3 Triglyceride Metabolism in The Liver

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Compr Physiol. Author manuscript; available in PMC 2019 February 15.
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Published in final edited form as:

Compr Physiol. ; 8(1): 1–8. doi:10.1002/cphy.c170012.
Triglyceride metabolism in the liver

Michele Alves-Bezerra and David E. Cohen
Joan & Sanford I. Weill Department of Medicine, Weill Cornell Medical College, New York, NY,
USA
Abstract
Triglyceride molecules represent the major form of storage and transport of fatty acids within cells
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and in the plasma. The liver is the central organ for fatty acid metabolism. Fatty acids accrue in
liver by hepatocellular uptake from the plasma and by de novo biosynthesis. Fatty acids are
eliminated by oxidation within the cell or by secretion into the plasma within triglyceride-rich very
low density lipoproteins. Notwithstanding high fluxes through these pathways, under normal
circumstances the liver stores only small amounts of fatty acids as triglycerides. In the setting of
overnutrition and obesity, hepatic fatty acid metabolism is altered, commonly leading the
accumulation of triglycerides within hepatocytes, and to a clinical condition known as non-
alcoholic fatty liver disease (NAFLD). In this review, we describe the current understanding of
fatty acid and triglyceride metabolism in the liver and its regulation in health and disease,
identifying potential directions for future research. Advances in understanding the molecular
mechanisms underlying the hepatic fat accumulation are critical to the development of targeted
therapies for NAFLD.
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Introduction
The liver is the central organ that controls lipid homeostasis by means of complex, but
precisely regulated biochemical, signaling and cellular pathways. Hepatocytes are the main
liver parenchymal cells, which control hepatic biochemical and metabolic functions in the
liver, including triglyceride metabolism. Additional cell types within the liver include
cholangiocytes, Kupffer cells, stellate cells, and endothelial cells, each of which has
specialized functions in hepatic pathobiology (261).
This review will focus on the hepatic metabolism of fatty acids (FA) and their neutral
storage form, triglycerides (TG), which occurs primarily in hepatocytes. Under normal
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circumstances on a daily basis, the liver processes large quantities of FA, but stores only
small amounts in the form of TG, with steady state TG contents of less than 5% (32). This is
because the rates of acquisition of FA by uptake from the plasma and from de novo synthesis
within the liver are balanced by rates of FA oxidation and secretion into plasma as TG-
Cross-References
Energy metabolism in the liver
Metabolism of lipids in chylomicrons and very low density lipoproteins (legacy)
Obesity
Steatosis in the liver
Triglyceride metabolism (legacy)
Alves-Bezerra and Cohen Page 2
enriched very low-density lipoprotein (VLDL-TG). The relatively small quantities of TG

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stored within the liver are localized in cytoplasmic lipid droplets.
NAFLD is the most common chronic liver disease and is characterized by excess TG
accumulation within the liver. It is associated with obesity, type 2 diabetes and dyslipidemia,
and commonly occurs in the setting of insulin resistance (22). NAFLD encompasses a
spectrum of liver histopathologies from simple hepatic steatosis, often referred to as non-
alcoholic fatty liver (NAFL), to non-alcoholic steatohepatitis (NASH) which hepatic
steatosis is accompanied by inflammation, cell death and fibrosis (5). Hepatic complications
of NASH include cirrhosis and hepatocellular carcinoma. Currently, there are no effective
therapies for NAFLD apart from changes in lifestyle that lead to improved physical fitness
and weight loss. In order to develop effective pharmacologic therapies, detailed knowledge
of the cellular and molecular mechanisms leading to altered hepatic lipid metabolism is
essential. In addition to describing the major pathways of hepatic FA and TG metabolism
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and their regulation, we will discuss recent advances in our understanding of how they are
altered in the setting of NAFLD.
Hepatic fatty acid metabolism

FA within the liver originate from either dietary or endogenous sources. Dietary TG are
emulsified by bile acids within the intestinal lumen following their hydrolysis primarily by
pancreatic lipase (166), which yields sn-2-monoacylglycerols and free FA as products.
Following emulsification, these lipid molecules are taken by enterocytes and resynthesized
into TG. TG are packaged into chylomicrons, secreted into the lymphatic system and
ultimately reach the plasma (112, 122). Much of the chylomicron TG are taken up by muscle
and adipose tissue due to the activity of lipoprotein lipase, which is expressed on the luminal
surfaces of capillary endothelial cells of these tissues (Fig. 1). TG remaining within the
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chylomicron remnants are delivered to the liver when these particles are taken up by receptor
mediated endocytosis, and FA are released during lysosomal processing of the particles (54).
When carbohydrates are abundant, the liver converts glucose into FA, a process referred to
as de novo lipogenesis (DNL) (120) (Fig.1). The control of DNL is primarily transcriptional.
Plasma insulin activates the endoplasmic reticulum membrane-bound transcription factor
sterol regulatory element binding protein 1C (SREBP1c), the N-terminus of which
translocates to the nucleus and upregulates all genes in the FA biosynthetic pathway (116).
The hepatic uptake of excess plasma glucose promotes the nuclear translocation of
carbohydrate response element binding protein (ChREBP), a transcription factor that also
upregulates transcription of the majority of FA biosynthetic genes plus pyruvate kinase (89,
205), which increases the availability of citrate for FA synthesis.
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Another important source of FA is direct uptake from the plasma. In human subjects, FA
from plasma constitute the main source of hepatic triglycerides during fasting (72). Under
fasting conditions when plasma insulin concentrations are low, a lipolytic program is
initiated in white adipose tissue, which increases the plasma FA pool that is available for
uptake by the liver (12, 214) (Fig. 1). FA are largely albumin-bound within the circulation.
Several steps are involved in hepatic FA uptake. These include dissociation of FAs from
albumin, transport across the hepatocyte plasma membrane, binding to intracellular proteins,

and esterification to coenzyme A (CoA). Plasma FA are also the principal source of VLDL-
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TG in both fasting and fed states (72).
Within the hepatocyte, FA are esterified to glycerol-3-phosphate (G3P) and to cholesterol in

order to generate TG or cholesteryl esters, respectively. These neutral lipids can be either
stored in cytoplasmic lipid droplets (LDs), or secreted into bloodstream as VLDL particles
(135) (Fig. 1). FA within the liver may also be used for the synthesis of other complex lipids,
including phospholipids (PL). During fasting, FA are used both as local energy supply as
well as substrate for ketone bodies production. Overall, hepatic FA metabolism is tightly
regulated by multiple interrelated transcriptional and signaling pathways that remain the
subject of intensive investigation and discovery.
Fatty acid uptake

Notwithstanding their ability to diffuse across a lipid bilayer (232), FA are taken up by
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hepatocytes via plasma membrane-associated proteins. A variety of proteins have been

associated with long-chain FA transport, including plasma membrane FA-binding protein
(FABPpm), FA translocase (FAT)/CD36, caveolin-1, and very long-chain acyl-CoA
synthetases (ACSVL/FA transport proteins, also named FATP/solute carrier family 27A1–6,
SLC27A1–6) (27, 253) (Fig. 2).
The roles of CD36 and FABPpm proteins in FA uptake are established for heart and skeletal
muscle, but their function in hepatocytes remains unresolved. Although expression levels are
low in the liver, CD36 mRNA levels are positively correlated with hepatic TG contents in a
rat model of hepatic steatosis (35) and its protein expression is increased in patients with
NAFLD (181). However, the pathophysiological relevance of this protein to the liver is
unclear. Whereas CD36 liver-specific deletion in mice leads to reduced hepatic lipid
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contents and decreased FA uptake in the setting of high fat feeding, suggesting the direct
contribution of CD36 to hepatic FA metabolism under these conditions (279), whole body
knockout of CD36 in mice significantly impairs FA uptake by heart, adipose tissue, and
muscle, but normal uptake is observed in the liver (52). There also seems to be a role for
CD36 in hepatic lipogenesis. CD36 is a target gene for the aryl hydrocarbon receptor (AhR),
and is required for the TG accumulation observed in the liver in response to its activation
(154). In addition, the pregnane X receptor (PXR) induces hepatic CD36 mRNA expression,
along with increased FA uptake and TG accumulation in the liver (294). Finally, the CD36 is
a transcriptional target of peroxisome proliferator activated receptor (PPAR) γ (259), which
is upregulated by PXR (294).
Functional evidence for protein-mediated fatty acid transport in the rat hepatocyte plasma
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membrane led to the identification of FABPpm as a protein that mediates this activity (247,
249). FABPpm was subsequently identified as mitochondrial aspartate aminotransferase (20,
28). The mRNA encoding this protein is increased in hepatocytes of genetically obese
mouse models (180), and the binding of oleate to rat hepatocyte plasma membranes is
reduced after treatment with specific antiserum (246). In cultured hepatocytes, ethanol
treatment increases mRNA and plasma membrane protein contents, and these changes
correlate with increased FA uptake (295). However, the relative contribution of FABPpm to
FA uptake by the liver remains unknown.

