Hindawi Publishing Corporation
Oxidative Medicine and Cellular Longevity
Volume 2012, Article ID 479348, 17 pages
Lisa Longato,1Kelsey Ripp,1Mashiko Setshedi,1Miroslav Dostalek,2Fatemeh Akhlaghi,2
Mark Branda,1Jack R.Wands,1andSuzanneM.de la Monte1,3
1Divisions of Gastroenterology and Neuropathology, Departments of Medicine, Pathology, Neurology, and Neurosurgery,
The Liver Research Center, Rhode Island Hospital and the Warren Alpert Medical School of Brown University,
Providence, RI 02903, USA
2Clinical Pharmacokinetics Research Laboratory, University of Rhode Island, Kingston, RI 02881, USA
3Pierre Galletti Research Building, Rhode Island Hospital, 55 Claverick Street, Room 419, Providence, RI 02903, USA
Correspondence should be addressed to Suzanne M. de la Monte, suzanne delamonte email@example.com
Received 15 October 2011; Revised 28 December 2011; Accepted 10 January 2012
Academic Editor: Florian Lang
Copyright © 2012 Lisa Longato et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Chronic alcohol-related liver disease (ALD) is mediated by insulin resistance, mitochondrial dysfunction,
inflammation, oxidative stress, and DNA damage. Recent studies suggest that dysregulated lipid metabolism with accumulation
of ceramides, together with ER stress potentiate hepatic insulin resistance and may cause steatohepatitis to progress. Objective. We
examined the degree to which hepatic insulin resistance in advanced human ALD is correlated with ER stress, dysregulated lipid
metabolism, and ceramide accumulation. Methods. We assessed the integrity of insulin signaling through the Akt pathway and
measured proceramide and ER stress gene expression, ER stress signaling proteins, and ceramide profiles in liver tissue. Results.
Chronic ALD was associated with increased expression of insulin, IGF-1, and IGF-2 receptors, impaired signaling through IGF-1R
and IRS1, increased expression of multiple proceramide and ER stress genes and proteins, and higher levels of the C14, C16, C18,
with dysregulated lipid metabolism, ceramide accumulation, and striking upregulation of multiple ER stress signaling molecules.
Given the role of ceramides as mediators of ER stress and insulin resistance, treatment with ceramide enzyme inhibitors may help
reverse or halt progression of chronic ALD.
Worldwide, alcohol abuse is one of the leading causes
of morbidity and mortality from chronic liver disease
[1, 2]. In its initial stages, alcohol-related liver disease
(ALD) is characterized by hepatic steatosis, which is largely
reversible. However, with continued chronic alcohol abuse,
liver disease can progress through stages of steatohepatitis,
followed by fibrosis, and then cirrhosis, and in the late
stages, it may culminate in liver failure or hepatocellular
carcinoma . Previous studies linked chronic ALD to the
combined effects of hepatic insulin resistance and toxicity
mediated by inflammation, oxidative stress, mitochondrial
dysfunction, or acetaldehyde-induced adduct formation [4–
7]. The consequences include impairments in hepatocellular
survival, energy metabolism, and capacity to regenerate
and remodel after injury. Together, these responses serve
as potent stimuli for stellate cell activation and attendant
Recent studies have linked insulin resistance with inflam-
mation and oxidative stress to dysregulated lipid metabolism
and increased ceramide generation [9–11]. However,
ceramides can further impair insulin signaling, mito-
chondrial function, and cell viability [12, 13]. Ceramides
comprise a family of simple sphingolipids generated from
fatty acid and sphingosine [13–15]. Although ceramides
2 Oxidative Medicine and Cellular Longevity
are present at low levels within biological membranes, they
participating in lipid raft formation. In addition, ceramides
exert diverse regulatory effects on cell signaling pathways
that mediate growth, proliferation, motility, adhesion,
differentiation, senescence, and apoptosis [12, 13, 16–18].
