Progress in Lipid Research 63 (2016) 50–69

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Progress in Lipid Research

journal homepage: www.elsevier.com/locate/plipres

Synthesis and degradation pathways, functions, and pathology of

ceramides and epidermal acylceramides
Akio Kihara
Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Ceramide (Cer) is a structural backbone of sphingolipids and is composed of a long-chain base and a fatty acid.
Received 22 February 2016 Existence of a variety of Cer species, which differ in chain-length, hydroxylation status, and/or double bond num-
Received in revised form 8 April 2016 ber of either of their hydrophobic chains, has been reported. Ceramide is produced by Cer synthases. Mammals
Accepted 20 April 2016
have six Cer synthases (CERS1–6), each of which exhibits characteristic substrate specificity toward acyl-CoAs
Available online 21 April 2016
with different chain-lengths. Knockout mice for each Cer synthase show corresponding, isozyme-specific pheno-
Keywords:
types, revealing the functional differences of Cers with different chain-lengths. Cer diversity is especially promi-
Acylceramide nent in epidermis. Changes in Cer levels, composition, and chain-lengths are associated with atopic dermatitis.
Ceramide Acylceramide (acyl-Cer) specifically exists in epidermis and plays an essential role in skin permeability barrier
Fatty acid formation. Accordingly, defects in acyl-Cer synthesis cause the cutaneous disorder ichthyosis with accompanying
Long-chain base severe skin barrier defects. Although the molecular mechanism by which acyl-Cer is generated was long unclear,
Skin barrier most genes involved in its synthesis have been identified recently. In Cer degradation pathways, the long-chain
Sphingolipid base moiety of Cer is converted to acyl-CoA, which is then incorporated mainly into glycerophospholipids. This
pathway generates the lipid mediator sphingosine 1-phosphate. This review will focus on recent advances in
our understanding of the synthesis and degradation pathways, physiological functions, and pathology of Cers/
acyl-Cers.
© 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2. Structural diversity of ceramides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2.1. Long-chain bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2.2. Fatty acids in ceramides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2.3. Diversity of ceramides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3. Synthesis of ceramides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.1. Serine palmitoyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.2. 3-Ketodihydrosphingosine reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.3. Ceramide production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.4. Ceramide synthases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.4.1. Ceramide synthases in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.4.2. CERS1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Abbreviations: ABC, ATP-binding cassette; acyl-Cer, acylceramide; ACS, acyl-CoA synthetase; ACSBG, acyl-CoA synthetase bubblegum; ACSF, acyl-CoA synthetase family; ACSL, acyl-
CoA synthetase long-chain; ACSM, acyl-CoA synthetase medium-chain; ACSS, acyl-CoA synthetase short-chain; ACSVL, acyl-CoA synthetase very long-chain; ALDH, aldehyde dehydroge-
nase; ARCI, autosomal recessive congenital ichthyosis; BAT, brown adipose tissue; CE, cornified envelope; Cer, ceramide; Cer (Sph), ceramide containing sphingosine; CHO, aldehyde; CLE,
corneocyte lipid envelope; COOH, carboxylic acid; dihydro-Cer, dihydroceramide; dihydro-Sph, dihydrosphingosine; dihydro-S1P, dihydrosphingosine 1-phosphate; ER, endoplasmic re-
ticulum; FA, fatty acid; FAS, fatty acid synthase; GalCer, galactosylceramide; GlcCer, glucosylceramide; KDS, 3-ketodihydrosphingosine; KO, knockout; LacCer, lactosylceramide; LC, long-
chain; LCB, long-chain base; LCBP, long-chain base 1-phosphate; LCFA, long-chain fatty acid; MC, medium-chain; 2-OH, 2-hydroxy; ω-OH, ω-hydroxy; 6-OH Sph, 6-hydroxysphingosine;
PC, phosphatidylcholine; phyto-Cer, phytoceramide; phyto-Sph, phytosphingosine; phyto-S1P, phytosphingosine 1-phosphate; PUFA, polyunsaturated fatty acid; SLS, Sjögren-Larsson
syndrome; SM, sphingomyelin; Sph, sphingosine; S1P, sphingosine 1-phosphate; SPT, serine palmitoyltransferase; STGD3, Stargardt disease type 3; tC16:1-CHO, trans-2-hexadecenal;
tC16:1-COOH, trans-2-hexadecenoic acid; tC16:1-CoA, trans-2-hexadecenoyl-CoA; ULC, ultra-long-chain; ULCFA, ultra-long-chain fatty acid; VLC, very-long-chain; VLCFA, very-long-
chain fatty acid; X-ALD, X-linked adrenoleukodystrophy.
E-mail address: kihara@pharm.hokudai.ac.jp.

http://dx.doi.org/10.1016/j.plipres.2016.04.001
0163-7827/© 2016 Elsevier Ltd. All rights reserved.
 A. Kihara / Progress in Lipid Research 63 (2016) 50–69 51

3.4.3. CERS2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.4.4. CERS3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.4.5. CERS4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.4.6. CERS5 and CERS6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.4.7. Regulation of ceramide synthases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4. Very-long-chain fatty acids in ceramides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.1. Roles of very-long-chain fatty acids in ceramides/sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.2. Generation of very-long-chain fatty acids via fatty acid elongation cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.3. Different substrate specificities and physiological functions of fatty acid elongases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.3.1. ELOVL1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.3.2. ELOVL2 and ELOVL5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.3.3. ELOVL3 and ELOVL7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.3.4. ELOVL4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.3.5. ELOVL6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.3.6. Fatty acid elongases in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.4. 3-Ketoacyl-CoA reductase and trans-2-enoyl-CoA reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.5. 3-Hydroxyacyl-CoA dehydratases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5. Acylceramide and its importance in skin barrier formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5.1. Skin barrier and structure of epidermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5.2. Formation of lipid lamellae in stratum corneum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5.3. Ceramides in epidermis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.4. Acylceramide synthetic pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.5. Protein-bound ceramide synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6. Ceramide degradation pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.1. Ceramide degradation pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.2. Ceramide degradation by ceramidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.3. Long-chain base 1-phosphate metabolic enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.3.1. Sphingosine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.3.2. Long-chain base 1-phosphate-degrading enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
6.4. The aldehyde dehydrogenase ALDH3A2 and Sjögren-Larsson syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
6.4.1. Aldehyde dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
6.4.2. Sjögren-Larsson syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
6.5. Acyl-CoA synthetases involved in ceramide degradation pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
6.6. Dual functions of the trans-2-enoyl-CoA reductase TECR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
6.7. Metabolism of phytosphingosine to odd-numbered fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
6.8. Generation of 2-hydroxy fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
7. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

1. Introduction Intracellular sphingolipids constitute ~ 10% of total lipids in mam-

Ceramide (Cer) is a hydrophobic backbone of sphingolipids.
Sphingolipids exist in all eukaryotes, but not in prokaryotes, except for
a limited number of bacteria species such as Sphingomonas [1]. Cer con-
tains two hydrophobic chains, a long-chain base (LCB) and a fatty acid
(FA), which are connected via an amide bond (Fig. 1A). Addition of a
polar head group at the C1 hydroxyl group of the LCB portion of Cer en-
dows the resulting sphingolipid with amphipathic properties. Charac-
teristic in vivo head group types differ among organisms [2–5]. In
mammals, it is either phosphocholine [in sphingomyelin (SM)] or
sugar chains (in glycosphingolipids) [5,6] (Fig. 1A). Hundreds of
glycosphingolipids differing in sugar classes and/or their linking
modes exist. The sugar residues found in mammalian sphingolipids
are glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, fu-
cose, and sialic acid [5,7,8] (Fig. 1B). The simplest glycosphingolipids are
glucosylceramide (GlcCer) and galactosylceramide (GalCer), which re-
spectively contain a glucose or galactose residue linked to Cer via β-
linkage (Fig. 1C). GlcCer and GalCer are collectively referred as cerebro-
side or mono-hexosylceramide. Sulfatide is a sulfated derivative of
GalCer (Fig. 1B and C). Gangliosides refer to glycosphingolipids contain-
ing sialic acid(s). Other derivatives of GalCer include the ganglioside
GM4 and the gala-series of glycosphingolipids (Fig. 1C). Addition of a
galactose residue to GlcCer produces lactosylceramide (LacCer), which
is a precursor of SM4, a sulfated derivative of LacCer, and numerous
glycosphingolipid series (the ganglio-series, asialoganglio-series,
isoganglio-series, globo-series, isoglobo-series, muco-series, lacto-
series, and neolacto-series) (Fig. 1C).
mals [9], although their levels vary among tissues. Sphingolipids are
enriched in the plasma membrane, especially in the outer leaflet [10].
Sphingolipids account for 20–30% of total plasma membrane lipids [9].
SM is the most abundant sphingolipid species in mammals; SM levels
are higher than even the sum of all glycosphingolipid levels in most tis-
sues. In erythrocytes, in which the only cellular membrane is the plasma
membrane, SM and glycosphingolipids account for 18% and 10% of total
lipids, respectively [11].
Levels of glycerophospholipids, another lipid class constituting
membranes, are much higher than those of sphingolipids [9]. Therefore,
only glycerophospholipids may be sufficient to build biological mem-
branes, as is the case for prokaryotes. Rather, the importance of
sphingolipids may lie in their conferring specialized functions and/or
properties to the membranes. During the course of evolution, eukary-
otes produced a variety of cell types differing in functions: creation of
a multiplicity of sphingolipids with individualized functions was likely
necessary to fulfill this wide range of roles. Loss of sphingolipids by mu-
tations in sphingolipid biosynthetic genes is lethal in all organisms ex-
amined to date [12–15], indicating that sphingolipids indeed possess
specialized functions and cannot be substituted by
glycerophospholipids.
Sphingolipids are involved in a variety of physiological functions in-
cluding skin barrier formation, myelin maintenance, immunity, blood
vessel stabilization, recognition of bacteria, bacterial toxins, and viruses,
insulin resistance, spermatogenesis, and auditory sense formation [5,7,
16–22]. They fulfill these roles through modulating cellular events
such as lipid microdomain formation, apoptosis, organelle and
52 A. Kihara / Progress in Lipid Research 63 (2016) 50–69

Fig. 1. Structures of sphingolipids and series of glycosphingolipids. (A) Structures of sphingolipids. R represents a polar head group: phosphocholine in SM or sugar chains in
glycosphingolipids. (B) Sugars used in mammalian glycosphingolipids. The simplest glycosphingolipids are GlcCer and GalCer, which contain a glucose and a galactose residue,
respectively. Sulfatide is a sulfated derivative of GalCer. (C) Glycosphingolipid series in mammals. Each number denotes the position of a bond: the C1 hydroxy group of the numbered
sugar residue links to the numbered position of the sugar residue to its left via an α-linkage (underlined) or a β-linkage (non-underlined).