FATP proteins (1–6) are FA-transporting proteins that are situated in the plasma membrane,
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as well as intracellular membranes. Because of their capacity to also activate long- or very-
long-chain FA (69), these proteins have been re-classified in the very-long-chain acyl-CoA
synthetase (ACSVL) family as ACSVL1–6 (106). FATPs couple FA uptake with intracellular
FA esterification into acyl-CoAs, a metabolic trapping mechanism that has been referred to
as vectorial acylation (77, 258). Although well characterized, neither FATP1 nor FATP4 are
normally expressed at substantial levels in the liver (239). Expression of FATP4, an ER
membrane-associated isoform (226, 273), is increased in the setting of hepatic steatosis and
may play a role in palmitate-mediated lipoapoptosis (226). FATP2 is highly expressed in
liver and kidney (85). In HepG2 cells, FATP2 localizes to the ER, but its overexpression
increases uptake of long-chain FA in cell culture (148). FATP5 is expressed in the liver, and
studies in knockout mice and cell culture systems support a role in FA uptake (70).
Caveolins (types 1–3) are integral membrane proteins that are essential components of
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caveolae, vesicular membrane microdomains that are rich in cholesterol and sphingolipids
(6). Caveolins were initially characterized with respect to cholesterol transport (204).
However, their roles in FA transport have been appreciated in experiments using HepG2
hepatoma cells (203), wherein Caveolin-1, CD36, FABPpm, and calcium-independent
membrane phospholipase A2 (iPLA2) form a heterotetrameric protein complex within
plasma membrane microdomains (248). These complexes promote FA accumulation within
caveolae-derived vesicular structures for transport into the cell (247). In support of this
mechanism, mice deficient in caveolin-1 show reduced hepatic TG content and impaired LD
accumulation (87).
Although multiple factors lead to hepatic steatosis, circulating FA are the main source of
hepatic lipids in NAFLD (72). The overflow of FA derived from excessive lipolysis in the
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adipose tissue contributes to the pathogenesis of insulin resistance in mice and humans
(159), leading to both type 2 diabetes (213) and NAFLD (27, 171). Although FA transporters
may exert a central role in the control of FA flux into the liver, additional studies are needed
to clarify specific functions and physiological contributions of each FA transporter, along
with their differential regulation in the context of NAFLD.
De novo lipogenesis
Under normal circumstances in humans, pathways of hepatic DNL appear to be utilized
sparingly (114). However, in addition to the enhanced FA uptake, increased DNL can
contribute substantially to hepatic steatosis (72). The first step in DNL pathway is catalyzed
by ATP-citrate lyase (ACLY), which converts citrate to acetyl-CoA, which is then
carboxylated to malonyl-CoA by acetyl-CoA carboxylase (ACC). ACC is followed by a
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series of reactions that convert malonyl-CoA into palmitate. Fatty acid synthase (FAS) is the
key rate-limiting enzyme in palmitate synthesis, and its activity is regulated by multiple
mechanisms (125, 143, 218, 228) (Fig. 2). Palmitate can be modified by elongases and
desaturases to generate a variety of FA species.
There are two ACC isoforms, cytosolic ACC1 and mitochondrial ACC2, which are encoded
by separate genes. The presence of distinct ACC enzymes enables the formation of two
distinct pools of malonyl-CoA. ACC1-generated malonyl-CoA is utilized as a substrate by

FAS, whereas the malonyl-CoA produced by ACC2 plays a key role in the negative
regulation of β-oxidation by inhibiting carnitine palmitoyltransferase 1 (CPT1) (266). ACC1
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is highly expressed in lipogenic organs, including the liver, wherein the gene expression and
enzyme activity are induced by carbohydrate-rich, low-fat diet, and are suppressed in the
setting of starvation or diabetes (139, 149). Whole body disruption of the ACC1 gene results
in embryonic lethality, but liver-specific knockout mice are viable and exhibit 70–75%
reductions in malonyl-CoA levels and 40–70% reductions in hepatic TG contents (170).
Despite similar malonyl-CoA levels as wild type animals, livers of ACC2 knockout mice are
characterized by reduced rates of β-oxidation and TG contents. These mice resist to diet-
induced obesity and diabetes (2, 3). In keeping with these observations, antisense-mediated
knockdown of both ACC1 and ACC2 led to improved hepatic insulin responsiveness in rats
with experimental NAFLD (217).
FAS expression is ubiquitous, but evidence of elevated transcriptional and enzymatic

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activities are observed in the liver of murine models of obesity, such as Zucker (fa/fa) rats
(227). The transcription of both ACC and FAS genes is enhanced in livers of NAFLD
subjects (146). Similar to ACC knockout mice, whole body disruption of FAS leads to
embryonic lethality (51). Surprisingly, liver-specific knockout of FAS results in
hypoglycemia and hepatic steatosis in mice fed a zero-fat diet (45). Because this phenotype
is reversed by treatment with a PPARα agonist, it has been suggested that newly synthesized
FA and their derivatives, including phospholipids (44), constitute a distinct pool that
provides endogenous ligands for PPARα. This in turn stimulates hepatic gluconeogenesis
and FA oxidation (45).
In leptin-deficient obese (ob/ob) mice, increased transcription of Acc1 and Fas is linked to
the increased activation of ChREBP (64). Additionally, SREBP-1c and ChREBP can
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synergistically induce the mRNA expression of Acc and Fas in the liver (65). Expression of
both transcriptional factors are upregulated in livers of mouse models of NAFLD (19),
however only SREBP-1c is upregulated in patients with NAFLD (115).
Among TG found in VLDL in normal subjects, less than 5% contain FA derived from DNL
(68). However, inhibition of DNL impairs production and secretion of VLDL-TG in rats
(95). The rate of incorporation of DNL-synthesized FA into VLDL-TG is increased in
normal subjects in the setting of high-carbohydrate diet (223, 224), alcohol consumption and
infectious states (88), and is positively correlated with increased plasma VLDL-TG content
(223). DNL is also increased in obese (67, 244) and NAFLD subjects (68, 88). These
observations suggest that hypertriglyceridemia is at least in part attributable to increased
availability of DNL-synthesized FA for TG synthesis and VLDL production (223). Ongoing
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research is needed to clarify mechanisms of altered DNL in a variety of pathogenic

conditions, including NAFLD.
Fatty acid activation

Activation by thioesterification to CoA to form fatty acyl-CoA molecules is an obligatory
step in the metabolism of long-chain FA. This reaction is catalyzed by members of the long-
chain acyl-CoA synthetase (ACSL) family of enzymes (Fig. 2). The five ACSL isoforms
found in mammalians have specific tissue distributions, subcellular locations and substrate

preferences (106). Additionally, distinct ACSL isoforms play key roles in partitioning fatty
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acyl-CoAs into different metabolic pathways (57, 106).
ACSL1, the best characterized isoform, is abundantly expressed in a variety of tissues.

