TEM-980; No.
of Pages 10
Review
Role of metabolic lipases and lipolytic
metabolites in the pathogenesis of
NAFLD
Claudia D. Fuchs, Thierry Claudel, and Michael Trauner
Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine
III, Medical University of Vienna, Vienna, Austria
Non-alcoholic fatty liver disease (NAFLD) is the most
frequent chronic liver disease in Western countries, rang-
ing from simple steatosis to steatohepatitis, cirrhosis, and
hepatocellular cancer. Although the mechanisms under-
lying disease progression are incompletely understood,
lipotoxic events in the liver resulting in inflammation and
fibrosis appear to be central. Free fatty acids and their
metabolites are potentially lipotoxic mediators triggering
liver injury, suggesting a central role for metabolic lipases.
These enzymes are major players in lipid partitioning
between tissues and within cells, and provide ligands
for nuclear receptors (NRs). We discuss the potential role
of intracellular lipases and their lipolytic products in
NAFLD. Because tissue-specific modulation of lipases is
currently impossible, targeting NRs with ligands may
open novel therapeutic perspectives.
Non-alcoholic fatty liver disease (NAFLD; see Glossary)
comprises a disease spectrum ranging from ‘simple’ hepatic
triglyceride (TG) accumulation (steatosis), to steatosis with
inflammation (steatohepatitis), more advanced fibrosis, cir-
rhosis, and liver cancer [1,2]. NAFLD is considered as the
hepatic manifestation of metabolic syndrome and is strongly
associated with type 2 diabetes mellitus (T2DM) and obesi-
ty. Obesity and T2DM are well known consequences of
modern lifestyle characterized by increased dietary calorie
intake of saturated and trans-unsaturated fatty acids (FAs),
as well as fructose [3], and a sedentary lifestyle with reduced
physical activity [3]. This contributes to an increasing prev-
alence for NAFLD that now affects up to 40–50% of the total
population in the EU and USA, with even higher preva-
lences (75% and more) in risk groups with T2DM and
obesity. Although the precise pathogenesis and progression
of NAFLD are still poorly understood [2], insulin resistance
(IR) in muscle, white adipose tissue (WAT), and liver
appears to play a central role [4,5]. Impaired insulin signal-
ing in WAT results in increased lipolysis, generating a flux of
FA from WAT to the liver, thus causing substrate overload
and further promoting hepatic IR, with subsequently en-
hanced de novo lipogenesis and TG accumulation [6,7].
Corresponding author: Trauner, M. (michael.trauner@meduniwien.ac.at).
1043-2760/
ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tem.2014.08.001
About 60% of FAs for hepatic TG accumulating in NAFLD
patients originate from WAT-derived FAs [2], whereas 25%
result from increased de novo lipogenesis controlled via the
transcription factors sterol regulatory element-binding pro-
tein-1c (SREBP-1c), carbohydrate response element-bind-
ing protein (ChREBP), and peroxisome proliferator-
activated receptor g (PPARg). The remaining 15% are
obtained from the diet [2].
Although IR may initiate hepatic steatosis as first hit,
additional (second) hits such as drugs, ischemia, or endo-
toxin result in inflammation with disease progression to
nonalcoholic steatohepatitis (NASH) and beyond [2]. How-
ever, this ‘second hit hypothesis’ is increasingly replaced by
the concept that benign steatosis (which may be non-pro-
gressive in the majority of patients) and NASH could be two
Glossary
G-protein-coupled receptors (GPCRs): GPCRs are proteins located at the cell
membrane. They bind to various extracellular substances (such as fatty acids,
bile acids) and therefore can transduce extracellular signals to intracellular
proteins.
Lipases: enzymes responsible for the hydrolysis of neutral lipids in free FAs
and glycerol. Triglycerides and phospholipids are common lipase substrates.
Intracellular/metabolic lipases are expressed specifically in various cells types
found in different organs such as adipose tissue (macrophages and white
adipocytes), liver (hepatocytes, Kupffer cells, stellate cells), intestine (enter-
ocytes), etc.
Non-alcoholic fatty liver disease (NAFLD): NAFLD comprises a wide spectrum
of liver injury ranging from simple, relatively benign steatosis to steatohepa-
titis (NASH), fibrosis, cirrhosis, and liver cancer. Estimated prevalence is in the
range 40–50% of the general population depending on country and region.
NAFLD diagnosis is based on minimal or absent alcohol consumption (<20–
30 g/day), steatosis on imaging, and exclusion of other liver diseases. Firm
diagnosis of NASH still relies on histological criteria (ballooning and
inflammation in addition to steatosis) revealed by liver biopsy. Simple
steatosis usually has a benign liver prognosis; however, patients displaying
histological inflammatory changes and/or fibrosis on liver biopsy have a
considerable risk to progress to end-stage liver disease and cancer. Treatment
of NAFLD is focused on lifestyle modification (diet and exercise), antioxidant
strategies (e.g., vitamin E), and treatment of associated metabolic conditions
such as obesity, insulin resistance (insulin sensitizers), and hyperlipidemia
(statins).
Nuclear receptors (NR): NRs belong to a family of zinc-finger proteins generally
binding DNA and cofactors modulating polymerase II activity therefore
regulating positively or negatively specific gene expression. Some but not all
NRs are activated after ligand binding (e.g., fatty acids for PPARs, bile acids for
FXR).