membrane structure organization, survival, migration, signaling, intra-
cellular protein trafficking, autophagy, adhesion, stress response, and
metabolic regulation [8,16,19,20,23–29]. Among them, Cer plays impor-
tant roles in skin barrier formation by forming multi-lamellar lipids
(lipid lamella) in the stratum corneum of epidermis [17,30,31]. GalCer
and sulfatide are enriched in myelin and are important for myelin func-
tion and maintenance [22]. The Cer metabolite sphingosine 1-
phosphate (S1P) is a lipid mediator and functions in immunity and
blood vessel stabilization among other capacities [16,32,33]. S1P binds
to cell surface receptors (S1P1–S1P5) and induces several cellular re-
sponses. In the immune system, S1P plays a central role in the egress
of T lymphocytes from thymus and secondary lymphoid tissues [16,
32,33]. This function of S1P has already been applied clinically, with
the immunomodulator fingolimod (FTY720) being used as a therapeu-
tic agent for multiple sclerosis [34,35].
2. Structural diversity of ceramides
2.1. Long-chain bases
Cer is composed of an LCB and a FA. The structures of LCBs varied
among organisms [36]; however, all LCBs contain two hydroxyl groups
at the C1 and C3 positions and one amino group at the C2 position. The
major mammalian LCBs are dihydrosphingosine (dihydro-Sph;
sphinganine), sphingosine (Sph), phytosphingosine (phyto-Sph), and
6-hydroxysphingosine (6-OH Sph) (Fig. 2A). The existence of
sphingadienine having two double bonds and dihydroxy-
dihydrosphingosine having four hydroxyl groups has also been report-
ed [37–39]. Since the saturated LCB dihydro-Sph is synthesized in the
de novo sphingolipid biosynthetic pathway, it is a common precursor
of all sphingolipids. Accordingly, dihydro-Sph exists in all tissues, al-
though its levels are low. Sph, which contains a trans double bond be-
tween C4 and C5 positions, is the major LCB in mammals and exists
ubiquitously among tissues. Phyto-Sph contains an extra hydroxyl
group at the C4 position and exists in restricted tissues such as epider-
mis, intestine, and kidney [38,40–42]. 6-OH Sph, which contains a
trans double bond between the C4 and C5 positions and an extra hy-
droxyl group at C6, exists only in epidermis [38,42]. Epidermis is unique
in that it contains all of these four types of LCBs. The chain-length of
LCBs is mostly C18 in mammals, although LCBs with other chain-
lengths also exist [36].
2.2. Fatty acids in ceramides
FAs are classified into short-chain FAs, medium-chain (MC) FAs,
long-chain (LC) FAs (LCFAs), and very-long-chain (VLC) FAs (VLCFAs),
 A. Kihara / Progress in Lipid Research 63 (2016) 50–69 53

Fig. 2. Components and structures of mammalian Cers. (A and B) Structures of LCBs (A) and FAs (B) and their tissue distributions. (C) Nomenclature of 12 ceramide classes. Each class is
composed of a specific FA and LCB, and its name is a combination of their corresponding abbreviations. (D) Of the 12 ceramide classes, the structures of NS [combination of non-hydroxy FA
(N) and Sph (S)] and EOS [combination of esterified ω-hydroxy FA (EO) and Sph(S)] are illustrated.
54 A. Kihara / Progress in Lipid Research 63 (2016) 50–69
depending on their carbon chain-lengths. Agreement on which ranges
of chain-lengths correspond to which category has not been completely
reached among researchers. In this review, the most widely accepted
classification is used: short-chain FAs (C2–C4), medium-chain FAs
(C5–C10), LCFAs (C11–C20), and VLCFAs (≥ C21) [43,44] (Fig. 2B).
Moreover, VLCFAs with ≥ C26 are sometimes referred as ultra-long-
chain (ULC) FAs (ULCFAs). The FAs found in glycerophospholipids are
mostly LCFAs [45]. On the other hand, VLCFAs, especially lignoceric
acid (C24:0-COOH) and nervonic acid (C24:1-COOH), are often found
in sphingolipids (Fig. 2B). For example, SMs containing C24 VLCFAs ac-
count for 20–50% of total SMs in mouse tissues, with the highest in liver
and the lowest in testis and skeletal muscle among 13 tissues examined
[44]. Another major chain-length found in the FA moiety of
sphingolipids is C16 (Fig. 2B). C16 SM comprises 20–60% of total
SMs in most tissues, whereas its levels are low in brain and skeletal
muscle (10–15%). In these tissues, C18 SMs abundantly exist instead
(~ 40%). C22 SMs account for 5–20% of total SMs [44]. The amounts
of C20 SMs are low in most tissues, usually 3–10%, but are exceptionally
high in heart (20%) [44]. Most FAs found in sphingolipids are satu-
rated or monounsaturated, in contrast to glycerophospholipids,
which often contain polyunsaturated FAs (PUFAs) at the sn-2 position
[44,45].
Sphingolipids with ULCFAs are low-level and barely detectable in
most tissues; however, epidermis and spermatogenic cells in testis spe-
cifically contain ULC-sphingolipids [21,38,42]. More specifically, Cers
containing C26–C36 ULCFAs exist in epidermis [46]. Among them,
≥ C30 Cers are often ω-hydroxylated (Fig. 2B) and esterified with
linoleic acid (C18:2-COOH), generating ω-O-acylceramides [or simply
acylceramides (acyl-Cers)]. The importance of acyl-Cers in skin barrier
formation is described below in detail. Although most of the FAs found
in sphingolipids are saturated or monounsaturated, ULC-sphingolipids
in testis are exceptionally polyunsaturated [43,44,47] (Fig. 2B). They
are fucose-containing glycosphingolipids having C26–C32 ULCFAs
with four to six double bonds [21,47]. These specialized sphingolipids
are required for spermatogenesis [21].
Most cellular FAs are non-hydroxy FAs. However, in addition to the
ω-hydroxy (ω-OH) FAs present in epidermis, some tissues such as epi-
dermis, intestine, and brain contain 2-hydroxy (α-hydroxy; 2-OH) FAs
[48–52] (Fig. 2B). These 2-OH FAs are exclusively used for sphingolipids
and are not found in glycerophospholipids.
2.3. Diversity of ceramides
In a narrow definition, the term Cer represents only Sph-containing
Cer [Cer (Sph)] and is distinguished from dihydroceramide (dihydro-
Cer) and phytoceramide (phyto-Cer), which contains dihydro-Sph and
phyto-Sph, respectively. However, in a wider definition, Cer represents
all combinations of the above-mentioned classes of LCBs and FAs. This
review uses Cer in this wider sense, and Cer (Sph) for the narrow
definition.
Cer diversity is especially prominent in epidermis. To discriminate
the epidermal Cer classes, a nomenclature is used in which abbrevia-
tions for types of LCBs (DS, dihydro-Sph; S, Sph; P, phyto-Sph; H, 6-
OH Sph) and FAs (N, non-hydroxy; A, α-hydroxy; EO, esterified ω-
hydroxy) are combined (Fig. 2C). For example, NS (which is the most
abundant Cer class in mammals) represents the combination of Sph
and a non-hydroxy FA (Fig. 2D). EOS, EOP, EOH, and EODS represent
acyl-Cers (Fig. 2C and D). Existence of 11 Cer classes other than EODS
in the stratum corneum of human epidermis was reported in 2008;
when differences in chain-length were taken into account, the number
of detected Cer species reached 342 [42]. Furthermore, more recent,
high-resolution mass spectrometric analysis detected EODS and anoth-
er class of Cers with LCBs containing four hydroxyl groups (dihydroxy-
dihydrosphingosine) in human stratum corneum, with an estimated
Cer diversity of N1000 species [38].
3. Synthesis of ceramides
3.1. Serine palmitoyltransferases
De novo sphingolipid biosynthesis occurs in the endoplasmic reticu-
lum (ER) from the first reaction, catalyzed by serine
palmitoyltransferase (SPT), to Cer synthesis. SPT catalyzes condensation
of L-serine with a FA using an acyl-CoA as a donor, generating 3-
ketodihydrosphingosine (KDS). This step is a rate-limiting step of
sphingolipid synthesis [53] (Fig. 3A). In mammals, SPTLC1, SPTLC2
(SPTLC2a), and SPTLC3 (SPTLC2b) encode large subunits of SPTs, whereas
SPTSSA (ssSPTa) and SPTSSB (ssSPTb) encode small subunits of SPTs
[53–55]. SPTs are composed of SPTLC1, either of SPTLC2 or SPTLC3, and ei-
ther of ssSPTa or ssSPTb. These combinations generate four types of SPTs
(SPTLC1/2/ssSPTa, SPTLC1/3/ssSPTa, SPTLC1/2/ssSPTb, and SPTLC1/3/
ssSPTb) (Fig. 3A), in which SPTLC2 and 3 are catalytic subunits. SPTs
need a pyridoxal 5′-phosphate as a coenzyme [53]. Although SPTLC1, 2,
and 3 share high sequence similarity, SPTLC1 lacks the active site lysine
residue that binds to pyridoxal 5′-phosphate. However, SPTLC1 is still im-
portant because it confers stability to SPTLC2 [56] and maybe also to
SPTLC3. The activities of SPTs are regulated to control cellular Cer/
sphingolipid levels. Orm family proteins (Orm1 and 2 in yeast and
ORMDL1–3 in mammals) are involved in this regulation, specifically by
negative regulation of SPTs through physical interactions [57,58].
The substrate specificities of SPTs toward acyl-CoAs are different,
leading to diversity in LCB chain-lengths. SPTLC1/2/ssSPTa prefers
C16:0-CoA and generates C18 KDS, the precursor of the most abundant
LCB class, C18 LCBs [55]. On the other hand, SPTLC1/2/ssSPTb and
SPTLC1/3/ssSPTa exhibit high activity toward C18:0-CoA and C14:0-
CoA, producing C20 and C16 KDSs, respectively. SPTLC1/3/ssSPTb ex-
hibits broad substrate specificity from C14- to C20-CoAs.
SPTLC1 and SPTLC2 are the causative genes of hereditary sensory
neuropathy type I, which is an autosomal dominant peripheral neurop-
athy [59,60]. Although SPTs normally use serine as a substrate, specific
point mutations in SPTLC1 or SPTLC2 cause the substrate specificities of
their gene products to become promiscuous and accept alanine and gly-
cine. Atypical LCBs are produced as a result: 1-deoxysphinganine (from
alanine) and 1-deoxymethylsphinganine (from glycine) [61]. Since
these deoxy-LCBs lack the hydroxyl group at C1, which is required for
the attachment of polar head groups, they cannot be converted to com-
plex sphingolipids. The accumulated deoxy-LCBs and/or deoxy-Cers
show neurotoxicity.
3.2. 3-Ketodihydrosphingosine reductase
The second step of sphingolipid biosynthesis is reduction of KDS,
generating the LCB dihydro-Sph (Fig. 3A). This step is catalyzed by
KDS reductase, which is encoded by KDSR (FVT-1) in mammals [62].
KDSR was reported as a candidate gene for spinal muscular atrophy in
bovines [63].
3.3. Ceramide production
Dihydro-Sph is converted to dihydro-Cer by Cer synthases using
acyl-CoAs as FA donors. Subsequently, dihydro-Cer is desaturated to
Cer (Sph) or hydroxylated to phyto-Cer in mammals (Fig. 3A). The
dihydro-Cer desaturase DEGS1 (DES1) catalyzes Cer (Sph) generation
[64]. On the other hand, DEGS2 (DES2) is responsible for phyto-Cer pro-
duction [64]. In addition to hydroxylase activity, DEGS2 also has weak
desaturase activity that can create Cer (Sph). In Degs1 knockout (KO)
mice, Cer (Sph) levels are largely reduced (~15% of wild type levels),
but the levels of dihydro-Cer, the substrate of Degs1, are concomitantly
increased [65]. Disruption of Degs1 causes pleiotropic effects, including
scaly skin, sparse hair, tremors, hematological abnormalities, growth re-
tardation, decreased liver function, and reduced life span (dying within
8 to 10 weeks of birth) [65], indicating the physiological importance of
 A. Kihara / Progress in Lipid Research 63 (2016) 50–69 55