Because Acsl1 is a target gene of PPARα in rat liver, this enzyme has been associated with
FA catabolism (220). ACSL1 interacts physically with CPT1 in the outer mitochondrial
membrane of rat hepatocytes, possibly functioning to channel fatty acyl-CoA products into
mitochondria for β-oxidation (155). However, this process is not fully understood because
liver-specific ACSL1 knockout mice exhibit only modestly decreased rates of β-oxidation
and reduced fatty acyl-chain incorporation into newly-synthesized TG, although the total
hepatic TG contents remain unchanged (161).
ACSL3 is expressed on lipid droplets in HuH7 hepatoma cells (94), and its expression is up-
regulated in livers of ob/ob mice and in mice fed a high-carbohydrate diet (34). Knockdown
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of ACSL3 in rat hepatocytes reduces DNL through diminished activation of lipogenic

transcriptional factors (i.e. PPARγ, ChREBP, SREBP-1c and LXRα) possibly due to the
reduced availability of FA species, acyl-CoA or their lipid intermediates (34).
Suggestive of a role for ACSL5 in TG synthesis, mRNA levels are increased by insulin and
by SREBP-1c activation (4). In support of this lipogenic role, overexpression of ACSL5 in
rat hepatoma cells increases uptake of exogenous FA, as well as its partitioning into TG, but
not into PL (174). Knockdown of ACSL5 in rat primary hepatocytes strongly influences the
partitioning of fatty acyl-CoAs between lipogenic and lipolytic pathways. In the absence of
this enzyme, the incorporation of exogenous and endogenous FA into complex lipids is
reduced, the formation of LD is decreased and the oxidation of FA is stimulated (33).
The reaction catalyzed by ACSL isoforms is reversed by the activities of acyl-CoA

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thioesterases (ACOTs) (Fig. 2). These enzymes catalyze the hydrolysis of acyl-CoA
molecules into FA and CoA. Despite their relatively well-investigated enzymatic activities,
the biological functions of the fifteen ACOT family members remain less well understood
(53, 142, 256). Acots 1–6, which constitute the type I enzymes, are upregulated in the livers
of fasted and high fat-fed mice under the transcriptional control of PPARα (79). These
observations suggest a role of type I ACOTs in lipid catabolism. Indeed, overexpression of
ACOT2 in the liver leads to increased fatty acid oxidation and ketogenesis, although the
mechanism underlying this effect remains unclear (182).
ACOTS 7–15 comprise the type II enzymes, and some of these contribute to hepatic FA
metabolism. ACOT11 (synonym, Thioesterase superfamily member 1, Them1) is highly
expressed in brown adipose tissue, where it functions to suppress energy expenditure (292)
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by decreasing rates of fat oxidation (196). Acot11 gene expression is also strongly induced
in the liver of obese mice (79). Whereas deficiency of this enzyme results in decreased
hepatic steatosis, improved glucose homeostasis, and resistance to diet-induced diabetes in
high fat fed mice (292), it is unclear whether these phenotypes are indirect effects of
increased energy expenditure.
ACOT13 (a.k.a. Them2) is a mitochondria-associated enzyme that is highly expressed in the

liver and other oxidative tissues. Them2 knockout mice are resistant to diet-induced hepatic

steatosis and exhibit improved glucose metabolism (130). Moreover, Them2-deficiency

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results in diminished expression of PPARα and HNF4α, possibly due to the role of this
enzyme in the control of FA ligands of these nuclear receptors (53) and the reduced FA
oxidation (135). Them2 also controls lipid and glucose metabolism by interacting with
phosphatidylcholine transfer protein (PC-TP) to suppress hepatic insulin signaling by
reducing the activation of both the insulin receptor substrate 2 (IRS2) and the mammalian
target of rapamycin (mTOR) (80).
ACOT15 (a.k.a. Them5) appears to play a key role in maintaining the integrity of
mitochondrial membranes. Mice lacking ACOT15 experience deficiencies in cardiolipin
remodeling in the inner mitochondrial membrane of hepatocytes. This phenotype leads to
mitochondrial dysfunction, including altered morphology, decreased mitochondrial FA
contents, reduced FA oxidation, and the development of liver steatosis (296).
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It is plausible that ACSL and ACOT enzymes coordinate the control of the intracellular
balance of fatty acyl-CoAs and FA, intracellular and intra-organelle concentrations of CoA,
and the availability of lipid substrates for a variety of metabolic pathways. Both types of
enzymes exhibit specific transcriptional patterns of expression that vary depending on cell
type and physiological state, and the coordinated regulation of ACSL and ACOT enzymes
has been recently described (79). Although elevated hepatic concentrations of long-chain
acyl-CoAs have been associated with increased plasma insulin levels and hepatic insulin
resistance (48), decreased hepatic fatty acyl-CoA concentrations in the setting of liver-
specific deletion of ACSL1 do not prevent the development of insulin resistance in high-fat
fed mice (161). As illustrated by these apparently contradictory observations, mechanisms
whereby ACSL and ACOT enzymes control FA metabolism within hepatocytes are
incompletely understood, and it remains uncertain whether their altered regulation
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contributes to insulin resistance and related metabolic abnormalities.
Intracellular fatty acid transport

Long-chain FAs and their acyl-CoA derivatives act not only as substrates for lipid synthesis
and oxidation, but are also involved in a range of diverse intracellular processes, such as
protein palmitoylation, intracellular signaling, and activation of transcription factors (83,
106, 222). Considering this multiplicity of cellular functions, their relative insolubility and
potential cytotoxicity (102, 103), it is logical that the intracellular concentrations and
localization of free FA and fatty acyl-CoAs are tightly regulated. This is accomplished at
least in part due to the activities of lipid binding proteins, several of which have been
implicated in the control of the intracellular concentration and the partitioning of long-chain
FA and acyl-CoA within hepatocytes. These include the liver fatty acid binding protein (L-
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FABP, synonym FABP1), acyl-CoA binding protein (ACBP; also named as acyl-CoA-
binding domain containing protein-1, ACBD1, or diazepam binding inhibitor, DBI), and
sterol carrier protein-2 (SCP2) (8, 103) (Fig. 2).
The FABP protein family comprises 10 members (237). FABP1 is abundant in the cytosol of
hepatocytes, comprising 2–5% of the cytosolic protein content (177) and reversibly binds
long-chain FA, fatty acyl-CoAs, lysophospholipids, cholesterol and other lipids (14, 36, 254,
255). Fabp1−/− male mice fed standard chow diet exhibit normal gain weight and liver

weights (172, 192). However, these animals have reduced total hepatic TG contents as well
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as decreased rates of FA esterification into TG, with preserved or increased esterification

into PL (172, 192). Because the overexpression of FABP1 in L-cell fibroblasts enhances the
intracellular traffic of short-, medium-, and long-chain FA from the cytosol to the nucleus
(119), it has been conjectured that FABP1 must enter nuclei to promote the entry of FA
(222). Indeed, FABP1 directly interacts with PPARα within nuclei of primary hepatocytes
(117). This interaction may represent a mechanism for the delivery of FA ligands that
activate this key transcription factor (222). FABP1 ablation is protective against diet-induced
obesity and hepatic steatosis and improves glucose metabolism in mice fed a Western (high
saturated fat, high cholesterol) diet (193). Taken together, these findings suggest a role for
FABP1 in FA partitioning into specific metabolic pathways.
ACBP binds medium- and long-chain acyl-CoA with high affinity (84, 207, 211). Levels of
ACBP mRNA and protein are highest in the liver (26). In the mouse, ACBP expression is
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modulated by nutritional status. Fasting results in decreased hepatic ACBP expression levels,
whereas high fat feeding increases it (21). Overexpression of ACBP in rat hepatoma cells
increases TG content (283). In addition, hepatic TG and PL concentrations are increased in
transgenic mice that overexpress ACBP in the liver (118). Under these conditions,
microsome-associated glycerol-3-phosphate acyltransferase (GPAT) activity is induced,
suggesting that ACBP has a role in the formation and partitioning of an acyl-CoA pool
towards glycerolipid synthesis (118).
Expression of SCP-2 is high in liver and intestine (15). This protein has high affinity for
binding both long-chain FA and acyl-CoAs, with Kd values in the same range as reported for
FABP and ACBP (103, 144, 177). Knockout mice exhibit reduced plasma FA
concentrations, decreased hepatic cholesteryl ester and TG contents, as well as increased
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rates of β-oxidation (225). SCP-2 ablation also results in alterations in the composition and
physical properties of lipid rafts from mice primary hepatocytes, including the enrichment in
PL contents (9). Moreover, hepatic SCP-2 and FABP1 act to facilitate hydrolysis of
cholesteryl ester molecules associated with high-density lipoprotein (HDL) particles and
increasing the biliary secretion of bile acids (270) and cholesterol (92).
Because the metabolic fates of FA and acyl-CoA molecules are linked to these intracellular
lipid biding proteins, it is tempting to speculate that they could be leveraged to manipulate
the flux of lipids between anabolic and catabolic pathways in order to defend against the FA-
mediated lipotoxicity, that contributes to the pathogenesis of NAFLD.
Triglyceride metabolism
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The assembly of TG molecules constitutes the principal means by which the liver stores and
exports FA. Under normal conditions, the liver stores little TG, but exports considerable
amounts in the form of VLDL particles that deliver FA to muscle and fat tissue, depending
on nutritional status. Whereas previously it was considered that excess TG stores in the
setting of NAFLD contributed to lipotoxicity (61), emerging concepts suggest that increased
TG storage and VLDL secretion are instead protective against FA-mediated hepatotoxicity.