Type 2 diabetes (T2DM): T2DM is characterized by hyperglycemia and
disturbances in carbohydrate and lipid metabolism resulting from defects in
insulin signaling termed insulin resistance (IR). Therefore the metabolic
complications of T2DM derive from deficient insulin action on target tissues.
Patients suffering from T2DM are at higher risk of cardiovascular, peripheral
vascular and cerebrovascular disease.
Trends in Endocrinology and Metabolism xx (2014) 1–10 1
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different entities with divergent pathogenic pictures [8], and
that ‘multiple hits’ may occur in parallel rather than in strict
sequence. In this context, lipid accumulation may be con-
sidered as a ‘bystander phenomenon’ [8]. Constant ectopic
exposure of hepatocytes to potentially toxic lipid metabolites
such as FAs, phosphatidic acid, lysophosphatidic acid, cer-
amides, and diacylglycerols (DAG) may result in ‘lipotoxic’
insults [2], characterized by endoplasmic reticulum (ER)
stress, inflammation, apoptosis, necrosis, ballooning, and
Mallory–Denk body formation in hepatocytes, all of which
are histopathological hallmarks of NASH [9]. Moreover,
these observations strengthen the assumption that not
TG but instead TG-derived metabolites may be toxic to
the liver, and TG breakdown via metabolic lipases contrib-
utes to development of NASH [9]. Conversely, specific FAs
are also known to have non-toxic properties such as ligand
function NRs and, in addition to signaling function, they act
also as energy substrates. NRs belong to a superfamily of
ligand-activated transcription factors [10]. Several key NRs
[e.g., PPARs, farnesoid X receptor (FXR) and liver X receptor
(LXR)] are expressed in the liver and activated by ligands
such as FAs, bile acids, and oxysterols, respectively [11]. In
addition, synthetic drugs designed to activate these recep-
tors such as fibrates (PPARa), glitazones (PPARg), and
obeticholic acid (FXR) are available [12]. Activation of these
NRs promotes FA catabolism (PPARa and FXR), storage
(PPARg), or endogenous synthesis (LXR) (see below). Be-
cause disturbances in these metabolic pathways favor
NAFLD development, targeting NRs with synthetic drugs
might counteract and/or reverse fatty liver development.
Potential role of lipases in the pathogenesis and
progression of NAFLD
Because the pathogenesis of NAFLD is characterized by
hepatic lipid accumulation [13], metabolic lipases (Box 1)
are in the center of interest of NAFLD/NASH pathogen-
esis. Free FAs derived from adipose tissue are taken up
via cell specific transporters such as cluster of differen-
tiation 36 (CD36) and fatty acid transport protein
(FATP) or via passive diffusion (Figure 1) [14]. Within
hepatocytes, FAs are esterified for storage in TGs. How-
ever, it appears that not TGs but FAs or other metab-
olites such as DAGs and ceramides are cytotoxic.
Because non-adipose tissue has a limited capacity to
store neutral lipids in the form of lipid droplets, excess
free FA spillover from the periphery or from intracellular
lipolysis results in hepatic lipid accumulation and lipo-
toxicity. Studies using mice overexpressing diacylgly-
cerol acyltransferase (DGAT) 2 (the enzyme catalyzing
the last step in TG formation) in the liver demonstrated
increased hepatic TG accumulation and hepatic phos-
phorylation of protein kinase R-like ER kinase (PERK)
[9], without inflammation or IR. Conversely, liver-specif-
ic knockdown of DGAT2 prevented TG accumulation in
the liver but resulted in lipotoxic injury caused by excess
of free FAs [9]. Based on these findings, it can be postu-
lated that lipid droplet/TG formation may protect
against toxic lipid-mediated liver injury [9]. However,
the anti-lipotoxic properties of TG may be limited be-
cause FAs can be released from TGs by lipases, thus
generating a new source of toxic lipid intermediates.
2
Trends in Endocrinology and Metabolism xxx xxxx, Vol. xxx, No. x
Box 1. Lipolysis and metabolic lipases
Physiologically under fasting conditions, and pathologically as a
result of IR, lipolysis of TGs in WAT by key lipases generates FAs
and glycerol, which are secreted into blood and transported to other
tissues such as liver and muscle.
Intracellular lipolysis consists of three steps, each releasing FAs,
and involves at least three different enzymes: adipose triglyceride
lipase (ATGL/PNPLA2), a member of the patatin-like phospholipase
domain-containing protein family, catalyzes the first step; convert-
ing TG into DAG. ATGL has a coactivator (comparative gene
identification 58, CGI-58) that considerably augments ATGL activity
[100]. Hormone sensitive lipase (HSL) is the enzyme responsible for
the second step and converts DAGs into monoacylglycerols (MAGs).
Finally, monoacylglycerol lipase (MGL) hydrolyzes MGs as last step
in lipolysis (Figure 1) [54]. Furthermore, MGL also plays an
important role in endocannabinoid signaling by creating arachido-
nic acid (a prostaglandin precursor) through hydrolysis of 2-
arachidonoylglycerol, a major endocannabinoid [101], therefore
playing a potential role in inflammation. ATGL and HSL account for
90% of TG hydrolysis in adipose tissue [100]. Hormonal (e.g.,
catecholamines) or neuronal [102] stimulation of lipolysis increases
FA release from WAT more than 100-fold. Although the enzymes
mentioned above are also expressed in non-adipose tissue such as
muscle and liver, much less is known about lipolytic mechanisms in
these organs [37]. Therefore, additional lipases may be necessary
for efficient lipolysis in non-adipose tissue such as liver [54].