Fig. 3. Ceramide synthetic pathways and Cer synthases. (A) Synthetic pathways of Cers, the catalytic enzymes of each reaction, and their related hereditary disorders. (B) Substrate
specificity (1), tissue distribution (2), and phenotypes of respective Cer synthase KO mice (3) are depicted for each CERS family member. WAT, white adipose tissue; HFD, high fat diet.

Cer (Sph). Expression of Degs2 mRNA is confined to skin, kidney, intes-
tine, and lung [66], consistent with the tissue-distribution pattern of
phyto-Cers. The molecular mechanism of 6-OH Cer creation remains
unclear.
Mammals cannot directly convert dihydro-Sph to Sph. Therefore, Sph
is generated only through breakdown of Cer (Sph). In the de novo
sphingolipid synthetic pathway, Cer synthases catalyze production of
dihydro-Cer from dihydro-Sph and acyl-CoA as described above. Cer
synthases can also use other LCBs generated through salvage pathways,
i.e., those generated by ceramidases in sphingolipid degradation path-
ways. When Cer synthases use Sph as a substrate, Cer (Sph) is directly
produced.
3.4. Ceramide synthases
3.4.1. Ceramide synthases in yeast
Cer synthases were firstly identified in yeast [67,68]. Yeast Saccharo-
myces cerevisiae contains the two Cer synthases Lag1 and Lac1, which
share high sequence similarity [67,68]. Lip1 is a small subunit of Lag1
and Lac1 that is required for their enzyme activities [69]. Deletion of
LAG1 or LAC1 does not affect growth, whereas double deletion of LAG1
and LAC1 (lag1Δ lac1Δ) or single deletion of LIP1 (lip1Δ) cause severe
growth defects [69,70]. In addition, lag1Δ lac1Δ cells contain only
trace amounts of Cers, produced via reverse reaction by ceramidases
[67]. Six mammalian Cer synthases (CERS1–6/LASS1–6) that differ in
substrate specificity toward acyl-CoAs have been identified based on
their sequence similarity to yeast Lag1 and Lac1 [17]. However, mam-
mals lack a Lip1 homolog.
3.4.2. CERS1
CERS1 (LASS1/UOG1) was the first Cer synthase to be identified in
mammals [71]. CERS1 exhibits high activity toward C18-CoA and is
expressed in brain, especially in neurons [25,71–75] (Figs. 3B and 4).
Cers1-deficient mice, whether the loss of function is spontaneously gen-
erated or constructed by gene targeting, exhibit defects in cerebellar de-
velopment, locomotion, and motor coordination [73,76].
56 A. Kihara / Progress in Lipid Research 63 (2016) 50–69
3.4.3. CERS2
CERS2 (LASS2/TRH3) prefers C22- and C24-CoAs as substrates [72,
75,77,78] (Figs. 3B and 4). CERS2 mRNA is widely expressed among tis-
sues, with the highest expression in liver and kidney [25,72,78]. This ex-
pression pattern is well-correlated with the distribution patterns of C22
and C24 VLC-sphingolipids [44]. In brain, CERS2 is highly expressed in
oligodendrocytes, which form myelin [74,79]. Myelin is a multilamellar
membrane enriched in lipids, and their electrical insulation properties
are important for neural functions [22]. C24 sphingolipids such as
GalCer, sulfatide, and sphingomyelin are abundant in myelin [22,74,
79,80]. In accordance with the preponderance of VLC-sphingolipids in
liver and myelin, disruption of Cers2 in mice causes liver dysfunction
such as hepatopathy and hepatocarcinomas and myelin disorders such
as myelin sheath defects and cerebellar degeneration [79,81,82]
(Fig. 3B). The myelin phenotype of Cers2 KO mice seems to be linked
to a decrease in C24 GalCer levels [79], since GalCer and its derivative
sulfatide are important for myelin function and stability [22,83–85].
3.4.4. CERS3
Although most Cer synthases exhibit relatively strict substrate spec-
ificity toward acyl-CoAs, CERS3 (LASS3) has broad substrate specificity,
exhibiting activity toward ≥ C18-CoAs [75,86] (Figs. 3B and 4). This
broad substrate specificity enables CERS3 to produce a wide range of
Cers including the ULC-Cers found in epidermis (saturated and mono-
unsaturated ULC-Cers) and in testis (polyunsaturated ULC-Cers) [21,
87]. CERS3 mRNA expression is restricted to these tissues [25,86,88].
Mutations in human CERS3 gene cause autosomal recessive congenital
ichthyosis (ARCI) [89]. The skin disorder ichthyosis is caused by skin
barrier defects and is characterized by dry, thickened, and scaly skin.
Disruption of Cers3 in mice also causes severe skin barrier defects and
neonatal lethality [87] (Fig. 3B). In the keratinocytes of ARCI patients
and in the epidermis of Cers3 KO mice, levels of ≥ C24 Cers, including
acyl-Cers, are reduced [87,89]. These observations indicate that ULC-
Cers and/or acyl-Cers are essential for skin barrier formation. In addi-
tion, the polyunsaturated ULC-Cers synthesized by CERS3 are important
in spermatogenesis: germ cell-specific Cers3 KO mice exhibit male infer-
tility [21] (Fig. 3B).
3.4.5. CERS4
CERS4 (LASS4/TRH1) exhibits activity toward C18- to C22-CoAs and
is expressed in skin and lung [72,75,77,90] (Figs. 3B and 4). In mouse
skin, Cers4 is expressed in suprabasal epidermal layers and sebaceous
Fig. 4. Interplay between Cer synthases and FA elongases in the production of Cers with different chain-lengths. Pathways and involved enzymes (Cer synthases and FA elongases) for
mammalian FA and Cer syntheses are illustrated. C and E represent CERS and ELOVL isozymes, respectively, and their sizes reflect the strength of their enzyme activities. SFA, saturated
FA; MUFA, monounsaturated FAs.
glands [91]. Cers4 KO mice exhibit progressive hair loss due to physical
blocking of the hair canal, with concomitant diminished levels of C18:0
and C20:0 Cers in the epidermis [91] (Fig. 3B).
3.4.6. CERS5 and CERS6
CERS5 (LASS5/TRH4) and CERS6 (LASS6) are redundant Cer
synthases exhibiting activity toward C16-CoA [25,72,75,90,92]
(Figs. 3B and 4). CERS5 and CERS6 are expressed ubiquitously with
high expressions observed for CERS5 in white adipose tissue, testis,
and lung and for CERS6 in intestine and kidney [25,72,90,93,94]. Cers5
KO mice are resistant to high fat diet-induced obesity and display im-
proved glucose tolerance and insulin sensitivity [94] (Fig. 3B). Cers6
KO mice exhibit defective hindlimb grasping and habituation in the
open field test [93]. In brain, Cers6 is expressed in hippocampus, cortex,
and Purkinje cells [93]. Cers6 KO mice also exhibit exacerbation of ex-
perimental autoimmune encephalomyelitis [95]. The Cers6 product
C16 Cer mediates anti-inflammatory effects, probably by suppressing
the migration and activation of neutrophils [95].
3.4.7. Regulation of ceramide synthases
Cer synthases are regulated by phosphorylation. Yeast Cer synthases
Lag1 and Lac1 are phosphorylated at the N-terminal regions by Ypk1,
the downstream kinase of target of rapamycin complex 2 (TORC2),
and at the C-terminal regions by the casein kinase 2 Cka2 [96,97]. The
Ypk1-dependent phosphorylation activates Cer synthase activity and
is increased upon sphingolipid depletion or heat shock [96]. The phos-
phorylation by Cka2 is also important for efficient ceramide biosynthe-
sis [97].
Cer synthases are ER membrane proteins. Yeast Lag1 and Lac1 con-
tain eight transmembrane helixes, and their N-termini and C-termini
face the cytosol [98]. On the other hand, mammalian CERS1–6 lack the
N-terminal cytosolic domain that includes the Ypk1 phosphorylation
sites and the first transmembrane helix present in yeast Cer synthases.
Accordingly, CERS1–6 are not phosphorylated at their N-termini. How-
ever, mammalian Cer synthases except CERS1 do contain the C-terminal
phosphorylation sites and are phosphorylated accordingly [75]. CERS2,
CERS4, CERS5, and CERS6 are phosphorylated by casein kinase 2, where-
as another kinase seems to be responsible for the phosphorylation of
CERS3. This phosphorylation is especially important for CERS2 activity,
whereas it modestly increases the activities of CERS4 and CERS5 and
mildly increases those of CERS3 and CERS6. Although CERS1 is not phos-
phorylated at the C-terminal region, it is phosphorylated at another site
 A. Kihara / Progress in Lipid Research 63 (2016) 50–69
upon activation of protein kinase C [99]. This phosphorylation protects
CERS1 from stress-induced degradation.
4. Very-long-chain fatty acids in ceramides
4.1. Roles of very-long-chain fatty acids in ceramides/sphingolipids
Sphingolipids contain more hydrogen donors and acceptors com-