Triglyceride synthesis
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In most mammalian cell types, the G3P pathway is the principal route for the synthesis of
TG, contributing over 90% of total TG synthesis (56, 62). The first and rate-limiting step of
this pathway is the esterification of long-chain acyl-CoA to G3P, which is catalyzed by
mitochondrial and microsomal G3P acyltransferase (GPAT) enzymes. Lysophosphatidic acid
(LPA) molecules produced in this reaction are then acylated to form phosphatidic acid (PA)
by the acylglycerol-3-phosphate acyltransferases (AGPAT) present in the ER membrane. PA
can be converted into cytidine diphosphate diacylglycerol (CDP-DG), which is a substrate
for the synthesis of certain glycerolphospholipids and cardiolipins (113, 231) or can be
dephosphorylated by phosphatidate phosphohydrolase (PAP, synonym Lipin) to form DG,
which serve as precursor molecules for the synthesis of TG, as well as phosphatidylcholine
(PC) and phosphatidylethanolamine (PE) (56, 73). DG acyltransferase (DGAT) catalyzes the
acylation of DG, constituting the final step of TG synthesis (55). Newly synthesized TG
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molecules are then directed from ER lipid bilayer to form cytosolic LDs (99, 278) (Fig. 3).
Four isoforms of GPAT are encoded by distinct genes (276). In the liver, the mitochondria-
associated GPAT isoforms (GPAT1 and 2) contribute 30–50% of total GPAT activity (56).
GPAT1 is highly expressed in the liver (157). It is induced after fasting/refeeding and in
response to insulin, and downregulated via cAMP-dependent signaling pathway during
nutrient deprivation (76, 186, 230). GPAT1-deficient mice exhibit increased rates of FA
oxidation and concentrations of plasma β-hydroxybutyrate. Moreover, hepatic TG contents
are decreased by 60%, suggesting that GPAT1 functions in the partitioning of FA towards
TG synthesis and away from oxidation in the liver (110). GPAT1 deficiency also leads to
reduced plasma TG levels, reduced VLDL secretion rates and decreased total body weights
(110, 191). Accordingly, the overexpression of GPAT1 in rat hepatocytes leads to increased
esterification of oleate into LPA, DG and TG, and to increased intracellular TG contents
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(158, 165, 188). Although highly expressed in the liver, the metabolic role(s) of GPAT2 in
the lipid metabolism remain unclear. This isoform is not regulated by either insulin or
fasting/refeeding (271), and mouse models with GPAT2 loss or gain of function have yet to
be reported.
GPAT3 and GPAT4 account for microsomal GPAT activity. These isoforms are modulated by
nutritional status and hormonal variations. Although initially considered to mainly mediate
PL synthesis (55), more recent studies have revealed their contributions to TG metabolism in
adipose tissue. Specific roles for GPAT3 and GPAT4 in hepatic lipid metabolism remain
largely undefined. The overexpression of GPAT4 in HepG2 cells leads to 20% increase in
intracellular TG content (189), and its knockout in mice results in 40–50% reduction in
hepatic TG (37, 262). It is noteworthy that hepatocytes from mice lacking GPAT4 are able to
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incorporate de novo synthesized FA into TG (275), suggesting that this isoform could be
relevant for the metabolism of exogenous FA.
The second step in TG synthesis is mediated by AGPAT enzymes. Although ten AGPAT
enzymes have been identified on the basis of sequence homology, the enzymatic activity for
LPA acylation has been confirmed for only a few (252). Certain isoforms initially described
as AGPAT were later reclassified into different acyltransferase groups. For example,
AGPAT6 and AGPAT8 are currently designated as GPAT4 and GPAT3, respectively (38, 49,

189). AGPAT1 and AGPAT2 are localized in the ER (74, 97, 150, 277) and are highly
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expressed in the liver (75, 240, 252, 277). However, the contribution of these enzymes to the
hepatic metabolism remains unknown.
Lipins-1–3 constitute the PAP enzyme family. Under lipogenic stimuli, these proteins
translocate from the cytosol to ER membrane in order to access their substrate PA and
contribute the TG biosynthetic pathway (31, 173). Additionally, lipin-1 can localize in the
nucleus of hepatocytes, where it acts as a transcriptional co-activator (234). Fasting and
glucocorticoid administration result in increased lipin-1 expression in mouse livers (90, 169,
195). This effect is mediated by PPARγ coactivator 1α (PGC-1α) and is required to sustain
the fasting-induced expression of PPARα and its downstream targets. Lipin-1 physically
interacts with PGC-1α and PPARα in the nucleus, leading to upregulation of oxidative fatty
acid metabolism by mitochondria (90). Accordingly, mice lacking lipin-1 exhibit hepatic
steatosis (90), enhanced stearoyl-CoA desaturase-1 (Scd1) gene expression, increased rates
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of VLDL secretion, and increased plasma concentrations of TG without changes in TG

synthetic rates (50). Reduced lipin-1 expression is observed in livers of obese diabetic mice,
and the overexpression of lipin-1 in this animal inhibits VLDL secretion and improves
hepatic insulin responsiveness (50). The effects of lipin-1 on VLDL secretion are
independent of PAP enzymatic activity and instead linked to its nuclear receptor interactions
(50). In addition to lipin-1, high levels of lipin-2 expression are also observed in the liver
(71) and hepatic lipin-3 transcription is increased in lipin-1-deficient mice (71). Moreover,
lipin-2 mRNA levels are induced in lipin-1-deficient mice, supporting the hypothesis that
lipin isoforms could compensate in order to preserve PAP activity and restore the levels of
TG synthesis (107).
In mouse liver, DGAT activity is detected in two microsomal membrane locations: luminal
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activity (DGAT1, measured as latent – luminal – DGAT activity) and cytosol-accessible

activity (predominantly DGAT2, measured as overt DGAT activity) (200). The former
contributes to the synthesis of TG that are packaged into VLDL. The latter contributes to the
synthesis of a TG pool to be stored in the cytoplasmic LDs (1, 200, 272). Interestingly, the
DGAT proteins are genetically unrelated. DGAT1 is part of the membrane bound O-acyl
transferase (MBOAT) protein family, which comprises a diverse group of acyltransferases,
including acyl-CoA:cholesterol acyltransferase (ACAT) and protein-cysteine N-
palmitoyltransferase (286). DGAT2 is a member of diacylglycerol acyltransferase (DAGAT)
family that also includes the monoacylglycerol acyltransferase (MGAT) and acyl-CoA wax
alcohol acyltransferases (AWAT) (286).
Although DGAT1 and DGAT2 are able to catalyze the same reaction, these enzymes do not
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appear to be redundant in their respective functions. DGAT1 mRNA is modestly expressed

in the liver in comparison to other organs, such as skeletal muscle and small intestine (41).
This enzyme is able to catalyze acyltransferase reactions using substrates other than DG. For
example, DGAT1 acylates MG, wax esters and retinol in vitro (285). DGAT1 predicted
topology combined with results obtained from loss of function studies supports the
involvement of this enzyme in both the overt and latent DGAT activities (286, 289).
Accordingly, overexpression of DGAT1 in rat hepatoma cells leads to increased intracellular
TG contents and also increased rates of VLDL-TG secretion (164). Dgat1 knockout mice

have slightly reductions in hepatic TG concentrations when fed a low fat diet, with more
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prominent decreases being observed in response to high fat feeding (47, 238). High fat fed
Dgat1 knockout high fat fed mice are also resistant to obesity and have improved insulin
responsiveness (238). Moreover, DGAT1 expression is increased in NAFLD subjects and
may contribute to hypertriglyceridemia (146).
By contrast, Dgat2 transcripts are abundant in the liver, and are decreased upon fasting and
increased after refeeding (179). DGAT2 exhibits in vitro specificity for the use of DG
molecules as substrates (42). The predicted topology of this protein suggests its contribution
to overt activity, and this was confirmed in HepG2 cells by studies using DGAT2 inhibitors
(98). Accordingly, DGAT2 is found in close association to cytosolic LDs and mitochondria-
associated membranes, where enzymes involved in TG synthesis and storage are also
localized (178, 242). Dgat2 knockout mice exhibit severe lipopenia and die in the first day
after birth (243). The liver-specific overexpression of DGAT2 in chow-fed mouse leads to
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the development of hepatic steatosis but no abnormalities in plasma glucose and insulin
signaling were observed, suggesting that hepatic TG accumulation is not a cause of insulin
resistance (183). In agreement with this observation, human DGAT2 genetic variants reduce
hepatic steatosis. However, the decrease in fat content does not lead to improved insulin
sensitivity (131).
In keeping with specialized cellular functions of the enzymes, gain and loss of function
studies in mice have revealed different phenotypes for DGAT1 and DGAT2, without
evidence of compensation (243). In fact, a more recent study revealed a unique role of
hepatic DGAT2 in its use of de novo-synthesized acyl-CoA and DG as substrates (281). The
TG formed by this reaction are stored in cytosolic LDs and ultimately undergoes lipase-
mediated hydrolysis. The resulting lipid intermediates (i.e., MG and DG) are available for
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re-esterification by overt DGAT1 activity present in the cytosolic compartment, followed by