A closely related enzyme of ATGL/PNPLA2, PNPLA3 (also known
as adiponutrin), might be such candidate. PNPLA3 is suggested to
have both lipase and lysophosphatidic acid acyltransferase (LPAAT)
functions, the latter contributing to TG formation rather than to
breakdown [15,103,104]. Interestingly, PNPLA3 expression in adi-
pose tissue and liver [15,105–107] decreases with fasting and
increases in response to insulin and glucose [15], which is more in
line with a suggested LPAAT function and subsequent role in
lipogenesis [15]. Additional studies to determine the role of PNPLA3
in hepatic lipid metabolism will be necessary to decipher whether
(or how) possible polymorphisms (see below) affect its lipase and/or
LPAAT function and alter the accumulation of key signaling lipid
metabolites such as DAG or ceramides in liver or elsewhere [15].
Because free FAs are metabolized via several different
pathways, it may be difficult to distinguish whether FAs
or their metabolites are the toxic component [9].
PNPLA3 polymorphisms
A genome-wide association study (GWAS) screen within
the Dallas Heart study has uncovered an association of
NAFLD with a single-nucleotide polymorphism (SNP)
(rs738409; C>G) in the PNPLA3 gene (Box 1) [15,16],
which was subsequently confirmed by several studies
from other centers [17–19]. The rs738409 SNP encodes
the I148 M substitution, which leads to loss of lipolytic
function. Although most SNP carriers have increased
hepatic fat content, several studies show that it is inde-
pendent of IR [20–22]. However, it has to be mentioned
that most of these studies were performed in relatively
insulin-resistant obese patients. Therefore, to identify
definitively whether PNPLA3 confers a risk independent
of IR, insulin sensitivity needs to be assessed in lean
NAFLD patients who are carriers of the wild type
PNPLA3 versus lean NAFLD patients carrying the
I148 M mutant [15].
Overexpression of the mutant I148 M PNPLA3 gene in
mice resulted in increased lipid droplet size and TG accu-
mulation, which is in line with the human data [15,23].
This observation strengthens the assumption that
TEM-980; No. of Pages 10
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TAG
ATGL PPARγ
DAG
HSL FFA
MAG
MGL
Glycerol
Adipocyte
Figure 1. Lipid partitioning and lipid nuclear receptor (NR) signaling via PPAR. Lipolysis of triglycerides (TGs) in white adipose tissue (WAT) via metabolic lipases adipose
triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL), initiates free fatty acid (FA) flux from WAT to the liver. In the liver, FAs
deriving from the periphery (or from endogenous sources) need to be activated with acyl-CoA, and are subsequently incorporated into TGs. Intrahepatic hydrolysis of TGs
via the metabolic lipases ATGL, HSL, and MGL create potential ligands (or ligand precursors) for nuclear receptors such as PPARa. In addition, PPARg in WAT is also
activated via free FAs, which stimulate TG hydrolysis.
PNPLA3 acts as a lipase. However, overexpression of wild
type PNPLA3 did not change hepatic lipid content in mice
[15,23,24]. Moreover, Pnpla3 knockout (KO) mice did not
develop hepatic steatosis or disturbances in glucose ho-
meostasis, even when challenged with various diets
[25,26]. Therefore, the function of PNPLA3 in vivo is still
controversial and needs to be understood better. Addition-
al studies using transgenic mice expressing the human
form of PNPLA3 and its variant I148 M under the control
of their natural promoter at the liver cell specific level may
help to clarify the exact role/function of PNPLA3 in
NAFLD development and progression.
Interestingly, not only steatosis but also disease pro-
gression towards steatohepatitis and fibrosis might be
initiated via disturbed lipid metabolism. The PNPLA3
polymorphism may reduce TG hydrolysis, which could
make the cells more susceptible to lipid peroxidation
and subsequent oxidative stress and inflammation [27].
The release of inflammatory cytokines could activate Kupf-
fer cells as well as hepatic stellate cells (HSCs), subse-
quently initiating fibrosis, possibly explaining the link
between genetic variants and fibrosis progression. More-
over, it was observed that PNPLA3 is highly expressed in
human HSC and that it exerts retinyl palmitate lipase
activity and therefore plays an essential role in HSC
activation and subsequent fibrosis development. The
I148 M PNPLA3 variant was identified as a loss of function
mutation and therefore reduces retinol release from HSC
[28]. Notably, PNPLA3 variants have also been linked to
fibrosis and are considered as marker of disease progres-
sion in several other liver diseases such as alcoholic liver
disease [29] and chronic hepatitis C [30]. Therefore further
studies need to clarify the role of I148 M PNPLA3 variant
in HSCs in the susceptibility toward hepatic fibrogenesis
and carcinogenesis [28]
Trends in Endocrinology and Metabolism xxx xxxx, Vol. xxx, No. x
Acetyl-CoA
TAG
synthesis
Fatp5 FA-CoA TAG
ATGL
FFA CD36
HSL
MGL
PPAR
FFA
α
RXR
Gene
expression
Nucleus
Hepatocyte
TRENDS in Endocrinology & Metabolism
ATGL and NAFLD
Animal studies have shown that absence of ATGL (Box 1)
in mice, in addition to TG accumulation in multiple tissues,
also leads to increased susceptibility to inflammation be-
cause of disturbances in PPARa signaling [31], emphasiz-
ing the important function of lipases for NAFLD. ATGL
deficiency in humans has been linked to neutral lipid
storage disease (NLSD) characterized by lipid deposition
in liver (and other organs such as skin, muscle, and leu-
cocytes) [32]. Two phenotypes can be distinguished, name-
ly NLSD with ichtiosis (NLSDI, also known as Chanarin–
Dorfman syndrome) and NLSD with myopathy (NLSDM).