pared to glycerophospholipids. Furthermore, the hydrophobic chains
in sphingolipids usually do not contain a cis-double bond, which pre-
vents tight packaging with neighborhood lipids, in contrast to
glycerophospholipids, which usually contain an unsaturated FA with
cis-double bonds at the sn-2 position [45]. These structural properties
enable sphingolipids to form lipid microdomains or lipid rafts in biolog-
ical membranes (especially in plasma membrane) together with choles-
terol [23]. Increases in chain-length and degree of saturation cause a
decrease in membrane fluidity and an increase in membrane order, re-
spectively, stimulating stability of membrane microdomains [100,101].
Thus, VLC-sphingolipids are important for lipid microdomain formation
and stabilization. Lipid microdomains act as a platform for signaling
proteins and facilitate efficient signaling [102]. It has been shown that
C24 sphingolipids are required for the activation of the Src family kinase
Lyn [103,104]. Apoptosis signaling is also modulated by VLC-
sphingolipids. Altering total sphingolipid composition to contain fewer
VLC- and more LC-sphingolipids increases susceptibility to apoptosis
[25,105]. VLC-sphingolipids seem to be also important for generation
and stabilization of highly curved membrane structures. Myelin is one
such structure: the Cers2 KO mice studies described above demonstrate
the importance in VLC-sphingolipids in myelin function and stability
[79,81]. VLCFAs have also been reported to be important in vesicular
trafficking (especially at the late endosome/multivesicular body),
where a highly curved membrane is generated during vesicle budding
and fusion, and in formation of the nuclear pore complex, around
which highly curved membranes also exist [27,106].
4.2. Generation of very-long-chain fatty acids via fatty acid elongation
cycles
Cellular FAs are derived either from palmitic acid (C16:0-COOH)
produced by FA synthase (FAS) or from LCFAs taken from food. After ac-
tivation to acyl-CoAs, some of these FAs are converted to VLCFAs via FA
elongation cycles occurring on the cytosolic side of the ER membrane
[43,44,107,108] (Fig. 4). The FA elongation cycle is composed of four
steps: condensation, reduction, dehydration, and reduction again [43,
44] (Fig. 5A). The chain-length of acyl-CoA is increased by two in each
cycle. The first, condensation step is the rate-limiting step of the FA
elongation cycle, and is catalyzed by FA elongases using a malonyl-
CoA as a two-carbon unit donor [109]. Acyl-CoA is converted to 3-
ketoacyl-CoA by this reaction. The second step is reduction of 3-
ketoacyl-CoA to 3-hydroxyacyl-CoA, which is catalyzed by 3-ketoacyl-
CoA reductase. The subsequent third step is dehydration of 3-
hydroxyacyl-CoA to trans-2-enoyl-CoA by 3-hydroxyacyl-CoA
dehydratases. The final step is reduction/saturation of trans-2-enoyl-
CoA to an acyl-CoA having two more carbons than the original acyl-
CoA, and is catalyzed by trans-2-enoyl-CoA reductase.
The FA elongation cycle and the involved enzymes are conserved
among eukaryotes [43,110,111]. S. cerevisiae and mammals respectively
possess three and seven FA elongase genes (yeast, ELO1, FEN1/ELO2, and
SUR4/ELO3; mammals, ELOVL1–7) (Fig. 5A). The difference in the num-
ber of the FA elongases seems to reflect the complex FA elongation path-
ways in mammals with respect to the simpler yeast pathways. In
mammals, saturated, monounsaturated, and polyunsaturated (n-3 and
n-6) FAs are all elongated up to C36 [44] (Fig. 4). On the other hand,
yeast contains ≤C26 saturated FAs and essentially two monounsaturat-
ed FAs—palmitoleic acid (C16:1-COOH) and oleic acid (C18:1-
COOH)—but no PUFAs [112]. Therefore, only saturated FAs are
57
elongated up to C26 in yeast. The third, dehydration step of the FA elon-
gation cycle is also catalyzed by multiple isozymes (HACD1–4) in mam-
mals (Fig. 5A), but by only one enzyme (Phs1) in yeast [107,113]. The
second and fourth steps of the FA elongation cycle are each catalyzed
by a single gene product in mammals (second step, KAR/HSD17B12;
fourth step, TECR/TER) [114]. In yeast, the second step is catalyzed pri-
marily by Ifa38/Ybr159w but also by the minor isozyme Ary1 [115],
and the fourth step is catalyzed by Tsc13 [116].
Each step of the FA elongation cycle seems not to occur indepen-
dently but, rather, to be regulated cooperatively with the other steps.
For example, the activity of ELOVL6, which catalyzes the first step of
the FA elongation cycle, is enhanced by KAR, which catalyzes the second
step [117]. Furthermore, decreased TECR, which is responsible for the
fourth step, by mutation or gene knockdown causes a decrease in the
enzyme activity of HACDs, which catalyze the third step [118]. These
regulations may be carried out through interactions among the protein
components of the FA elongation machinery [113,119,120].
Production of VLCFAs is essential for embryogenesis and survival in
mammals. Disruption of KAR in mice causes lethality at embryonic day
9.5 [121]. VLCFAs are also essential for yeast growth. Mutations that
cause complete abrogation of VLCFA production, such as fen1Δ sur4Δ,
ifa38Δ ary1Δ, phs1Δ, and tsc13Δ mutations, are all lethal [107,115,116,
122]. The lethality of such mutations in yeast is closely related to the
organism's inability to produce sphingolipids [123–125]. The yeast Cer
synthase Lag1/Lac1 seems unable to use LC acyl-CoAs as substrates.
When the mammalian CERS5, which produces C16 and C18 Cers, is
expressed in tsc13Δ cells, TSC13 disruption is no longer lethal [125].
4.3. Different substrate specificities and physiological functions of fatty acid
elongases
4.3.1. ELOVL1
Just like Cer synthases, mammalian ELOVLs exhibit different sub-
strate specificities toward acyl-CoAs [43,44,104,126]. ELOVL1 is
expressed ubiquitously among tissues and is important for production
of C22–C26 saturated and monounsaturated VLCFAs [104,127] (Figs. 4
and 5B). In most tissues, ELOVL1 mainly generates C24 VLCFAs, which
are converted to C24 Cers by the Cer synthase CERS2 [127]. Knockdown
of CERS2 causes a reduction in this ELOVL1 activity, indicating that
CERS2 positively regulates ELOVL1 [104]. On the other hand, ELOVL1
is regulated by another Cer synthase, CERS3, in the epidermal stratum
granulosum, where CERS3 but not CERS2 is expressed [127,128].
CERS3 enables ELOVL1 to produce C26-CoAs beyond C24-CoAs in stra-
tum granulosum [127]. The produced C26-CoAs are either converted
to C26 Cers or further elongated to ≥ C28-CoAs by ELOVL4. Elovl1 KO
mice have greatly decreased levels of ≥ C26 Cers including acyl-Cers,
and as a result die within one day of birth due to transepidermal
water loss [127] (Fig. 5B).
Although LCFAs are degraded in mitochondria by β-oxidation,
VLCFAs are degraded in peroxisomes [129,130]. The ATP-binding cas-
sette (ABC) transporter ABCD1 transports VLC acyl-CoAs to peroxi-
somes for degradation [131]. Mutations in ABCD1 cause X-linked
adrenoleukodystrophy (X-ALD) accompanied by accumulation of
lignoceric acid (C24:0-COOH) and cerotic acid (C26:0-COOH) [131,
132]. To date, no therapeutic agent for X-ALD has been successfully de-
veloped. ELOVL1 is one potential pharmacological target, since it is re-
sponsible for the production of these VLCFAs. Indeed, knockdown of
ELOVL1 in X-ALD fibroblasts reduces the levels of C26:0-COOH [133].
Furthermore, Lorenzo's oil, a 4:1 mixture of glyceryl trioleate and glyc-
eryl trierucate, inhibits ELOVL1 activity [134] and has been used in an
experimental treatment of X-ALD [135].
4.3.2. ELOVL2 and ELOVL5
ELOVL2 and ELOVL5 exhibit activity toward polyunsaturated acyl-
CoAs (ELOVL2, C20- and C22-CoAs; ELOVL5, 18- and 20-CoAs) [42,
104,126,136–139] (Figs. 4 and 5B). The expression of ELOVL2 mRNA is
58 A. Kihara / Progress in Lipid Research 63 (2016) 50–69

Fig. 5. FA elongation cycle and FA elongases. (A) The FA elongation cycle consists of four reactions (condensation, reduction, dehydration, and reduction); mammalian enzymes and related
hereditary disorders are indicated for each reaction. (B) Substrate specificity (1), tissue distribution (2), and phenotypes of the respective FA elongase KO mice (3) are depicted for each
ELOVL family member. S/MU, saturated and monounsaturated; PU, polyunsaturated.