TG transport into the ER. These intermediates can also undergo translocation to the ER
lumen followed by acylation catalyzed by latent DGAT1 activity (Fig. 3A). Overt and latent
activity of DGAT1 contribute to lipoprotein lipidation during VLDL assembly and utilize
pre-formed, but not de novo-synthesized acyl-CoA as preferred substrate (Fig. 3B).
Therefore, DGAT2 and DGAT1 use independent pools of acyl-CoA for TG synthesis, and
act in a sequential and integrated way for TG packaging into nascent VLDL particles (281).
Although hepatic TG accumulation is the major determinant of NAFLD (61), lipid and
lipidomic analyses have revealed changes in the compositions of a variety of lipids,
including FA, DG, LPA, PA, PL and ceramides (78, 127, 168, 216, 260). The specific
contributions of hepatic lipid species to NAFLD remain the subject of active investigation
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(136).
VLDL assembly and secretion

TG-rich VLDL particles represent the mechanism by which FA are exported from the liver
and delivered to muscle for oxidation and adipose tissue for storage, respectively. VLDL
assembly is a two-step process that begins in the ER lumen. In the first step, microsomal
triglyceride transfer protein (MTP) acts to incorporate a small amount of TG in apoB100 as
it is being translated by ribosomes and translocated across the ER membrane (101). In the

second step, additional TG is packaged into the nascent apoB100-containing particles as

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they are traverse from the ER to the Golgi apparatus to form VLDL particles (Fig. 4) (54).
The link between increased VLDL secretion and metabolic diseases, such as insulin
resistance and diabetes, is well stablished (54, 251, 297). The development of insulin
resistance leads to hepatic TG accumulation owing to both enhanced FA uptake into the liver
and increased DNL. The greater availability of TG together with higher MTP activity
promote the overproduction of VLDL particles (10) and greater plasma TG concentrations
(54). A positive correlation between plasma TG concentrations and the hepatic steatosis is
not always observed (63). Obese leptin-deficient ob/ob mice exhibit decreased VLDL
production rates despite marked hepatic steatosis (163). In nondiabetic obese NAFLD
subjects, VLDL-TG secretion rates are elevated up to 2-times in comparison to normal
subjects (82). In this setting, higher rates of TG accumulation fail to increase VLDL-TG
proportionately, suggesting a limited capacity for increasing VLDL production and secretion
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(81).
Reductions in VLDL secretion can also lead to hepatic steatosis. This occurs in the setting of
genetic defects in apoB100 and MTP, which lead to hypobetalipoproteinemia and
abetalipoproteinemia, respectively (274). Mipomersen and lomitapide are drugs that
respectively target apoB100 and MTP in the interest of reducing plasma LDL cholesterol
concentrations by blocking VLDL secretion (58, 263). A mechanism-based side effect of
these medications is hepatic steatosis. Finally, a polymorphism in Transmembrane 6
superfamily member 2 (TM6SF2) has been identified by human genome-wide association
studies to be associated with hepatic steatosis (147, 236). The polymorphism results in
reduced VLDL secretion rates, because of impaired lipidation of nascent VLDL particles
(236). Whether the control of VLDL secretion can be leveraged in the management of
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NAFLD remains unclear.
Lipid droplet biology

LDs are dynamic cellular structures that transiently store lipids. Second to white adipose
tissue, the liver has the next greatest capacity to store TG in LDs, such that overnight fasting
leads to hepatic LD formation that accommodates FA derived from adipose tissue lipolysis
(267). The neutral lipid composition of LDs differs among different hepatic cell types.
Whereas hepatocytes harbor TG-enriched LD cores, stellate cell LDs store predominantly
vitamin A as retinyl esters (23). The TG-rich cores of LDs in hepatocytes are surrounded by
a coat comprised of PL and proteins. The PL are primarily PC and form a monolayer around
the neutral lipid core (13). CTP-phosphocholine cytidylyltransferase (CCT) is the rate-
limiting enzyme in the Kennedy pathway for PC biosynthesis. Indicative of its role in LD
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biogenesis, CCT is recruited from the cytosol to expanding LDs in a variety of cell types in
order to provide PC for LD surface expansion (278). However, the precise role of CCT on
LD formation within hepatocytes is uncertain.
In addition to preserving the LD structure, LD-associated proteins regulate the formation,

expansion and contraction of LDs. These proteins include selected enzymes of lipid
synthesis, as ACSL3 and DGAT2 (Fig. 4). Changes in the composition and activity of the
protein components of the LD have been implicated as contributing to metabolic disease (40,

99, 100, 105). The predominant hepatocellular LD proteins are the members of perilipin
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(PLIN; former perilipin/ADRP/TIP47, PAT) protein superfamily (PLIN1–5). These proteins

in adipocytes are involved both in the stabilization of LD structure as well as in the control
of availability of substrates for several LD-associated enzymes (141). Perilipin 1 (PLIN1;
synonym Perilipin A) is normally only expressed in LDs from adipocytes, whereas PLIN2
(synonym ADRP) and PLIN3 (synonym TIP47) are associated with LDs in hepatocytes
(245). Plin1 mRNA is expressed in livers of subjects with NAFLD (104), and PLIN1 protein
is found in LDs of steatotic hepatocytes (245). A key role for PLIN2 in hepatocytes is
supported by the observation that it is the most abundant LD protein in the HuH7 hepatocyte
cell line (94), and its levels are also increased in steatotic livers of both humans and mice
(185) in response to PPARγ activation (197). Overexpression of PLIN2 in rat hepatic stellate
cells results in lipid accumulation within LDs (96), while PLIN2-deficient mice exhibit a
60% reduction in hepatic TG content and are resistant to diet-induced hepatic steatosis (46).
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Liver-specific ablation of PLIN2 in mice results in reduced LD size and is protective against
diet-induced NASH (190). PLIN1 is frequently associated with larger LDs in the liver of
NAFLD subjects while PLIN2 is predominantly found in smaller LDs (93). The specific
targeting of these proteins raises the suggestion of distinct PLIN1 and PLIN2 functions.
Plin3 expression is also induced in livers of high fat-fed mice and its knockdown reduces
hepatic TG content, attenuates steatosis, and improves insulin sensitivity and glucose
tolerance (39). In hepatocytes, the degradation of PLIN2 and PLIN3 are steps required for
lipolysis during nutrient deprivation. The removal of PLIN proteins from LD occurs via
chaperone-mediated autophagy (CMA) and facilitates the LD association of components of
the lipolytic machinery, including ATGL (134). Moreover, the combined knockdown of
Plin2 and Plin3 results in increased insulin resistance in mouse AML12 hepatocytes
stimulated with oleate (17). PLIN5 is highly expressed in oxidative tissues, including liver.
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Hepatic Plin5 mRNA levels are increased after fasting in response to activation of PPARα
and/or PPARβ/δ (140). PLIN5 expression is also enhanced in steatotic livers of humans and
obese mice, and the knockout of this protein results in decreased hepatic TG content and
smaller-sized LDs (268).
In hepatocytes, LDs are tightly associated with ER membrane cisternae, allowing the
physical interaction between LD components and microsomal proteins, such as apoB100
(194) (Fig. 4). The fat-inducing transmembrane proteins (FIT1 and 2) are localized in the
ER and have the ability to bind DG and TG, with FIT2 showing higher affinity (108). The
overexpression on FIT2, the most abundant hepatic FIT isoform, in mouse liver results in
increased TG content and LD accumulation (128). These data suggest that FIT2 functions in
the binding of neutral lipids synthesized within ER and their channeling to nascent LDs (99).
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The DFF45-like effector (CIDE) proteins, Cidea, Cideb, and Fsp27, are associated both to
the ER and LDs and have been linked to LD metabolism (100). Recent studies have
demonstrated that these proteins act to promote lipid exchange and fusion among LDs (282).
However, they are differentially distributed among hepatocytes based on sizes of LD that
they harbor. Cideb localizes both to hepatocytes with small and with large LDs, whereas
Cidea and Fsp27 specifically localize to hepatocytes containing large LDs (282).