Whereas NLSDM is caused by mutations in ATGL, NLSDI
is based on mutations in the ATGL cofactor CGI 58 [32].
NLSDI is characterized by hepatomegaly, liver steatosis
and neurological disorders, and NLSDM by skeletal my-
opathy [32]. Interestingly, these patients do not develop
impaired glucose tolerance or IR, emphasizing that ectopic
fat accumulation does not necessarily cause IR. Apart from
these rare mutations, the role of polymorphisms of these
genes in NAFLD is still to be explored. To clarify the role of
ATGL in liver physiology and disease, this enzyme was
knocked down either genetically [33] or via adenovirus
injection [34]. In comparison to wild type (WT) mice,
liver-specific Atgl KO mice developed hepatic steatosis
but the inflammatory response were slightly lower in liver
Atgl KO than in WT mice [33]. Interestingly, under ER
stress conditions it was also observed that (global) Atgl KO
mice develop hepatic steatosis but no inflammation [35],
suggesting that loss of hepatic ATGL activity may protect
from the disease progression. Of note, in contrast to global
Atgl KO mice, liver-specific Atgl knockdown did not show
increased insulin sensitivity [33]. Knockdown of Cgi58 at
the hepatocellular level results in a much more severe
NAFLD phenotype than hepatic Atgl knockdown [33,36].
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In contrast to Atgl liver-specific KO mice, Cgi58 liver-
specific KO mice not only develop steatosis but also severe
inflammation and fibrosis [36], emphasizing an important
role of CGI 58 in NAFLD development.
HSL deficiency
In humans, loss of HSL is accompanied by a smaller size of
adipocytes accumulating DGs [37]. To date only two indi-
viduals with HSL deficiency have been identified. These
patients showed IR, low high-density lipoprotein (HDL)
cholesterol, hypertriglyceridemia, and ectopic fat accumu-
lation in the liver [38]. These findings are in line with the
phenotype of Hsl KO mice [39], where impaired PPARg
activity causes decreased de novo lipogenesis and subse-
quent reduction in lipid storage [40]. Interestingly, aged Hsl
KO mice show a decrease in adipose tissue mass, which was
ascribed to disturbances in PPARg signaling [40] as a result
of reduced free FAs, which are PPARg ligands [41]. An
additional factor contributing to reduction of WAT in Hsl
KO mice may be cell death caused by increased inflamma-
tion, macrophage infiltration, and the formation of ‘crown-
like structures’ [37] characterized by macrophages sur-
rounding hypertrophic adipocytes [42]. HSL expression is
very low in the liver [39] and Hsl KO mice do not develop
steatosis. Therefore, from the hepatic point of view loss of
HSL does not appear to be a major factor in NAFLD devel-
opment. However, when HSL is overexpressed in livers of
ob/ob mice fed a high-fat diet (HFD) hepatic TG content is
reduced by about 50%, suggesting that HSL (despite its
minor role in fatty liver development) might be a potential
drug target in treatment of NAFLD [43]
Because hepatic TG hydrolysis provides substrates for
very low density lipoprotein (VLDL)–TG synthesis (ATGL
as well as HSL levels are very low, and neither overexpres-
sion nor knockdown affects hepatic VLDL secretion), it is
tempting to speculate that other lipases may take over this
important role in the liver [44]. It is suggested that trigly-
cerol hydrolase (TGH) could be the important component in
VLDL assembling. In mice overexpressing human TGH in
the liver, increased secretion of ApoB was observed and, in
line, global deletion of TGH resulted in reduced plasma TG
as well as ApoB levels [44]. Interestingly, no concomitant
liver phenotype was observed in the Tgh KO mouse. Lack of
steatosis in this mouse model was linked to increased FA
oxidation and low FA flux from adipose tissue to the liver
[44].