tissue-specific: testis and liver have the highest expression, followed by
pancreas and placenta [104]. Elovl2 KO mice exhibit male infertility due
to decrease in ULC-PUFAs [140], an essential component of spermato-
genesis as described above. ELOVL5 is expressed ubiquitously among
tissues [104]. Levels of arachidonic acid (C20:4-COOH) and
docosahexaenoic acid (C22:6-COOH) are reduced in the liver of Elovl5
KO mice, and their reductions cause enhanced expressions of several
lipid-related genes through activation of sterol regulatory element-
binding protein SREBP-1c, leading to hepatic steatosis [141] (Fig. 5B).
4.3.3. ELOVL3 and ELOVL7
ELOVL3 and ELOVL7 are active toward saturated and unsaturated
C16- to C20-CoAs, with the highest activity toward C18-CoAs [104,
126,142–145] (Figs. 4 and 5B). ELOVL3 mRNA is expressed in restricted
tissues/cells such as hair follicles and sebaceous glands in skin, brown
adipose tissue (BAT), and testis [104,142]. Their expression in BAT is
greatly heightened upon cold stress [142]. Elovl3 KO mice display a
sparse hair coat and mild skin barrier defects [142] (Fig. 5B). ELOVL7
mRNA is expressed in a broad range of tissues, with the highest
expression in prostate [104,144]. The involvement of ELOVL7 in prostate
cancer growth has been reported [144].
4.3.4. ELOVL4
ELOVL4 is responsible for saturated and unsaturated ULCFA produc-
tion [104,120,126,146–148] (Figs. 4 and 5B). Two types of disorders
have been reported to arise from ELOVL4 mutations. One is dominantly
inherited macular dystrophy, termed Stargardt disease type 3 (STGD3)
[149], and the other is a recessively inherited neurocutaneous disorder
characterized by ichthyosis, intellectual disability, and spastic quadri-
plegia [150]. Phosphatidylcholines (PCs) containing ULC-PUFAs at the
sn-1 position exist in retina and brain, whereas saturated and monoun-
saturated ULC-Cers including acyl-Cers exist in epidermis [43,44]. Since
these unique ULC lipids play important functions in their respective tis-
sues, their decreases due to ELOVL4 mutation lead to the corresponding,
tissue-related disorders. As are other proteins involved in the FA elonga-
tion cycle, ELOVL4 is localized in the ER, and its localization requires a C-
terminal ER retention signal [151]. STGD3 mutations cause loss of this sig-
nal, resulting in mislocalization of the truncated ELOVL4 protein
 A. Kihara / Progress in Lipid Research 63 (2016) 50–69
(ELOVL4ΔC). Moreover, ELOVL4ΔC interacts with wild type ELOVL4, caus-
ing its mislocalization as well. This cascading dysfunction is the basis of the
dominant transmission pattern of STGD3 [151]. Elovl4 KO mice exhibit
neonatal lethality due to skin barrier defects [147,152] (Fig. 5B). These
mice lack most acyl-Cers and ≥C28 non-acyl Cers [147,152].
4.3.5. ELOVL6
ELOVL6 exhibits activity toward saturated and monounsaturated
C12–C16 acyl-CoAs in vitro [104,126,137,138,153] (Figs. 4 and 5B). In
Elovl6 KO mice, C16:0-COOH and C16:1-COOH levels are increased,
whereas stearic acid (C18:0-COOH) and C18:1-COOH levels are de-
creased [154]. These changes in FA profile cause amelioration of
obesity-induced insulin resistance and nonalcoholic steatohepatitis
[154,155] (Fig. 5B).
4.3.6. Fatty acid elongases in yeast
Yeast contains three FA elongases: ELO1, FEN1/ELO2, and SUR4/ELO3.
Elo1 is involved in the elongation of LCFAs [156], whereas Fen1 and Sur4
redundantly function in producing VLCFAs [122]. Double deletion of
FEN1 and SUR4 (fen1Δ sur4Δ) genes causes lethality [122]. In sur4Δ
cells, C26 VLCFAs are diminished and C24 VLCFAs are concomitantly ac-
cumulated [157]. On the other hand, in fen1Δ cells, overall VLCFA
amounts are decreased to ~ 1/3 of levels in wild type cells [157]. Fen1
and Sur4 are ER-resident, integral membrane proteins with seven trans-
membrane helixes [107]. The positions of the Lys residues in transmem-
brane helix 6 of Fen1 and Sur4 determine the chain-lengths of their
products (C24 for Fen1 and C26 for Sur4) [107].
4.4. 3-Ketoacyl-CoA reductase and trans-2-enoyl-CoA reductase
Two reductases, the 3-ketoacyl-CoA reductase KAR and trans-2-
enoyl-CoA reductase TECR, catalyze the second and fourth steps of the
mammalian FA elongation cycle, respectively, using NADPH as a reduc-
ing agent [114] (Fig. 5A). A point mutation in TECR where Pro-182 is
substituted with Leu in the gene product (TECR P182L) causes nonsyn-
dromic mental retardation [158]. This mutation reduces the activity and
stability of TECR, thereby impairing VLCFA synthesis and, in turn, de-
creasing VLC-sphingolipid levels [118]. However, TECR P182L is a rela-
tively weak mutation, and the corresponding decrease in VLC-
sphingolipid levels is small [118]. Since TECR is solely responsible for
the fourth step of the FA elongation cycle, its complete functional distur-
bance should cause complete loss of VLCFAs and embryonic lethality.
Considering that only neural function is affected by the weak mutation
TECR P182L, the nervous system may require VLCFAs more dearly than
any other tissues. The importance of VLC-sphingolipids in myelin main-
tenance and function described above may be related to this
requirement.
4.5. 3-Hydroxyacyl-CoA dehydratases
Mammals contain four 3-hydroxyacyl-CoA dehydratase genes:
HACD1/PTPLA, HACD2/PTPLB, HACD3/PTPLAD1, and HACD4/PTPLAD2
[113] (Fig. 5A). Among them, HACD2 mRNA is expressed ubiquitously
and seems to be the major 3-hydroxyacyl-CoA dehydratase in mammals
[159]. HACD1 mRNA is expressed only in heart and skeletal muscle
[160]. Mutations in HACD1 are associated with recessive congenital my-
opathy characterized by early-onset muscle weakness in human and
dogs [161,162]. Hacd1 KO mice also display a myopathic phenotype
with reduced muscle weight and muscle fiber growth [163]. HACD1
functions in myoblast fusion during muscle development and regenera-
tion [163]. HACD3 mRNA is expressed in a broad range of tissues with
high expression in brain, liver, kidney, and placenta [113]. The expres-
sion of HACD4 mRNA is restricted to leukocytes [113]. To date, substrate
specificities of HACDs toward 3-hydroxyacyl-CoA substrates with dif-
ferent chain-lengths and double bond numbers have not yet been eluci-
dated. Furthermore, the functions of HACD3 and HACD4 are unclear.
59
Yeast contains only one 3-hydroxyacyl-CoA dehydratase gene, PHS1
[107]. Phs1 is an ER membrane protein with six transmembrane helixes,
and the active site residues (Tyr-149 and Glu-156) reside in transmem-
brane helix 5 [108,164].
5. Acylceramide and its importance in skin barrier formation
5.1. Skin barrier and structure of epidermis
The skin permeability barrier serves a vital function in land animals
by protecting against internal water loss. The skin barrier also impor-
tantly prevents the invasion of pathogens, allergens, and toxic com-
pounds. Accordingly, decreases in skin barrier formation lead to
several cutaneous disorders (e.g., ichthyosis, atopic dermatitis, and pso-
riasis), infectious disease, and dry skin [165–168]. Skin is composed of
the outer epidermis and inner dermis. The skin barrier is primarily
formed in the outermost cell layer of the epidermis stratum corneum,
where intercellular spaces are filled with multilayered lipids (lipid la-
mellae) [165,167,168] (Fig. 6). The high hydrophobicity of the lipid la-
mellae inhibits internal water loss and invasion of external materials.
Mammalian epidermis consists of four cell layers: stratum basale,
stratum spinosum, stratum granulosum, and stratum corneum [17,
165] (Fig. 6). Approximately 95% of epidermal cells are keratinocytes.
Keratinocytes proliferate in stratum basale and migrate outward while
differentiating into cell layer-specific cells. Stratum granulosum is char-
acterized by the presence of keratohyalin granules containing
profilaggrin, the precursor of filaggrin. Mutations in the filaggrin gene
(FLG) cause ichthyosis vulgaris [166,169], and they are also frequently
found in atopic dermatitis patients [170,171]. Filaggrin is a keratin inter-
mediate filament-associated protein, and its degradation product acts as
a natural moisturizing factor [171].
In the upper layers of epidermis, cornified envelope (CE) forms on
the periphery of keratinocytes (Fig. 6). CE is important for mechanical
and permeability barriers, and is produced through crosslinking of pro-
teins such as involucrin, envoplakin, periplakin, loricrin, and small
proline-rich proteins by transglutaminases beneath the plasma mem-
brane [172,173]. During the cornification process, the lipid bilayer of
the plasma membrane is replaced with a monolayer of protein-bound
Cers, in which ω-OH Cers are crosslinked with CE proteins such as
involucrin, envoplakin, and periplakin [172,174,175] (Fig. 6). The mem-
brane structure containing protein-bound Cers is called the corneocyte
lipid envelope (CLE), and functions to connect corneocytes with lipid
lamellae.
5.2. Formation of lipid lamellae in stratum corneum
Lipid lamellae are mainly composed of Cers (~ 50%), cholesterol
(~25%), and free FAs (~15%) [31,168]. The major free FAs of human ab-
domen stratum corneum are C16:0-COOH (36.8%), followed by C18:1-
COOH (33.1%) and C18:2-COOH (12.5%) [176]. The precursor lipids of
lipid lamellae are stored in intracellular lamellar bodies, which exist in
the upper layers of stratum spinosum and in stratum granulosum. The
ABC transporter ABCA12 transports the lipids into lamellar bodies
[177]; its gene, ABCA12, is an ARCI-causative gene [166,167,178]. ARCI
is classified into harlequin ichthyosis, lamellar ichthyosis, and congeni-
tal ichthyosiform erythroderma [179]. Mutations in the ABCA12 gene
can cause harlequin ichthyosis, which is accompanied by the most se-
vere barrier defect among ARCIs [178].
At the interface of stratum granulosum and stratum corneum, lamel-
lar bodies are fused with the plasma membrane [165] (Fig. 6). The
contained lipids are released to form lipid lamellae. The major precur-
sors for Cers in lipid lamellae are GlcCers; the minor precursors are
SMs. These are respectively converted to Cers by β-glucocerebrosidase
and sphingomyelinase upon release into the extracellular spaces [30,
31,168]. Mutations in the β-glucocerebrosidase gene (GBA) cause
Gaucher disease, one of the sphingolipid storage diseases
60 A. Kihara / Progress in Lipid Research 63 (2016) 50–69

Fig. 6. Generation of acyl-Cer and protein-bound Cer in epidermis. The structure of epidermis is illustrated as well as the enzymes and reactions responsible for creating acyl-Cer and
protein-bound Cer. SB, stratum basale; SS, stratum spinosum; SG, stratum granulosum; SC, stratum corneum; G, glucose.