Cidea expression is increased in livers of diabetic mice (137) and a coding region
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polymorphism is associated with obesity in human subjects (60, 290). Moreover, expression
of both Cidea and Fsp27 are upregulated in mice with hepatic steatosis induced by PPARγ
activation (176, 264, 288). Fsp27 is a target gene of PPARγ and its transcription is decreased
in ob/ob mice with liver-specific knockdown of PPARγ, accompanied by decreased TG
content in the liver (175, 176). Cideb is highly expressed in the liver and Cideb knockout
mice are resistant to obesity and liver steatosis induced by high-fat diet (160). Moreover,
these animals have decreased levels of circulating TG and FA, and exhibit improved hepatic
insulin sensitivity. These effects are mediated by the hepatic downregulation of SREBP-1c,
resulting in decreased lipogenesis and increased FA oxidation (160). Cideb also interacts
with apoB100, and mice lacking Cideb exhibit reduced VLDL secretion and increased
hepatic steatosis (284). In addition to its role in VLDL assembly, Cideb has been associated
in VLDL export from ER to the Golgi. This occurs through the interaction with components
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of coat complex II (COPII), namely Sar1 and Sec24, which are present in VLDL transport
vesicles (257).
Because NAFLD is characterized by excessive deposition of TG within cytosolic LDs and

because proteomic studies have revealed that numerous LD-associated proteins are
modulated in human and mice steatotic livers (138, 250), detailed knowledge of the biology
hepatocyte LDs could reveal novel targets for therapeutic intervention.
Lipolysis
The LD-associated adipose triglyceride lipase (ATGL, also known as PNPLA2) is the rate-
limiting step in TG lipolysis within adipocytes. The resulting DG molecules are then
hydrolyzed by the hormone sensitive lipase (HSL) to release monoglycerides (MG). In the
final step, the monoacylglycerol lipase (MGL) cleaves MG into glycerol and FA (151) (Fig.
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5). Gain and loss of function studies have revealed that hepatic ATGL is required for the
lipolysis of TG stored in LDs, controls substrate availability for FA oxidation, and modulates
the progression of hepatosteatosis (198, 208, 280). Full activation of ATGL depends on co-
activation by the comparative gene identification-58 (CGI-58) (152). Liver-specific ablation
of CGI-58 results in NAFLD phenotypes in mice that include hepatic steatosis and fibrosis
(109). In the setting of hepatic steatosis, lipolysis mediated by hepatic ATGL is decreased
when PLIN5 binds competitively to CGI-58, displacing ATGL (268, 269). Insulin-resistant
NAFLD patients who exhibit higher degrees of liver steatosis when compared to NAFLD
subjects without insulin resistance, also exhibit decreased CGI-58 mRNA levels. These
observations suggest that insulin-resistance could induce hepatic TG accumulation through
CGI-58-mediated reductions in ATGL-dependent lipolysis (132). Less is known concerning
MGL and HSL activities in hepatocytes, although Hsl expression is down-regulated in livers
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of NAFLD patients (146).
In the setting of insulin resistance, enhanced rates of lipolysis within white adipose tissue are
a major contributor to the increased FFA plasma levels and hepatic steatosis (159). However,
the contribution of TG hydrolysis within hepatocytes to aberrant lipid accumulation in the
liver is less clear. Several lipases are present in close association with LDs in adipocytes (30,
66) (Fig. 5), but their contributions to LD TG hydrolysis remain topics of discovery.

The patatin-like phospholipase domain-containing 3 (PNPLA3/adiponutrin) is a TG

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hydrolase/transacylase that localizes to LD, ER and cytoplasm (124, 212). A single-

nucleotide variant of the Pnpla3 gene (I148M) confers increased risk of NAFLD in humans
(210). In this connection, livers from transgenic mice expressing this allele show
accumulation of inactive PNPLA3 in lipid droplets and increased steatosis when fed a high-
sucrose diet (235). Although the genetic association of PNPLA3 with NALFD is robust, key
details are still lacking regarding the metabolic activity of this protein.
Autophagy
Autophagy is a lysosome-dependent process that selectively targets, transports and regulates
the storage of essential components, including lipids, proteins and carbohydrates. Three
types of autophagy have been described in hepatocytes (59). In macroautophagy, LDs or
other organelles are engulfed to form autophagosomes, which fuses to lysosome to generate
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an autolysosome. After hydrolysis by lysosomal enzymes, lipid products are released to the
cytosol and recycled by other cellular processes (167). The autophagosome formation relies
on several autophagy receptors in addition to the autophagy-related proteins (ATGs) (241).
In microautophagy, small vesicles originated from lysosomal membrane invagination
mediate the engulfment of cytosolic components (167). In chaperone-mediated autophagy
(CMA), lysosomal degradation of specific proteins is regulated upon recognition by CMA
receptor (133).
Macroautophagy of LDs (also named lipophagy) is stimulated in hepatocytes during

starvation, leading to the release of FA in the cytosol after TG breakdown (233).
Additionally, products of lipid anabolism have inhibitory effects in lipophagy (167).
Although the mechanisms controlling lipophagy are not well understood, this process is
driven by the small GTPase Rab7 protein, which recognizes LDs and mediates their
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engulfment by autophagosomes (221).
Decreased autophagy has been reported in obesity-related disorders, including

hepatosteatosis (219). Altered membrane lipid composition due to high-fat feeding reduces
the activity of lysosomal proteins and decreases autophagic activity in mouse liver (145,
209). Impaired CMA leads to hepatosteatosis through at least three mechanisms. First,
reduced CMA activity decreases mitochondrial function and β-oxidation. Second, CMA-
deficient mice exhibit increased expression of lipogenic enzymes, including DGAT2 (219).
Finally, CMA is required for the lipolysis of hepatic TG through hsp70-mediated
degradation of PLIN2 and PLIN3 (134). Hence, impaired autophagy in the setting of
steatosis exacerbates lipid accumulation within the liver.
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Fatty acid oxidation

FA derived from hydrolysis of hepatic TG stores, circulating lipids or DNL, can be oxidized
by multiple pathways. Mitochondrial β-oxidation is the primary route for the oxidation of
the majority of FA found in hepatocytes, including short- (<C4), medium- (C4-C12), and
long-chain (C12-C20) FA. β-oxidation of very long- (C20-C26) and branched-chain FA
begins in the peroxisomes. Additional pathways for FA oxidation include α-oxidation and
ω-oxidation within the ER and are mediated by cytochrome P450 4A family members (153,

187) (Fig. 5). The expression of genes involved in mitochondrial and extramitochondrial FA
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oxidation is regulated largely by PPARα activity. PPARα is expressed at high levels in liver,
and both whole-body and hepatocyte-specific ablation of PPARα in mice lead to reduced
transcription of hepatic genes related to mitochondrial β-oxidation, such as very long chain
acyl-CoA dehydrogenase (VLCAD), long chain acyl-CoA dehydrogenase (LCAD), and
ACSL1 (7), and genes involved in peroxisomal β-oxidation, including peroxisomal fatty
acyl-CoA oxidase (AOX) and cytochrome P450 4A (156, 184). These animals also
experience hypoketonemia and increased hepatic steatosis (111, 184).
It is noteworthy that AOX ablation leads to sustained activation of PPARα, likely due to
accumulation of PPARα ligands and induction of cytochrome P450 4A (86). Impaired
peroxisomal FA oxidation results in the accumulation of dicarboxylic acids within the liver
and leads to mitochondrial damage and microvesicular steatosis (153). Alterations in
dicarboxylic acid levels are also observed in conditions of FA overload and have been
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correlated with increased risk of NAFLD development, although the cellular mechanism
remains unknown (215, 287). PGC1α and BAF60a form a complex that regulates PPARα
transcriptional activity in the liver. Overexpression of BAF60a increases fatty acid oxidation
and reduces hepatic steatosis in obese mice (162). Similar phenotypes have been observed in
livers of mice overexpressing the PPARα coactivator PGC1β (18).
Notwithstanding the relationship between PPARα activity and hepatic lipid metabolism, the
correlation between PPARα expression levels and hepatic steatosis is inconsistent, with
reports demonstrating unchanged, increased and decreased expression (16). This is also the
case for rates of hepatic mitochondrial β-oxidation. The transcription of CPT1a, is down-
regulated in NAFLD (146). Although previous studies revealed decreased hepatic
mitochondrial FA oxidation in the setting of hepatic steatosis (123, 202, 206), recent work
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has demonstrated increased oxidation rates in livers of subjects with hepatic steatosis,
metabolic syndrome and NAFLD (16). In livers of ob/ob mice, mitochondrial and
peroxisomal oxidative capacities are elevated (29). Mechanisms underlying increased FA
oxidation in NAFLD have been reviewed (16). Hypotheses include enhanced activation of
PPARα due to increased hepatic FA uptake or biosynthesis.
ER-mediated control of lipid homeostasis