FAs as lipotoxic mediators
Hepatic lipotoxicity is known to induce hepatocyte death,
referred to as lipoapoptosis [45]. Free FAs [especially
saturated FAs (SFAs)] have hepatotoxic properties. The
difference in toxicity between SFAs and unsaturated FAs is
related to the possibility that unsaturated FAs are easily
esterified and incorporated into neutral lipids [45]. Hepa-
tocytes can undergo apoptosis either via an extrinsic or an
intrinsic pathway. The extrinsic pathway is activated via
death ligands, whereas the intrinsic pathway is initiated by
intracellular stress of organelles such as ER, lysosomes, and
mitochondria [45]. Hepatocytes treated with SFAs dis-
played increased expression of the proapoptotic proteins
BIM and p53 upregulated modulator of apoptosis (PUMA)
4
Trends in Endocrinology and Metabolism xxx xxxx, Vol. xxx, No. x
SFA
TLR2 or TLR4
Cell membrane or FATP5
SFA
BIM/PUMA (pro-apoptoc proteins)
BCL2 family (an-apoptoc proteins)
BAX/BAX Caspase 3/6/7
Apoptosis
TRENDS in Endocrinology & Metabolism
Figure 2. Saturated fatty acids (SFAs) induce lipoapoptosis. SFAs activate pro-
apoptotic proteins (as such BIM and PUMA), leading to inhibition of the anti-
apoptotic proteins of the BCL 2 family and subsequent activation of pro-apoptotic
proteins BAK and BAX. Once activated, BAK and BAX cause mitochondrial
dysfunction, which leads to caspase activation and apoptotic cell death.
(Figure 2). Genetic deletion of Puma leads to resistance to
SFA induced apoptosis in vitro. Free FA-mediated activa-
tion of BIM and PUMA is accompanied by induction of the
multi-domain proapoptotic member of the BCL2 family
BAX. This initiates a mitochondrial pathway of cell death,
including activation of caspases 3, 6, and 7, resulting in
apoptosis. Of note, BAX expression is increased in a murine
NASH model, and is related to structural and functional
mitochondrial abnormalities found in patients with NASH
[45]. Interestingly, the effects of SFAs are counteracted by
unsaturated FAs [44,46]. As mentioned above, unsaturated
FAs have the potential to initiate TG formation and there-
fore have anti-lipotoxic properties. Furthermore, SFAs such
as palmitic acid (PA) have been shown to act as proinflam-
matory lipid mediators and activate inflammatory path-
ways in different cell types such as macrophages,
adipocytes, myocytes, and hepatocytes [47], which leads
to activation of NF-kB and JNK1/AP1 pathways and cyto-
kine release [47]. Although it has been reported that the
proinflammatory effects of SFAs are mediated via Toll-like
receptor 4 (TLR4) and TLR2 [47], recent studies demon-
strated that SFAs indirectly activate TLRs by interacting
with TLR coreceptors [47] such as CD36 or LDL receptor
[48,49], and/or stimulate TLR4 dimerization [50]. Emerging
evidence suggests that fetuin A could act as an endogenous
TLR4 ligand and subsequent connector through which FAs
may induce TLR4 driven insulin resistance [51] (Figure 3).
An additional possibility could be that SFAs release a tissue
damage signal which may function via TLRs [47]. Never-
theless, SFAs are also able to induce inflammation indepen-
dently of TLR pathways and via activation of stress kinases
and subsequent reactive oxygen species (ROS) production.
Increased ROS levels deriving from SFA stimulation may
account for the activation of nucleotide binding domain
leucine-rich repeats containing family, pyrin domain-con-
taining 3/apoptotic speck protein containing a caspase re-
cruitment domain (NLRP3/ASC) inflammasome complex,
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Review Trends in Endocrinology and Metabolism xxx xxxx, Vol. xxx, No. x

ATGL HSL MGL

TAG DAG MAG Glycerol
Adipocyte FFA FFA FFA

(B) SFA
SFA
(A) FetA
ω3-FAs Insulin
Hepatocyte
TLR2 or TLR4
GPR120 FATP5

Cell IR
SFA IRS
membrane PKCε Ser-P
ω3-FAs
DAG
TAK
PI3K
β arr2
TAB1
ATGL
TAG FFA PPARa NF-κB JNK Ceramides PKCε AKT

DAG
FA oxidaon
FOXO1
Inflammaon (gluconeogenesis)

(cytokine producon)
mTOR GSK-3
(protein synthesis) (glycogen synthesis)
TRENDS in Endocrinology & Metabolism

Figure 3. Impact of fatty acid (FA) signaling on inflammation and insulin resistance. (A) Omega-3 fatty acids (v3-FA) are able to bind the G-protein-coupled receptor GPR120,
leading to receptor internalization and binding of b arrestin 2 (b arr2). Furthermore, TAK1 binding protein (TAB) will be complexed by b arr2. Complex formation with TAK1
(TGFb-activated kinase 1) and TAB is inhibited, resulting in TAK1 inhibition and repression of downstream inflammatory signaling. (B) Saturated FAs (SFAs) activate the
inflammatory cascade via TLR4- or TLR2-dependent or -independent pathways. Moreover, SFAs are precursors for diacylglycerol (DAG) as well as ceramide biosynthesis.