(sphingolipidoses) [180]. Gaucher patients and GBA KO mice alike dis-
play ichthyosis phenotype due to failure of GlcCer-to-Cer conversion
in stratum corneum [181,182].
5.3. Ceramides in epidermis
Because of the tissue's specialized role in skin barrier formation, Cers
in epidermis are much more abundant and diverse than in other tissues
[17,30,31,167]. The composition of Cer classes in human stratum
corneum is NP (22.1%), NH (14.5%), AH (10.8%), NDS (9.8%), AS
(9.6%), AP (8.8%), NS (7.4%), EOS (6.5%), EOH (4.3%), ADS (1.6%), EOP
(1.1%), and EODS (0.4%) [31,38]. Of these, acyl-Cers (EOS, EOH, EOP,
and EODS) account for ~ 12% of stratum corneum Cers. The chain-
lengths of the FA portions of non-acyl-Cers in human stratum corneum
are mostly C24–C30, whereas those of acyl-Cers are C30–C36 [46]. Inter-
estingly, levels of Cers containing odd-numbered FAs are especially high
(~30% of total Cers) in stratum corneum [46].
Acyl-Cers are epidermis-specific and are quite important for skin
barrier formation [17,30,31,167]. Accordingly, mutations in the genes
involved in acyl-Cer production such as CERS3, ELOVL4, and CYP4F22
cause ichthyosis symptoms with severe barrier defects in human pa-
tients and mouse models [87,89,147,150,152,183,184]. Atopic dermati-
tis is also accompanied by mild skin barrier defects. In atopic dermatitis
patients, total Cer levels in skin are reduced to ~2/3 of those in healthy
controls [185], and Cer compositions are altered [185–187]. Atopic der-
matitis patients exhibit decreased EOS, EOH, EOP NH, and NP among the
stratum corneum Cer classes [187], as well as shortened Cer chain-
lengths [167,187,188]. Changes in Cer composition are also observed
in patients afflicted with psoriasis [189], which is an immuno-
inflammatory disease characterized by red, itchy, and scaly patches
and dryness. EOS, NP, and AP levels are decreased in psoriatic scales
[189].
5.4. Acylceramide synthetic pathway
Acyl-Cers are unique in their structures. Although normal Cers con-
tain two hydrophobic chains (an LCB and a FA), acyl-Cers have three hy-
drophobic chains (an LCB, an ω-OH ULCFA with C30–36, and C18:2-
COOH) (Fig. 6). Therefore, production of acyl-Cers requires more en-
zymes than does that of normal Cers. ULC acyl-CoAs are generated by
sequential actions of the FA elongases ELOVL1 (up to C26) and
ELOVL4 (≥ C28) [104,127,147] (Fig. 6). After removal of CoAs, the
resulting ULCFAs are ω-hydroxylated by the cytochrome P450 member
CYP4F22, generating ω-OH ULCFAs [190]. CYP4F22 is a type I ER mem-
brane protein, and its active site is exposed to the cytosolic side of the ER
membrane [190]. CYP4F22 is an ARCI-causative gene [183,184]: al-
though such patients are rare, acyl-Cers were almost absent in the one
CYP4F22-mutant ARCI patient examined to date [190]. After conversion
of ω-OH ULCFAs to ω-OH ULC acyl-CoAs by acyl-CoA synthetases
(ACSs), the generated ω-OH ULC acyl-CoAs are used for ω-OH Cer syn-
thesis by the Cer synthase CERS3 [87]. The de novo synthesized dihydro-
Sph appears to be the major LCB donor for ω-OH Cer synthesis, but other
 A. Kihara / Progress in Lipid Research 63 (2016) 50–69
LCBs generated from the salvage pathways can be used. The LCB part
of ω-OH Cers containing dihydro-Sph is converted to other types of
LCBs (Sph by DEGS1, phyto-Sph by DEGS2, or to 6-OH Sph by an un-
known enzyme). A scaly skin phenotype is observed in Degs1 KO
mice [65], indicating the importance of Sph-based Cers/acyl-Cers in
skin barrier formation. Finally, an ester linkage is formed between
C18:2-COOH and the ω-OH group of ω-OH Cer to create an acyl-
Cer; the acyltransferase or transacylase responsible has yet to be
identified. C18:2-COOH seems to be supplied from triglycerides
[191], since KO mice for the diacylglycerol acyltransferase Dgat2,
which is involved in triglyceride synthesis, exhibit severe skin barri-
er defects [192].
All of the enzymes involved in acyl-Cer production (except for the
unidentified acyltransferase/transacylase) are localized in the ER, indi-
cating that acyl-Cer production takes place there [190]. Produced acyl-
Cers are then converted to acyl-GlcCers in the cis-Golgi by the UDP-
glucose Cer glucosyltransferase UGCG [193,194], followed by transport
into lamellar bodies by the ABC transporter ABCA12 [31,177].
Epidermis-specific Ugcg KO mice display severe skin barrier defects
[195]. In these mice, levels of GlcCers/acyl-GlcCers are reduced, but in-
stead of the acyl-GlcCers disappearing, unusual acyl-SMs are produced.
The amounts of acyl-Cer EOS and protein-bound Cers are unchanged.
Acyl-SMs cannot substitute for acyl-GlcCers in lamellar body formation:
abnormal arrangements of lamellae were observed in the lamellar bod-
ies of Ugcg KO mice [195]. Finally, acyl-GlcCers in lamellar bodies are re-
leased into extracellular spaces at the interface of stratum granulosum
and stratum corneum and are reverted to acyl-Cers by β-
glucocerebrosidase [30,168] (Fig. 6).
5.5. Protein-bound ceramide synthesis
Protein-bound Cers, components of the CLE, are produced from acyl-
Cers [175,196]. Thus, acyl-Cers are important not only as lipid lamella
components but also as precursors of protein-bound Cers for skin barri-
er formation. The Cer moieties of protein-bound Cers are OS (ω-OH
acyl-Sph derived from EOS), OH (ω-OH acyl-6-OH Sph derived from
EOH), or OP (ω-OH acyl-phyto-Sph derived from EOP); their respective
percentages in stratum corneum are 54%, 31%, and 15% [31,38].
In the course of protein-bound Cer production, the C18:2-COOH por-
tions of acyl-Cers (or acyl-GlcCers) are subjected to peroxidation and
subsequent epoxyalcohol derivatization by successive actions of two
lipoxygenases (the 12R-lipoxygenase ALOX12B and the epidermal
lipoxygenase-3 ALOXE3) [196,197] (Fig. 6). Both ALOX12B and ALOXE3
are ARCI-causative genes [198]. Furthermore, severe skin barrier defect
phenotypes are observed in Alox12b and Aloxe3 KO mice [199–201].
These results indicate the pivotal role of the CLE in skin barrier forma-
tion. The oxygenation by the two lipoxygenases seems to act as a signal
for subsequent esterase-catalyzed hydrolysis of the oxidized C18:2-
COOH, generating ω-OH Cers. The exposed ω-OH group may be then
crosslinked with CE proteins such as involucrin, envoplakin, and
periplakin, producing the CLE [174,175]. In vitro experiments initially
suggested that this reaction is catalyzed by the transglutaminase
transglutaminase 1 (TGM1), which is known to function in forming
the CE (via isopeptide bond formation among CE proteins) [202]. How-
ever, later study showed that ARCI (lamellar ichthyosis) patients with
absent or low TGM1 activity/protein levels had normal levels of
protein-bound Cers [203]. Therefore, protein-bound Cers may be pro-
duced by other enzymes or via another mechanism. The removal of glu-
cose from acyl-GlcCers by β-glucocerebrosidase may occur after the
creation of protein-bound GlcCers, since protein-bound GlcCers are in-
creased in β-glucocerebrosidase-deficient type 2 Gaucher mice and in
KO mice lacking the gene encoding the activator protein for β-
glucocerebrosidase (saposin-C) [204,205]. However, it is entirely possi-
ble that the order of the crosslinking with respect to the glucose remov-
al is not strict.
61
6. Ceramide degradation pathways
6.1. Ceramide degradation pathways
In sphingolipid biosynthetic pathways, Cer is converted to the com-
plex sphingolipids SM and glycosphingolipids. Alternatively, Cer can be
converted to Cer 1-phosphate by Cer kinase [206]. Cer 1-phosphate is
involved in several cellular processes including phagolysosome forma-
tion, mast cell degranulation, and inflammation [6,207].
In their degradation pathways, Cers are first converted to LCBs and
FAs by ceramidases [6,208]. The released FAs can be used for lipid syn-
thesis or energy production via β-oxidation. On the other hand, the re-
leased LCBs are exclusively recycled back to sphingolipids, since they
are used only as components of sphingolipids. Therefore, LCBs must be
converted to other metabolizable compounds in order for homeostasis
to be maintained in terms of the concentrations of LCBs and
sphingolipids. Eukaryotic cells share metabolic pathways for converting
LCBs to acyl-CoAs via LCB 1-phosphates (LCBPs), fatty aldehydes, and
FAs [5,209,210] (Fig. 7A). The generated acyl-CoAs are then used as
the FA components of lipids (mainly ester-linked glycerophospholipids
and partly ether-linked glycerophospholipids, triglycerides, and
sphingolipids) or degraded to CO2 via β-oxidation [5,125,209–212]
(Fig. 7B). C16:0-CoA is generated from Sph and dihydro-Sph, whereas
odd-numbered C15:0-CoA is created from phyto-Sph through Cer deg-
radation pathways [209,213,214] (Fig. 7A). Some of the produced acyl-
CoAs are elongated or desaturated before being incorporating into lipids
[211–214].
As described above, most LCBs are either recycled to sphingolipids or
metabolized to glycerophospholipids. What percentages of LCBs under-
go the latter pathway varies among tissues. HeLa cells, PC12 (neuronal)
cells, HepG2 hepatocytes, and IEC-6 intestinal epithelial cells were la-
beled with [3H]Sph, and 23–42% of it was found to be metabolized to
ester-linked glycerophospholipids (HeLa, 23%; PC12, 32%; HepG2, 21%;
and IEC-6, 42%) [125]. In addition, a trace amount was metabolized to
ether-linked glycerophospholipids in PC12 and HepG2 cells [125].
6.2. Ceramide degradation by ceramidases
The first step of Cer degradation pathways—i.e., cleavage of Cer to a
FA and an LCB—is catalyzed by ceramidases (Fig. 7A). Mammals contain
five ceramidases having different optimal pH: the acid ceramidase
ASAH1/AC, the neutral ceramidase ASAH2/NC, and the alkaline
ceramidases 1–3 (ACER1, ACER2, and ACER3) [208]. Among them, the
lysosome-localized ASAH1 is the physiologically most important. Muta-
tions in ASAH1 cause the sphingolipid storage disease Farber disease,
characterized by deformed joints, progressive hoarseness, subcutane-
ous nodules, and neurologic deterioration [215]. Disruption of mouse
Asah1 causes early embryonic lethality [216].
6.3. Long-chain base 1-phosphate metabolic enzymes
6.3.1. Sphingosine kinases
In Cer degradation pathways, LCBs are converted to LCBPs: S1P from
Sph, dihydro-Sph 1-phosphate (dihydro-S1P) from dihydro-Sph, and
phyto-Sph 1-phosphate (phyto-S1P) from phyto-Sph (Fig. 7A). The
two redundant sphingosine kinases SPHK1 and SPHK2 catalyze the pro-
duction of LCBPs in mammals [217,218]. Although KO mice having sin-
gle gene deletion of Sphk exhibit no obvious phenotype, Sphk1 Sphk2
double KO mice are embryonic lethal due to impaired neurogenesis
and angiogenesis [219]. SPHK1 has higher substrate specificity than
SPHK2; it exhibits activity toward Sph and dihydro-Sph, whereas
SPHK2 is active toward not only all LCBs but also the LCB analog
fingolimod/FTY720 [217,218,220], which is therapeutic for multiple
sclerosis [34,35]. Fingolimod is activated by phosphorylation, then acts
by binding to S1P receptors, mainly S1P1 [16,34,35].
62 A. Kihara / Progress in Lipid Research 63 (2016) 50–69

