatty acid biosynthetic genes (lipogenesis) and increased plasma triglyceride and phospholipid levels in both species. Complementary studies in cell culture and animals suggested that the increase in plasma lipids occurs via LXR-mediated induction of the sterol regulatory element-binding protein 1 (SREBP-1) lipogenic program. Keywords • LXR • lipogenesis • SREBP • fatty acid • triglyceride
AIM: To clarify whether nonalcoholic fatty liver disease (NAFLD) increases the risk of cardiovascular disease. METHODS: We carried out a prospective observational study with a total of 1637 apparently healthy Japanese men and women who were recruited from a health check-up program. NAFLD was diagnosed by abdominal ultrasonography. The metabolic syndrome (MS) was defined according to the modified National Cholesterol Education Program (NCEP) ATP III criteria. Five years after the baseline evaluations, the incidence of cardiovascular disease was assessed by a self-administered questionnaire. RESULTS: Among 1221 participants available for outcome analyses, the incidence of cardiovascular disease was higher in 231 subjects with NAFLD at baseline (5 coronary heart disease, 6 ischemic stroke, and 1 cerebral hemorrhage) than 990 subjects without NAFLD (3 coronary heart disease, 6 ischemic stroke, and 1 cerebral hemorrhage). Multivariate analyses indicated that NAFLD was a predictor of cardiovascular disease independent of conventional risk factors (odds ratio 4.12, 95% CI, 1.58 to 10.75, P = 0.004). MS was also independently associated with cardiovascular events. But simultaneous inclusion of NAFLD and MS in a multivariate model revealed that NAFLD but not MS retained a statistically significant correlation with cardiovascular disease. CONCLUSION: Although both of them were predictors of cardiovascular disease, NAFLD but not MS retained a statistically significant correlation with cardiovascular disease in a multivariate model. NAFLD is a strong predictor of cardiovascular disease and may play a central role in the cardiovascular risk of MS.
Tumor necrosis factor (TNF)alpha, a pivotal cytokine involved in inflammation, is produced primarily by Kupffer cells in the liver. It has been shown that inactivation of Kupffer cells prevents alcohol-induced liver injury; therefore, the purpose of this study was to determine if neutralizing anti-TNF-alpha antibody is also effective. Male Wistar rats were exposed to ethanol (11 to 12 g · kg-1 · d-1) continuously for up to 4 weeks via intragastric feeding using an enteral feeding model. Before ethanol exposure, polyclonal anti-mouse TNF-alpha rabbit serum was injected (2.0 mg/kg intravenously). There were no significant differences in body weight, mean ethanol concentration, or cyclic patterns of ethanol in urine when ethanol-and ethanol plus antibody-treated groups were compared. Expression of TNF- alpha and macrophage inflammatory protein 2 (MIP-2) messenger RNA (mRNA), determined using reverse transcription-polymerase chain reaction, was three- to four-fold higher in livers of ethanol-treated rats than in those of rats fed an ethanol-free, high-fat control diet. In addition, MIP-2 levels were also elevated when detected by Northern blot analysis. Anti-TNF-alpha antibody did not affect expression of mRNA for interleukin (IL) 1alpha, IL-6, transforming growth factor beta1, or TNF-alpha. However, MIP-2 mRNA expression, which is regulated by TNF-alpha, was decreased significantly by anti-TNF-alpha antibody treatment. Serum aspartate transaminase levels were elevated in ethanol-treated rats to 136 +/- 12 IU/L after 4 weeks but only reached 90 +/- 5 IU/L (P < .05) in rats treated with anti-TNF-alpha antibody. The hepatic inflammation and necrosis observed in ethanol-fed rats were attenuated significantly by antibody treatment, and steatosis was not. These results support the hypothesis that TNF-alpha plays an important role in inflammation and necrosis in alcohol-induced liver injury and that treatment with anti-TNF-alpha antibody may be therapeutically useful in this disease. (Hepatology 1997 Dec;26(6):1530-7)
Fatty liver disease that develops in the absence of alcohol abuse is recognized increasingly as a major health burden. This report summarizes the presentations and discussions at a Single Topic Conference held September 20-22, 2002, and sponsored by the American Association for the Study of Liver Diseases. The conference focused on fatty liver disorders. Estimates based on imaging and autopsy studies suggest that about 20% to 30% of adults in the United States and other Western countries have excess fat accumulation in the liver. About 10% of these individuals, or fully 2% to 3% of adults, are estimated to meet current diagnostic criteria for nonalcoholic steatohepatitis (NASH). Sustained liver injury leads to progressive fibrosis and cirrhosis in a fraction, possibly up to one third, of those with NASH, and NASH may be a cause of cryptogenic cirrhosis. NASH is now a significant health issue for obese children as well, leading to cirrhosis in some. The diagnostic criteria for NASH continue to evolve and rely on the histologic findings of steatosis, hepatocellular injury (ballooning, Mallory bodies), and the pattern of fibrosis. Generally recognized indications for biopsy include establishing the diagnosis and staging of the injury, but strict guidelines do not exist. Liver enzymes are insensitive and cannot be used reliably to confirm the diagnosis or stage the extent of fibrosis. Older age, obesity, and diabetes are predictive of fibrosis. The pathogenesis of NASH is multifactorial. Insulin resistance may be an important factor in the accumulation of hepatocellular fat, whereas excess intracellular fatty acids, oxidant stress, adenosine triphosphate (ATP) depletion, and mitochondrial dysfunction may be important causes of hepatocellular injury in the steatotic liver. Efforts are underway to refine the role of insulin resistance in NASH and determine whether improving insulin sensitivity pharmacologically is an effective treatment. An altered lifestyle may be a more effective means of improving insulin sensitivity. The research agenda for the future includes establishing the role of insulin resistance and abnormal lipoprotein metabolism in NASH, determining the pathogenesis of cellular injury, defining predisposing genetic abnormalities, identifying better noninvasive predictors of disease, and defining effective therapy.
We demonstrate that mice lacking the oxysterol receptor, LXRα, lose their ability to respond normally to dietary cholesterol and are unable to tolerate any amount of cholesterol in excess of that which they synthesize de novo. When fed diets containing cholesterol, LXRα (−/−) mice fail to induce transcription of the gene encoding cholesterol 7α-hydroxylase (Cyp7a), the rate-limiting enzyme in bile acid synthesis. This defect is associated with a rapid accumulation of large amounts of cholesterol in the liver that eventually leads to impaired hepatic function. The regulation of several other crucial lipid metabolizing genes is also altered in LXRα (−/−) mice. These results demonstrate the existence of a physiologically significant feed-forward regulatory pathway for sterol metabolism and establish the role of LXRα as the major sensor of dietary cholesterol.