thereby increasing interleukin 1b (IL1b) release [52],
which was shown to impair insulin signaling, suggesting
an additional SFA-mediated mechanism leading to IR. In
line, Nlrp3, Asc, and caspase1 KO mice are protected from
HFD-induced inflammation and IR [52]. Paradoxically,
Asc KO mice developed a more severe NASH phenotype
with a methionine- and choline-deficient (MCD) diet owing
to abnormal microbiota composition [53]. Furthermore,
SFAs lead to increased production of ROS which activate
redox-sensitive serine kinases that subsequently inhibit
insulin signaling [54]. Although SFAs appear to be lipo-
toxic because of proinflammatory and IR properties, other
classes of FAs such as long chain polyunsaturated omega-3
FAs may have protective functions [47] (Figure 3). Omega-
3 FAs have been suggested as ligands for the G-protein-
coupled receptor (GPCR) 120, as well as for PPARa and
PPARg [55], and therefore may exert anti-inflammatory
effects in macrophages [56]. Importantly, SFAs are also
precursors for other lipid products such as DAGs
(Figure 3). Intracellular concentrations of DAG is
increase during IR [57] leading to increased protein kinase
C (PKC) e activation and impairment of insulin signaling
[58] with subsequent development of NAFLD [59]. DAG-
dependent PKCe activation leads to insulin receptor ki-
nase inhibition and reduced tyrosine phosphorylation of
insulin receptor substrate (IRS) 1 and 2, resulting in
repressed activation of AKT. Reduced AKT function is
accompanied with attenuated glycogen synthesis and de-
creased suppression of gluconeogenesis, which in turn
leads to increased glucose release [59] (Figure 3). Of note,
it has also been shown that intracellular DAG concentra-
tion can increase independently of impaired insulin sig-
naling [60]. Moreover, localization and intracellular
compartmentation of DAGs may play a major role in
determining its lipotoxic properties because it has been
demonstrated that internalization of DAGs into lipid dro-
plets prevents PKCe activation at the plasma membrane
and subsequently impairs insulin receptor signaling [61].
In addition to localization, the structure of DAGs is also of
particular importance for lipotoxicity. It has been demon-
strated that only the 1,2-DAG stereoisomer is able to
activate PKCe [62], whereas both saturated as well as
unsaturated acyl side chains had the same effect on kinase
activation [63].
In addition to potential lipotoxic TG degradation pro-
ducts, cholesterol may also be an important trigger for
NAFLD development. Patients suffering from NAFLD dis-
played increased cholesterol synthesis [64]. Interestingly,
cholesterol can activate LXRa and subsequent de novo
lipogenesis via increased oxysterol levels (see below). This

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TEM-980; No. of Pages 10
Review
observation indicates that enhanced cholesterol intake can
cause steatosis development [65]. Furthermore, increased
dietary cholesterol uptake leads to hepatic inflammation
and oxidative stress in mice [66], suggesting that increased
cholesterol does not only act as disease initiator but can also
promote its progression beyond the more severe stages. Free
cholesterol was found to accumulate in HSC, where it
upregulates TLR4 protein levels and subsequently
represses the inhibitory TGF-pseudoreceptor BMP and acti-
vin membrane-bound inhibitor homolog (BAMBI). The con-
sequential increase in TGF signaling promotes HSC
activation and liver fibrosis [67].
FAs and NR signaling
PPAR signaling
FAs, especially polyunsaturated FAs (PUFAs) [68], are
ligands for the NRs PPARa (NR1C1), PPARg (NR1C3),
and PPARd (NR1C2), and can also activate GPCRs. PPARa
is highly expressed in oxidative tissues such as liver, cardiac
and skeletal muscle, whereas PPARg is mainly found in
adipocytes, enterocytes, and macrophages, and PPARd is
ubiquitously expressed [11,69]. PPARa increases FA oxida-
tion and reverse cholesterol transport, PPARg promotes
lipid storage, adipocyte differentiation, and normalizes glu-
cose levels [9], whereas PPARd increases FA and glucose
catabolism but also plays a role in fertility and cancer [70].
Interestingly, free FAs originating from adipose tissue
lipolysis are not able to activate hepatic PPARa directly
but are ligands for PPARd in the liver [68]. However,
activation of cardiac and hepatic gene transcription via
PPARa largely depends on lipolytic breakdown of intracel-
lular TG depots [71] by ATGL, HSL, and MGL (Figure 1)
[31] in these tissues. Therefore, FA-induced activation of
PPARa drives a feed-forward loop protecting against FA-
derived lipotoxicity by promoting their oxidation in heart
and liver [68]. This observation suggests that lipid droplets
do not only serve as energy stores but also as a reservoir for
lipid mediators that can act as PPARa ligands [71]. There-
fore, FA-activated PPARs play a protective role against
hepatic lipotoxicity [72]. Metabolic lipases such as ATGL
release the intracellular ligands, providing a potential
mechanistic link and drawing attention to lipases as po-
tential ‘ligand providers’ for anti-lipotoxic pathways. In
addition, it was also shown that not only FAs derived from
intracellular lipolysis but also 1-palmitoyl-2-oleoyl-sn-
glycerol-3-phosphocholine (POPC) is an endogenous ligand
for PPARa [73]. In addition to their endogenous ligands,
synthetic drugs are also able to activate PPARs, including
fibrates for PPARa or glitazones for PPARg [74] (see
therapeutic implications).
FXR signaling
In addition to PPARs, FXR (NR1H4) is activated by PUFAs
[75]. FXR, which is mainly known for its regulatory func-
tion in bile acid metabolism, is also a key regulator of
hepatic lipid metabolism [12,76]. Moreover FXR and the
FXR target gene small heterodimer partner (SHP) play key
roles in lipid metabolism by downregulating SREBP1c
after inhibition of LXRa signaling [77] and repress
microsomal triglyceride transfer protein (MTTP) induction
mediated by liver receptor homolog 1 (LRH-1) [78].