Fig. 7. Cer degradation pathways. (A) Pathways, reactions, and enzymes involved in the degradation of dihydro-Cer, Cer (Sph), and phyto-Cer in yeast and mammals. (B) Overall flow of
Cer degradation pathways leading to other classes of lipids. In Cer degradation pathways, LCBs are metabolized mainly to ester-linked glycerophospholipids but partly to other lipids such
as triglyceride (TG), sphingolipids, and ether-linked glycerophospholipids or CO2. In SLS patients, the conversion of the fatty aldehydes to FAs is impaired. Accordingly, metabolism of LCBs
to ester-linked glycerophospholipids is abrogated, and instead that to ether-linked glycerophospholipids becomes a major metabolic route. F-Ald, fatty aldehyde; F-Alc, fatty alcohol.

S1P is a well-known lipid mediator in extracellular fluid (such as
plasma), whereas in cells it is a metabolic intermediate of sphingolipids
[6,209,210]. Only limited cell types secrete S1P extracellularly: the
major sources of plasma S1P are erythrocytes and vascular endothelial
cells [16,221–223]. Fingolimod phosphate is mainly generated by
SPHK2 in platelets before being secreted into plasma [16,224]. On the
other hand, sphingolipid metabolism via LCBPs occurs in all tissues ex-
cept for platelets and erythrocytes, which lack S1P lyase [210,221,225,
226]. Accordingly, SPHKs are expressed in all tissues [227].
SPHK1 is localized intracellularly mainly in the cytosol and partly in
the plasma membrane [228–232]. SPHK2 is also primarily localized in
cytosol, but incidence in the plasma membrane, internal membranes,
and nucleus has also been reported [230,231]. Although SPHK1 and
SPHK2 are mostly located in the cytosol, it is membranes where they
must function, given that their substrate LCBs are too hydrophobic to
freely diffuse into the cytosol. Since the lysosomal acid ceramidase
Asah1 is the major ceramidase as described above, Sph seems to be
mainly generated in lysosomes. However, the site of S1P production
still remains unclear. It is possible that cytosolic SPHKs are transiently
recruited to lysosomes for S1P production, or that lysosomally
generated Sph is transported to the plasma membrane or other mem-
branes, whereupon SPHKs phosphorylate it.
6.3.2. Long-chain base 1-phosphate-degrading enzymes
LCBPs are either dephosphorylated to LCBs by S1P phosphatases or
cleaved to fatty aldehydes [trans-2-hexadecenal (tC16:1-CHO) from
S1P, hexadecanal (C16:0-CHO) from dihydro-S1P, and 2-
hydroxyhexadecanal (2-OH C16:0-CHO) from phyto-S1P] and
phosphoethanolamine by S1P lyase [6,210] (Fig. 7A).
Phosphoethanolamine is metabolized to phosphatidylethanolamine
via CDP-ethanolamine [211]. Two S1P phosphatases (SGPP1/SPP1 and
SGPP2/SPP2) and one S1P lyase (SGPL/SPL) have been identified in
mammals [233–235]. All of these S1P-degrading enzymes are localized
in the ER [225,235,236]. SGPP1 and SPL are expressed ubiquitously,
whereas expression of SGPP2 is limited to certain tissues such as kidney
and heart [225,235,237]. Sgpp1 KO mice exhibit focal desquamation and
morphological aberration in epidermis, and most die before weaning
[238]. Moreover, S1P levels are increased in their keratinocytes, and
their keratinocyte differentiation is accelerated [238].
 A. Kihara / Progress in Lipid Research 63 (2016) 50–69
The S1P lyase-catalyzed reaction is irreversible, and is the rate-
limiting step of Cer degradation pathways. Generally, biomolecules
must be kept in narrow ranges of concentrations; otherwise cellular
functions can become impaired, leading to disorders. Therefore, the bal-
ance between synthesis and degradation must always be properly
maintained. This is no less true of Cer degradation pathways. Their
blockage by the disruption of Sgpl1 in mice causes accumulation of
S1P as well as increases in Cer and SM levels [239]. Sgpl1 KO mice exhibit
several phenotypes, such as lymphopenia, thymus atrophy, myeloid cell
hyperplasia, disturbance of lipid homeostasis in liver, lesions in lung,
heart, urinary tract, and bone, and early mortality (20–50 days after
birth) [239–242].
6.4. The aldehyde dehydrogenase ALDH3A2 and Sjögren-Larsson syndrome
6.4.1. Aldehyde dehydrogenases
Fatty aldehydes produced by S1P lyase are converted to FAs [C16:0-
COOH from C16:0-CHO, trans-2-hexadecenoic acid (tC16:1-COOH)
from tC16:1-CHO, and 2-hydroxypalmitic acid (2-OH C16:0-COOH)
from 2-OH C16:0-CHO] by aldehyde dehydrogenases (ALDHs). Multiple
ALDHs exist in mammals (19 in human and 21 in mouse), divided into
eleven subfamilies (ALDH1–9, −16, and −18) [243,244]. Of these, the
ALDH3 subfamily is responsible for the oxidation of fatty aldehydes.
Humans possess four ALDH3 subfamily genes [ALDH3A1, ALDH3A2,
ALDH3B1, and (the pseudogene) ALDH3B2], whereas the mouse genome
has five Aldh3 genes (Aldh3a1, Aldh3a2, Aldh3b1, Aldh3b2, and Aldh3b3)
[243,244]. ALDH3A1/Aldh3a1 is a cytosolic protein highly expressed in
cornea and exhibits high activity toward MC aldehydes [245,246].
ALDH3A2/Aldh3a2, ALDH3B1/Aldh3b1, Aldh3b2, and Aldh3b3 prefer-
entially oxidize LC aldehydes, although they show weak activities to-
ward MC aldehydes [246,247]. Among them, only ALDH3A2/Aldh3a2
is localized in the ER [248], where LCBP-to-glycerophospholipid conver-
sion occurs. In contrast, ALDH3B1/Aldh3b1 and Aldh3b3 are localized in
the plasma membrane, and Aldh3b2 in lipid droplets [246,247]. Accord-
ingly, ALDH3A2/Aldh3a2 is the major ALDH involved in Cer degradation
pathways [213]. In one of our previous studies, Aldh3a2-null CHO-K1
cells were labeled with [3H]Sph or [3H]dihydro-Sph, and the metabo-
lism of Sph/dihydro-Sph to ester-linked glycerophospholipids was
largely impaired [213]. Instead, a fraction of unmetabolized fatty alde-
hydes were reduced to fatty alcohols and incorporated into ether-
linked glycerophospholipids.
Ether-linked glycerophospholipids include plasmanyl (1-alkyl-2-
acyl) and plasmenyl (1-alkenyl-2-acyl) types, the latter of which are
collectively called plasmalogens (Fig. 7B). The polar head groups of
ether-linked glycerophospholipids are exclusively
phosphoethanolamine (plasmanyl/plasmenylethanolamine) and
phosphocholine (plasmanyl/plasmenylcholine) [249]. Plasmalogens ac-
count for ~18% of total human phospholipids and are highly abundant in
erythrocytes, kidney, lung, testis, skeletal muscle, and myelin [249].
They play several physiological functions including removal of oxidants,
cell–cell or cell-extracellular matrix interactions, lipid microdomain for-
mation, intracellular cholesterol homeostasis, spermatogenesis, and
myelination [250]. In the ether-linked glycerophospholipid biosynthetic
pathway, 1-acyl-dihydroxyacetone phosphate is first created from acyl-
CoA and dihydroxyacetone phosphate, and then the acyl-chain of 1-
acyl-dihydroxyacetone phosphate is replaced with a fatty alcohol by
alkyl dihydroxyacetone phosphate synthase [249]. Fatty alcohols are
mainly generated from acyl-CoAs by fatty acyl-CoA reductases [249,
251]. Although most fatty aldehydes generated through ceramide deg-
radation pathways are converted to FAs by ALDHs, a small amount of
them is converted to fatty alcohols and incorporated into ether-linked
glycerophospholipids, even in ALDH3A2-active cells [125,209,252]
(Fig. 7B). However, the contribution of ceramide degradation pathways
to the total fatty alcohol generation for ether-linked
glycerophospholipid synthesis seems to be low.
63
6.4.2. Sjögren-Larsson syndrome
ALDH3A2 is a causative gene of the neurocutaneous disorder
Sjögren-Larsson syndrome (SLS), which is characterized by mental re-
tardation, spastic di- or tetraplegia, and ichthyosis [253]. Aldehyde mol-
ecules are reactive, such as by forming Schiff bases with primary amines,
and thus toxic to cells. Therefore, the SLS pathology is considered to be
caused by accumulated fatty aldehydes reacting with certain important
proteins in epidermis and the nervous system. Several metabolic path-
ways can also generate the substrates of ALDH3A2, fatty aldehydes,
other than Cer degradation pathways, such as those of leukotriene B4,
diet-derived phytol, plasmalogens, and fatty alcohols [254]. It is still un-
clear which fatty aldehyde or which metabolic pathway is the main con-
tributor to the pathogenesis of SLS. However, Cer degradation pathways
are a likely candidate. Cer degradation is ubiquitous and occurs actively,
suggesting that tC16:1-CHO is generated constantly at a relatively high
level. Furthermore, the site of tC16:1-CHO generation via Cer degrada-
tion and that of ALDH3A2 localization are both the ER [225,248]. The
Sph metabolite tC16:1-CHO is an α,β-unsaturated aldehyde and hence
a particularly reactive fatty aldehyde. Although normal aldehydes can
only react with primary amines to form Schiff bases, α,β-unsaturated al-
dehydes can react with general nucleophiles such as lysine, cysteine,
and histidine side-chains via 1,4-Michael addition [255].
6.5. Acyl-CoA synthetases involved in ceramide degradation pathways
Conversion to acyl-CoAs by ACSs is necessary for FAs to be incorpo-
rated into lipids, degraded via β-oxidation, or elongated via the FA elon-
gation cycle [256]. Therefore, the FAs generated in Cer degradation
pathways must also be activated for further metabolism. ACSs are divid-