Among its many functions, the ER regulates the synthesis of lipids and proteins, as well as
intracellular calcium storage (24). Under physiological conditions, unfolded and misfolded
proteins accumulate in the ER lumen. In response to this ER stress, the cell initiates multiple
lines of defense. One is the activation of ER-associated degradation (ERAD), a ubiquitin/
proteasome protein degradation pathway that relies on E3 ligase complex activation (11).
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Second, the unfolded protein response (UPR) is initiated (229). This comprises three main
signaling pathways mediated by microsomal transmembrane proteins: PKR-like ER kinase
(PERK; also known as eukaryotic translation initiation factor 2-α kinase 3, eIF2αk3),
inositol requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) (229). UPR
activation alleviates the accumulation of misfolded proteins into the ER through the
inhibition of protein translation and the induction of chaperones to increase protein folding
capacity, as well as expanding the ER membrane (11).

Lipid accumulation is also associated with chronic ER stress in hepatocytes (293). Lipid-
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mediated induction of ER stress can be triggered by multiple mechanisms. Obese mice

exhibit altered microsomal membrane composition in the liver, with increased PC:PE ratios
and variations in PL acyl-chain saturation degree (91, 121). Hence, changes in membrane
fluidity leads to altered activity of membrane-associated proteins. For instance, sarco-
endoplasmic reticulum Ca2+-ATPase (SERCA) activity is reduced in steatotic mouse livers.
In this model, decreased ER Ca2+ content impairs intraluminal Ca2+-dependent chaperones
function and is a cause for ER stress (91).
UPR-related proteins directly sense membrane lipid composition in non-hepatic cells. In

mouse fibroblasts, PERK and IRE1 are responsive to increased lipid saturation even in the
absence of their ER-spanning transmembrane domain (265). However, it is uncertain
whether direct lipid sensing mechanisms modulate hepatic ER stress. Because ER stress in
turn promotes lipid accumulation in hepatocytes, chronic ER stress may directly contribute
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to the pathogenesis of NAFLD (291). In cell culture systems, prolonged exposure to oleate
or palmitate induces ER stress when these saturated FA are incorporated to ER membrane
PL (25). A concomitant reduction in VLDL-TG secretion is attributable to enhanced
apoB100 degradation by proteasomal and non-proteasomal mechanisms (43, 199). In this
connection, treatment with 4-phenyl butyric acid (PBA), a chemical chaperone, improves ER
homeostasis and VLDL secretion after FA exposure (43). Although reduced VLDL secretion
due to chronic ER stress results in TG accumulation in McA-RH7777, its potential
contribution for the progression of hepatic steatosis was not demonstrated in animal models
(199).
ER stress also promotes lipid accumulation in hepatocytes that is attributable to increased

lipogenesis (11). In obese mice, hepatic induction of UPR pathway reduces Insig-1 levels
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and leads to the activation of SREBP, increased expression of lipogenic enzymes and
steatosis (129). UPR-mediated activation of PERK may also contribute to hepatic steatosis
by increasing expression of VLDL receptors in hepatocytes (126). Moreover, ER stress
reduces autophagy through the cross-talk with components of ERAD and UPR in different
cell types, exacerbating the hepatic steatosis (11, 201)
In summary, excess lipid load potentially initiates a vicious cycle of hepatic lipid
accumulation due to abnormal activity of ER stress-activated pathways, leading to altered
lipid homeostasis and TG deposition in the liver (11, 291, 293).
Conclusion
The liver is a main organ for the metabolism of fatty acids and triglycerides and altered
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activity of metabolic pathways in the setting of overnutrition leads to a common chronic

liver disease known as NAFLD. Despite this biomedical relevance, many important
questions regarding basic aspects of hepatic lipid metabolism remain unanswered. These
include: (a) Which FA transporters are responsible for the increased intracellular
concentration of FA in the setting of overnutrition? (b) What is the precise mechanism by
which DNL is increased in obese and NAFLD subjects? (c) What is the precise molecular
mechanism underlying the partitioning of FA and acyl-CoA among metabolic pathways,

which presumably involve ACOT, ACSL and lipid-binding proteins? (d) How are rates of FA
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oxidation regulated in the setting of overnutrition? In summary, a deeper understanding is

needed regarding the precise mechanisms whereby fatty acid uptake and synthesis are tightly
balance against oxidation and secretion within the liver. This would be expected to lead to a
better understanding of how triglycerides accumulate in many, but not all, individuals who
consume excess calories and to new therapeutic strategies for the management of NAFLD.
Acknowledgements
This work was supported by NIH grants R37DK048873, R01DK056626 and R01DK103046 to D.E.C. M.A.B. is
the recipient of NASH Fatty Liver Disease Postdoctoral Research Fellowship from the American Liver Foundation.
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Didactic Synopsis
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Major teaching points:
• A variety of endogenous and exogenous sources provide fatty acids that can
be assembled into to triglycerides within the liver in health and disease;
• Multiple hepatocellular mechanisms regulate fatty acid uptake, synthesis,

transport, and oxidation;
• Triglycerides can be stored within hepatocytes, undergo lipolysis, or be

exported into the bloodstream depending upon physiological and pathological
conditions;
• Mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes, and

lipid droplets are examples of organelles that participate in fatty acid and
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triglyceride metabolism;
• The metabolism of fatty acids and triglyceride is regulated by transcriptional

and post-transcriptional mechanisms;
• Alterations in fatty acid and triglyceride metabolism lead to non-alcoholic

fatty liver disease (NAFLD), which is a common consequence of
overnutrition.
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Figure 1. Major sources for hepatic fatty acids.

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The three major sources for hepatic fatty acids (FA) are dietary lipids, adipose tissue
derived-FA and de novo-synthesized FA. After a meal, dietary lipids are hydrolyzed within
the intestinal lumen. Upon intestinal uptake, FA are re-esterified to form TG molecules,
which are packaged into chylomicrons and delivered primarily to muscle and adipose tissue.
The remaining TG present in chylomicron remnants is then transported to the liver and
processed intracellularly, leading to FA release within hepatocytes. Carbohydrates,
particularly glucose, are utilized in hepatic de novo lipogenesis (DNL) for the production of

FA. In order to be metabolized, FAs are activated to form acyl-CoA molecules, which can
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undergo oxidation or be incorporated into complex lipids. Locally synthesized TG can be

stored in intracellular lipid droplets (LDs) or packed into VLDL and secreted into the
plasma. Upon fasting, intracellular TG stores are mobilized from adipocytes and hepatocytes
to release FA products. Hepatic DNL may also contribute to form an acyl-CoA pool
available for energy production, undergoing oxidation by mitochondria, or for VLDL-TG
production. In the setting of overnutrition and insulin resistance, hepatic FA levels are
increased due to enhanced lipolysis within adipocytes, which leads to increased circulating
levels of FA in the bloodstream, and increased hepatic DNL. Excess FA cannot be consumed
by oxidative pathways and FA are instead directed towards the synthesis of TG, leading to
increased hepatic TG storage and VLDL overproduction. Arrow thickness denotes rates of
metabolic activities.
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Figure 2. Hepatic fatty acid transport and metabolism.