6
Trends in Endocrinology and Metabolism xxx xxxx, Vol. xxx, No. x
Interestingly, a recently identified variant of transmem-
brane 6 superfamily member 2 (TM6SF2) that confers
susceptibility to NAFLD was shown to lower hepatic VLDL
secretion and increase liver TG [79]. Thus, it is tempting to
speculate that FXR may impact on regulation of TM6SF2.
Furthermore, FXR is also known to inhibit ChREBP [80],
another lipogenic transcription factor potentially involved
in fatty liver development [60]. Finally, human FXR also
upregulates PPARa expression [81], suggesting that some
FXR effects on lipid homeostasis might be mediated via
PPARa-induced FA oxidation [12].
LXR signaling
LXRs (LXRa and LXRb) are sterol-activated transcription
factors which are regulated via oxysterols and other cho-
lesterol derivatives [82]. In addition to their role in reverse
cholesterol transport and bile acid synthesis, LXRs also
affect glucose and lipid metabolism. The lipogenic activity
of LXR results from the activation of SREBP1c (master
regulator of de novo lipogenesis) as well as direct regula-
tion of some SREBP1c downstream targets such as fatty
acid synthase (FAS) and stearyl-CoA desaturase (SCD1)
[82]. In line, feeding mice with an LXR agonist led to
increased hepatic TG formation and VLDL secretion
[82]. Ob/ob mice lacking LXRa and LXRb displayed re-
duced hepatic steatosis and improved insulin sensitivity in
comparison to ob/ob mice. However, loss of hepatic de novo
lipogenesis in ob/ob mice deficient for LXRs was accompa-
nied by increased adipose-tissue lipid storage, which was
ascribed to tissue-selective action of LXR [82]. Therefore
the mice remained obese despite improved insulin sensi-
tivity [82].
Therapeutic implications
Metabolic lipases control the balance between FA storage
and FA release, thus regulating FA flux from periphery to
the liver. To protect the liver from potentially toxic FA
spillover and development of NAFLD, targeted suppres-
sion of lipase activity in adipose tissue (especially visceral
fat) might represent a therapeutic aim because general
suppression of lipase activity has many risks including
impaired PPAR signaling. The lipolysis inhibitor nicotinic
acid (niacin) reduces free FA levels in the serum by acti-
vating the high-affinity receptor for niacin, GPR109A, in
WAT [83]. Owing to multiple side effects such as flushing
and lack of efficiency (AIM-HIGH study [84]), nicotinic acid
is not suitable as therapy of NAFLD/NASH. Orlistat, an
inhibitor of extracellular but not intracellular lipases, was
tested in some small NAFLD trials, but the effects on liver
histology and body weight were not superior to placebo
[85–87].
NRs such as PPARs and FXR, which have metabolic,
anti-inflammatory, and anti-fibrotic effects, may be valid
targets. PPARa agonists such as fibrates are used to
treat dyslipidemia and hypertriglyceridemia [88,89].
However, studies with PPARa agonists in NAFLD have
generated divergent results. Fenofibrate treatment did
not show beneficial effects on steatosis, assessed by MR
spectroscopy (MRS), in a study with only 15 patients
[90], whereas another small (n = 16) study reported a
reduction in steatosis by fenofibrate in combination with
TEM-980; No. of Pages 10
Review
dietary intervention [88]. Unfortunately, no placebo
group was included and histological data were not
obtained. Another small (n = 16) biopsy-controlled study
revealed a reduction in hepatic alanine transaminase
(ALT) levels, but no histological improvement of key
features such as hepatocellular ballooning, steatosis,
inflammation, or fibrosis was observed [88]. Finally,
short-term (4 weeks) treatment with gemfibrozil resulted
in reduction of serum transaminases [88]. In light of
these data, there is currently very limited evidence to
support a role for fibrates in the treatment of NAFLD/
NASH.
However, treatment with the PPARad dual agonist
GFT505 has lead to promising results [91,92]. After 4–
12 weeks of treatment, serum transaminases, as well as
alkaline phosphatase and g-glutamyl transpeptidase
(gGT), were reduced in a small Phase II trial with patients
displaying dyslipidemia, diabetes, and IR. A biopsy-con-
trolled Phase II study evaluating GFT505 in NASH is
currently ongoing (http://clinical.trials.gov, identifier
NCT01694849) [91].