ed into six subfamilies [ACS short-chain (ACSS), ACS medium-chain
(ACSM), ACS long-chain (ACSL), ACS very-long-chain (ACSVL), ACS
bubblegum (ACSBG), and ACS family (ACSF)], depending on their sub-
strate specificities and sequence similarities [257]. The human genome
contains 26 ACS genes (ACSS1–3, ACSM1–5, ACSL1, 3–6, ACSVL1–6,
ACSBG1, 2, and ACSF1–4) [257]. Among them, eight ACSs (ACSL1, 3, 4,
5, 6, ACSVL1, 4, and ACSBG1), which exhibit activity toward LCFAs
[256], are involved in Cer degradation pathways [213,258].
6.6. Dual functions of the trans-2-enoyl-CoA reductase TECR
Dihydro-Sph is metabolized to C16:0-CoA via four reactions (phos-
phorylation, cleavage, oxidation, and CoA addition) as described above
(Fig. 7A). Since Sph contains a trans-double bond, it needs an additional
saturation step for conversion to C16:0-CoA. After creation of trans-2-
hexadecenoyl-CoA (tC16:1-CoA) by ACSs from the ALDH product
tC16:1-COOH, the resulting tC16:1-CoA is saturated to C16:0-CoA by
the trans-2-enoyl-CoA reductase TECR [125] (Fig. 7A). As mentioned
above, TECR is also involved in the fourth step of the FA elongation
cycle. Thus, TECR functions in two lipid metabolic pathways: Cer degra-
dation pathways and FA elongation cycle.
6.7. Metabolism of phytosphingosine to odd-numbered fatty acids
Phyto-Sph has an extra hydroxyl bond at the C4 position compared
to dihydro-Sph. The hydroxyl bond at C4 in phyto-Sph is shifted to the
C2 position by removal of a two-carbon unit by S1P lyase: the product
is 2-OH C16:0-CHO [214] (Fig. 7A). Existence of this hydroxyl bond
makes the metabolic pathway of phyto-Sph different from those of
dihydro-Sph and Sph. Although dihydro-Sph and Sph are metabolized
to glycerophospholipids as C16:0-COOH or its derivatives (C18:0-
COOH and C16:1-COOH), phyto-Sph is metabolized to
glycerophospholipids as pentadecanoic acid (C15:0-COOH) or its deriv-
atives [heptadecanoic acid (C17:0-COOH) and pentadecenoic acid
(C15:1-COOH)] [209,214]. After phyto-S1P is cleaved to 2-OH C16:0-
CHO by S1P lyase, it is oxidized to 2-OH C16:0-COOH by ALDHs. The
resulting 2-OH C16:0-COOH [or its acyl-CoA derivative 2-
64 A. Kihara / Progress in Lipid Research 63 (2016) 50–69
hexadecanoyl-CoA (2-OH C16:0-CoA)] is subjected to α-oxidation, cre-
ating C15:0-COOH [214].
Cer degradation pathways are conserved among eukaryotes includ-
ing yeast, in which dihydro-Sph and phyto-Sph are two major LCBs. The
yeast S. cerevisiae contains two alkaline ceramidases, Ydc1 and Ypc1
[259,260]. Ypc1 exhibits high activity toward dihydro-Cer and weak ac-
tivity toward phyto-Cer [259,260]. On the other hand, Ydc1 is highly
specific to dihydro-Cer [260]. As in mammals, in yeast the produced
dihydro-Sph is sequentially metabolized to dihydro-S1P (by LCB kinase
Lcb4), C16:0-CHO (by LCBP lyase Dpl1), C16:0-COOH (by ALDH Hfd1),
and C16:0-CoA (by ACSs Faa1 and Faa4), followed by incorporation
into glycerophospholipids [213] (Fig. 7A). Although Sph is not a natural
LCB in yeast, exogenously added Sph can be metabolized to both
sphingolipids and glycerophospholipids [261]. In the latter metabolic
pathway, again as in mammals, Sph is metabolized to C16:0-CoA by
the above enzymes plus the trans-2-enoyl-CoA reductase Tsc13 (i.e., a
homolog of TECR), followed by incorporation into glycerophospholipids
[125,213].
Phyto-Sph is metabolized to C15:0-COOH (or its derivative C17:0-
COOH) in yeast as in mammals [214]. In yeast mutants defective in
phyto-Sph production (i.e., by deletion of the dihydro-Sph C4-
hydroxylase gene SUR2) or metabolism (i.e., by deletion of the LCBP
lyase gene DPL1), the levels of odd-numbered PCs are reduced to
~ 40% of those in wild type cells [214]. Thus, the phyto-Sph metabolic
pathway is the major source of cellular odd-numbered FAs in yeast. Al-
though the details of the α-oxidation reaction remain unclear, the novel
yeast gene MPO1 has been implicated in the α-oxidation step of the
phyto-Sph metabolic pathway [214]. Mpo1 is an ER protein [214], indi-
cating that the α-oxidation step takes place in the ER in yeast. Mpo1 be-
longs to the large protein family DUF962 (DUF: domain of unknown
function), whose function had been unknown until MPO1 was identi-
fied. DUF962 family members exist in bacteria, fungi, plants, and pro-
tists, whereas mammals contain none, suggesting that α-oxidation
pathways differ in yeast and mammals. In mammals, the 2-
hydroxyacyl-CoA lyase HACL1, which is localized to the peroxisomes,
was shown to exhibit activity to convert 2-OH octadecanoyl-CoA (2-
OH C18:0-CoA) to heptadecanol (C17:0-CHO) in vitro [262]. Therefore,
it is possible that 2-OH C16:0-COOH derived from phyto-Sph is sequen-
tially metabolized to 2-OH C16:0-CoA by ACSs, to C15:0-CHO by HACL1,
and to C15:0-COOH by ALDHs in mammals.
The purpose of phyto-Sph metabolism to odd-numbered FAs may
not be to generate odd-numbered FAs per se, but to convert 2-OH FAs,
which are difficult to metabolize, to easily metabolizable non-hydroxy
FAs. Only sphingolipids contain 2-OH FAs, and 2-OH FA-containing
sphingolipids exist in restricted tissues such as stomach, intestine,
brain, kidney, and epidermis [38,48,263]. Therefore, unless they are
first converted to other metabolizable compounds, the metabolic fate
of 2-OH FAs is limited to conversion to sphingolipids. On the other
hand, once 2-OH FAs are metabolized to odd-numbered FAs, they can
be metabolized to several lipids and be degraded via β-oxidation as
even-numbered FAs. Although the β-oxidation products of even-
numbered FAs are always acetyl-CoAs, β-oxidation of odd-numbered
FAs generates acetyl-CoAs and propionyl-CoA, the latter of which can
be further metabolized to succinyl-CoA for breakdown in the citric
acid cycle [264].
6.8. Generation of 2-hydroxy fatty acids
Although most cellular FAs are even-numbered FAs, the existence of
odd-numbered FAs has long been known. Their levels are quite low in
most tissues (b1% of those of even-numbered FAs) [265]. However, in
specific tissues such as brain and epidermis, odd-numbered FA levels
are unusually high [46,49]. At first, a de novo FA synthetic pathway
that used propionate as a starting material was proposed to be the
major source of odd-numbered FAs [266]. However, later it turned out
that 2-OH FAs are major sources of odd-numbered FAs [267]. In brain,
the levels of cerebrosides and sulfatides containing C23:0-COOH are es-
pecially high, reflecting the abundance of sphingolipids containing
cerebronic acid (2-OH C24:0-COOH) in myelin [49].
The phyto-Sph metabolic pathway is not the sole pathway that gen-
erates 2-OH FAs. Rather, most 2-OH FAs are generated via direct hydrox-
ylation of FAs by the FA 2-hydroxylase FA2H [263,268]. A large
proportion of GalCers and sulfatides contains 2-OH FAs (i.e., contains
AS type Cers) in brain [49,50,52]. The 2-OH group seems important for
GalCers and sulfatides to exert their functions in myelin, perhaps en-
hancing lipid-lipid interactions via hydrogen bonds and thereby stabi-
lizing the myelin sheath. Accordingly, FA2H mutations in humans
cause hereditary spastic paraplegia [269,270], and Fa2h KO mice exhibit
late-onset axon and myelin sheath degeneration [52,271].
Epidermis contains high amounts of 2-OH FA-containing Cers:
i.e., AS, AP, AH, and ADS types of Cers. Disruption of Fa2h in mice, how-
ever, does not cause changes in levels of 2-OH FA-containing Cers in epi-
dermis [272]. Expression of Fa2h in skin is restricted to the sebaceous
glands, and Fa2h KO mice exhibit delayed fur development and cycle al-
opecia [272]. Therefore, another, unknown FA 2-hydroxylase must exist
in epidermis.
7. Concluding remarks
Cers are highly diverse molecules, with recently developed high-
sensitivity mass spectrometry detecting over 1000 species [38]. Most
of the genes involved in the synthesis, degradation, and modification
of Cers have been identified in the last 15 years. Through analyses of
these genes, such as screenings for mutations in hereditary diseases, ex-
aminations of changes in their expression or activity patterns in meta-
bolic diseases and cancer, and phenotypic analyses using KO mice,
vast knowledge about the physiological and pathological importance
of Cers and sphingolipids has been accumulated. We now realize that
even small differences between Cer species—i.e., in chain-length, hy-
droxylation status, and double bond number—bear great significance
on how they fulfill their species-specific functions. These findings can
lead to clinical applications in the future, through the development of
inhibitors or activators of these Cer-related gene products.
Acknowledgments
This work was supported by funding from Advanced Research and
Development Programs for Medical Innovation (AMED-CREST) from
the Japan Agency for Medical Research and Development (AMED), by
funding from Creation of Innovation Centers for Advanced Interdisci-
plinary Research Areas Program from the Ministry of Education, Culture,
Sports, Science and Technology of Japan, and by a Grant-in-Aid for Sci-
entific Research (A) 26251010 from the Japan Society for the Promotion
of Science (JSPS).
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