Within the plasma membrane, FA translocase (FAT)/CD36, plasma membrane FA-binding
protein (FABPpm), and Caveolin-1 mediate the uptake of fatty acid (FA) that is bound to
circulating albumin. Alternatively, hepatic FA can be obtained by the internalization of
chylomicron remnant or by de novo lipogenesis. The latter occurs through the activity of
three key enzymes: ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty
acid synthase (FAS). In the cytosol, FAs are bound to the fatty acid-binding protein-1
(FABP1) and sterol carrier protein-2 (SCP2), which may control their cellular distribution.
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FA transport proteins (FATP2, 4 and 5) and long-chain acyl-CoA synthetases (ACSL1, 3 and
5) mediate the activation of long-chain FA to acyl-CoA molecules and their channeling to
metabolic pathways. Although associated with mitochondria, ACSL5 may function to
promote triglyceride biosynthesis. In the cytosol, acyl-CoAs are bound to acyl-CoA-binding
protein (ACBP) or SCP2. Acyl-CoA thioesterases (ACOT)/thioesterase superfamily
members (Them1, 2 and 5) appear to counteract ACSL activity by catalyzing the hydrolysis
of acyl-CoA molecules into FA and CoA. This may provide additional means of controlling
the balance between FA and acyl-CoA within hepatocytes.

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Figure 3. Hepatic triglyceride metabolism.

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Acyl-CoA molecules can be esterified to glycerol-3-phosphate (G3P) by isoforms of

glycerol-3-phosphate acyltransferase (GPAT). In hepatocytes, the isoforms are
predominantly GPATs 1 and 4. The produced lysophosphatidic acid (LPA) is acylated by
acylglycerol-3-phosphate acyltransferases (AGPATs) to form phosphatidic acid (PA), which
can be dephosphorylated by phosphatidic acid phosphatase (PAP) to form diacylglycerol
(DG). Both PA and DG can be directed towards phospholipid (PL) synthesis. Additionally,
diacylglycerol acyltransferases (DGATs 1 and 2) synthesize triglyceride (TG) by acylation

of DG. (A) Fas synthesized de novo are likely to be channeled to VLDL-TG production
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through DGAT1 activity. (B) Exogenous FA appear to be directed towards TG synthesis for
storage in lipid droplets by the activity of DGAT2. In addition, lipid droplet-TG can undergo
hydrolysis, with FA re-routed into VLDL by DGAT1.
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Figure 4. Triglyceride storage and secretion in hepatocytes.

Triglyceride (TG) can be synthesized from diacylglycerol (DG) by diacylglycerol
acyltransferases (DGAT1 and 2). DGAT1 preferably provides TG to VLDL during lipidation
in the lumen of the endoplasmic reticulum. This process is mediated by microsomal
triglyceride transfer protein (MTP), which facilitates the association between TG and
apoB100. Transmembrane 6 superfamily member 2 (TM6SF2) may also contribute to
VLDL lipidation via yet unknown mechanisms. The nascent VLDL particle is then
transferred to the Golgi apparatus through the VLDL transfer vesicle (VTV), followed by a
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second MTP-mediated lipidation step. VLDL particles are secreted via a vesicle-mediated
mechanism. TG can also be formed by the activity of DGAT2, which mainly contributes to
storage in lipid droplets. Lipid droplets are delimitated by proteins and a
phosphatidylcholine (PC)-enriched surfactant monolayer. Among the lipid-droplet
associated proteins, perilipins (PLIN2, 3 and 5) and comparative gene identification-58
(CGI-58) contribute to lipid droplet structure and/or the regulation of lipid droplet-
associated enzymes; CTP-phosphocholine cytidylyltransferase (CCT) and acyl-CoA
synthetase 3 (ACSL3) may be required for the biosynthesis of lipids; DFF45-like effector

(CIDE) proteins, Cidea, Cideb, and Fsp27, and the microsomal fat-inducing transmembrane
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protein 2 (FIT2) are required for lipid droplet formation, however their specific cellular roles
are incompletely understood. Cideb also contributes to VLDL production and secretion via
its interaction with Sar1 and Sec24, which are present in VTV.
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Figure 5. Lipolysis and fatty acid oxidation in hepatocytes.

The initiation of hepatic lipolysis depends on the activation of adipose triglyceride lipase
(ATGL) through the binding to the comparative gene identification-58 (CGI-58). ATGL
catalyzes the hydrolysis of triglyceride (TG), releasing diacylglycerol (DG). This lipid is
further hydrolyzed by hormone sensitive lipase (HSL), releasing monoacylglycerol (MG).
Monoacylglycerol lipase (MGL) mediates the breakdown of MG into fatty acid (FA) and
glycerol. Alternatively, autophagic pathways, such as macroautophagy and chaperone-
mediated autophagy (CMA), promote the hydrolysis of LD. FA can also be generated from
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acyl-CoA by the activity of acyl-CoA thioesterase 13 (ACOT 13; synonym: thioesterase

superfamily member 2, Them2). In the mitochondria outer membrane, long-chain acyl-CoA
synthetase 1 (ACSL1) converts long-chain FAs to acyl-COAs. ACSL1 interacts physically
with carnitine palmitoyltransferase 1 (CPT1). It is likely that ACSL1 channels acyl-CoA to
CPT1 to control the availability of substrates for mitochondrial β-oxidation. Additionally,
acyl-CoAs can be directed to β-oxidation in the peroxisome, where fatty acyl-CoA oxidase
(AOX) represents a rate-limiting step, or ω-oxidation and α-oxidation in the endoplasmic
reticulum, mediated by P450 4A family members. Also, part of the acyl-CoA pool can be

directed for TG synthesis and VLDL assembly, sustaining the transfer of hepatic lipids to
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other organs.
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3 Triglyceride Metabolism in The Liver

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Triglyceride metabolism in the liver

enriched very low-density lipoprotein (VLDL-TG). The relatively small quantities of TG

stored within the liver are localized in cytoplasmic lipid droplets.

Hepatic fatty acid metabolism

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

TG in both fasting and fed states (72).

Within the hepatocyte, FA are esterified to glycerol-3-phosphate (G3P) and to cholesterol in

Fatty acid uptake

hepatocytes via plasma membrane-associated proteins. A variety of proteins have been

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

FAS expression is ubiquitous, but evidence of elevated transcriptional and enzymatic

research is needed to clarify mechanisms of altered DNL in a variety of pathogenic

Fatty acid activation

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

acyl-CoAs into different metabolic pathways (57, 106).

ACSL1, the best characterized isoform, is abundantly expressed in a variety of tissues.

of ACSL3 in rat hepatocytes reduces DNL through diminished activation of lipogenic

The reaction catalyzed by ACSL isoforms is reversed by the activities of acyl-CoA

ACOT13 (a.k.a. Them2) is a mitochondria-associated enzyme that is highly expressed in the

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

steatosis and exhibit improved glucose metabolism (130). Moreover, Them2-deficiency

contributes to insulin resistance and related metabolic abnormalities.

Intracellular fatty acid transport

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

as decreased rates of FA esterification into TG, with preserved or increased esterification

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Compr Physiol. Author manuscript; available in PMC 2019 February 15.

of VLDL secretion, and increased plasma concentrations of TG without changes in TG

activity (DGAT1, measured as latent – luminal – DGAT activity) and cytosol-accessible

appear to be redundant in their respective functions. DGAT1 mRNA is modestly expressed

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

re-esterification by overt DGAT1 activity present in the cytosolic compartment, followed by

VLDL assembly and secretion

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

second step, additional TG is packaged into the nascent apoB100-containing particles as

NAFLD remains unclear.

Lipid droplet biology

In addition to preserving the LD structure, LD-associated proteins regulate the formation,

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

(PLIN; former perilipin/ADRP/TIP47, PAT) protein superfamily (PLIN1–5). These proteins

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

Because NAFLD is characterized by excessive deposition of TG within cytosolic LDs and

of NAFLD patients (146).

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

The patatin-like phospholipase domain-containing 3 (PNPLA3/adiponutrin) is a TG

hydrolase/transacylase that localizes to LD, ER and cytoplasm (124, 212). A single-

Macroautophagy of LDs (also named lipophagy) is stimulated in hepatocytes during

engulfment by autophagosomes (221).

Decreased autophagy has been reported in obesity-related disorders, including

Fatty acid oxidation

Compr Physiol. Author manuscript; available in PMC 2019 February 15.

ER-mediated control of lipid homeostasis

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mediated induction of ER stress can be triggered by multiple mechanisms. Obese mice

UPR-related proteins directly sense membrane lipid composition in non-hepatic cells. In