The therapeutic effects of the PPARg ligands (glita-
zones) in NAFLD can be explained by their actions in
adipose tissue and muscle. PPARg was shown to activate
directly ATGL activity in adipose tissue at the transcrip-
tional level in vitro and in vivo [93], a finding that was
verified by increased ATGL mRNA and protein expression
in adipose tissue of mice treated with the PPARg agonist
rosiglitazone [93]. Therefore, as a lipogenic master regula-
tor, PPARg may regulate lipid partitioning via ATGL
regulation in WAT (Figure 3). Moreover, PPARg activation
increases insulin sensitivity and reduces FA flux to the
liver. In liver, PPARg is expressed in HSCs (maintaining
them in a quiescent state, and showing anti-fibrotic prop-
erties in preclinical studies), and macrophages (where it
displays anti-inflammatory effects) [88]. Another impor-
tant mechanism is the capacity of glitazones to induce the
production of adiponectin, an insulin-sensitizing and anti-
steatogenic adipokine, which increases FA oxidation in
liver and muscle [93]. Studies with PPARg agonists in
diabetic and non-diabetic patients with biopsy-proven
NASH showed improvements in IR, liver enzyme levels,
and in hepatic fat accumulation (assessed by MR), some-
times with improvement of some histological NASH fea-
tures such as steatosis and inflammation, but not
ballooning and fibrosis [88]. A randomized double-blind
placebo-controlled study of 55 patients with impaired glu-
cose tolerance or T2DM and biopsy-proven NASH, testing
the effects of caloric restriction in combination with 6
month pioglitazone versus placebo treatment, uncovered
a reduction in steatosis, inflammation, and ballooning by
pioglitazone [94]. Importantly, fibrosis did not significantly
improve compared to placebo. Another pioglitazone study
in non-diabetic patients with biopsy-proven NASH
revealed reduced liver enzyme levels, hepatic steatosis,
and inflammation, again without improvement of fibrosis
[95]. Although the glitazone trials are very heterogeneous
in outcome, the two most robust and reproducible findings
are the reduction of serum ALT levels and the grade of
steatosis [93]. However, the optimal duration of therapy is
not yet established. A prolonged 3 year study did not
Trends in Endocrinology and Metabolism xxx xxxx, Vol. xxx, No. x
further improve liver histology, suggesting a plateau effect
of histological improvement when the maximal insulin-
sensitizing effect is reached [93]. Furthermore, glitazone-
based improvement of ALT and hepatic IR returned to
baseline after 3 months of treatment cessation, and in few
patients steatohepatitis recurred a 1 year follow-up liver
biopsy [96]. The main restriction for the use of glitazones
came from their rather poor safety profile, and persistent
weight gain that arises mainly from increased peripheral
fat mass [97]. Indeed, glitazones promote preadipocyte
differentiation in subcutaneous fat depots, while inhibiting
adipocyte maturation in visceral fat, thus shifting free FA
storage from liver to more protective fat depots such as
subcutaneous fat [98]. However, weight gain as a cosmetic
(not metabolic) side effect is not desirable. In addition,
based on the increase in cardiovascular events, congestive
heart failure, and bone fractures in women, as well as
bladder cancer risk, glitazones were withdrawn from the
market in France and Germany [93].
PPARd, which is also expressed in HSC and macro-
phages, was shown to have anti-fibrotic features in vivo.
However, human data on NAFLD treatment are lacking
[88].
FXR ligands also improved fatty liver and related IR in
various rodent models of obesity and NAFLD (reviewed in
[12]). Moreover, different FXR agonists such as PX-104
(non-steroidal) and INT747, a chenodeoxycholic acid deriv-
ative also known as obeticholic acid, have entered clinical
trials for NAFLD treatment. A double-blind, placebo-con-
trolled proof-of-concept study evaluated the effects of
INT747 in diabetic patients with presumed NAFLD [99].
Administration of a low (25 mg) and a high (50 mg) INT747
for 6 weeks improved insulin sensitivity as well as liver
inflammation and fibrosis [99]. INT747 was also tested in a
biopsy-controlled NIH/NDDK-sponsored Phase IIb trial
investigating its effects on histologically proven NASH
[FXR ligand NASH Treatment (FLINT) trial; http://clin-
icaltrials.gov/ct2/show/NCT01265498] which was prema-
turely stopped because of positive effects on liver histology.
Concluding remarks and future perspectives
Metabolic lipases are the enzymes responsible for TG
breakdown in WAT and liver. Lipolysis in WAT creates
a free FA flux to the liver, which promotes steatosis devel-
opment in IR conditions. Saturated FAs induce hepatocyte
death and inflammation, which are key players for NASH
development. However, PUFAs exert anti-inflammatory
and anti-steatotic effects. Increased hepatic ATGL activity
generates free FAs, which are PPARa ligands. Pharmaco-
logical activation of FXR and PPARa results in increased
FA catabolism in the liver, whereas PPARg stimulation
favors TG storage, therefore limiting hepatic lipotoxicity.
Indeed, clinical trials with FXR and PPARagd ligands
have resulted in promising results for NAFLD/NASH
treatment. In contrast to NRs, lipases are not yet suitable
as direct pharmacological targets. However, the develop-
ment of tissue-specific lipase modulators (i.e., inhibiting
ATGL and HSL in WAT), while increasing ATGL activity
in the liver, may be desirable. Analysis of tissue-specific
Atgl KO mice might validate this concept (Box 2). In
addition, small peptides targeting PNPLA3 variants
7
TEM-980; No. of Pages 10
Review
Box 2. Outstanding questions
 Which non-invasive markers can stratify the risk for progression in
NAFLD/NASH?
 Does PNPLA3 function as a lipase or an acyltransferase?
 How does PNPLA3 promote fibrosis development in progression
of liver disease?
 What is the role of genetic variants of metabolic lipases (other
than PNPLA3) in NAFLD/NASH pathogenesis and progression?
 Which as yet unknown/metabolic lipases account for the remain-
ing lipolytic activity in liver (not accounted by ATGL, HSL, and
PNPLA3)?
 How can we target genetic variants of metabolic lipases
pharmacologically?
associated with NAFLD/NASH progression reverting the
enzyme activity to normal could provide an innovative
approach to treat NAFLD.
Acknowledgments
This work was supported by grants SFB 30 and SFB 35 (F3008-B19 and
F3517-B20) from the Austrian Science Foundation (to M.T.).
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