Ceramides are generated via 3 main pathways: (1) de
novo synthesis by coupling sphinganine to a long-chain fatty
acid, yielding dihydroceramide; (2) hydrolysis of complex
sphingolipids such as sphingomyelin or glycosphingolipids;
(3) recycling after acylation of sphingosine, utilizing the “sal-
vage pathway” [16, 17]. Ceramides cause insulin resistance
by activating proinflammatory cytokines, inhibiting trans-
mission of signals through phosphatidyl-inositol-3 kinase
(PI3K) and Akt [19–22], and activating protein phosphatase
2A (PP2A) [23, 24]. Furthermore, ceramides can promote
apoptosis by activating protein kinase C, PP1, caspases,
and cathepsin D [16, 18]. Therefore, dysregulated lipid
generation of ceramides that exacerbate insulin resistance,
inflammation, tissue injury, and cell death.
Emerging evidence suggests that ceramide production
can be modulated by ethanol exposure, and that some of
the hepatotoxic and degenerative effects of ethanol may be
mediated by ceramide accumulation in liver. For example,
ethanol increases ceramide levels by activating acidic sphin-
gomyelinase , and ethanol-exposed hepatocyte cultures
exhibit reduced levels of sphingomyelin and sphingosine,
and increased levels of ceramide . The findings that (1)
mice deficient in acidic sphingomyelinase are resistant to
ethanol-induced hepatic steatosis , and (2) ceramides
contribute to the inhibitory effects of ethanol on AMPK
phosphorylation  suggest that ceramides generated in
ethanol exposed livers may cause liver injury.
Increased ceramide production can lead to endoplasmic
reticulum (ER) stress and thereby contribute to the pro-
gression of ALD. ER stress can potentiate insulin resistance
and lipolysis leading to increased ceramide production [29–
32] and worsening of inflammation and insulin resistance.
ER stress is caused by disruption of homeostatic mecha-
nisms, causing unfolded proteins to accumulate,and reactive
oxygen species (ROS) to form . Normally, the ER
adapts to stress by activating the unfolded protein response
(UPR) [34, 35], which quickly increases the levels of ER
stress sensor proteins including inositol-requiring enzyme 1
(IRE1), PKR-like ER-localized eIF2α kinase (PERK), and the
activating transcription factor 6α (ATF-6α; ER membrane-
anchored transcription factor). PERK and IRE1 activate
ER stress networks by transmitting signals in response to
protein misfolding or unfolding. PERK promotes a global
arrest of protein synthesis by stimulating phosphorylation of
eukaryotic translation initiation factor 2α (eIF2α), selective
translation of ATF4, and upregulation of the transcription
factor C/EBP homologous protein CHOP. IRE1α promotes
alternative splicing of XBP1, leading to increased transcrip-
tion of chaperones and ER-associated protein degradation
(ERAD) machinery. Activated ATF-6 promotes increased
synthesis of chaperones and other components of the folding
and ERAD machinery. Prolonged activation of the UPR
induces a pathological response leading to inflammation,
injury, steatosis, and apoptosis [34, 36].
The above discussion highlights the importance of
insulin resistance in ALD, and the probable interrelationship
among insulin resistance, increased ceramide generation,
and ER stress. We hypothesize that progressive ALD is
loop whereby insulin resistance leads to lipolysis and toxic
ceramide generation, which promotes ER stress, and both
toxic ceramides and ER stress worsen insulin resistance.
The present work examines the potential contributions of
sustained insulin resistance, ER stress, and dysregulated
lipid metabolism with increased activation of proceramide
mechanisms and ceramide accumulation in human livers
with chronic ALD.
2.1. Materials. Antibodies to ER stress markers were pur-
chased from Cell Signaling (Danvers, MA). The Akt Total
and Phospho 7-Plex Panels, Taqman Gene expression master
mix, and Amplex Red Sphingomyelinase assay kit were
purchased from Invitrogen (Carlsbad, CA). Bead-based
magnetic immunoassays for cytokines quantification were
from Biorad (Hercules, CA). MaxiSorp 96-well plates used
for ELISAs were from Nunc (Thermo Fisher Scientific;
Rochester, NY). Superblock-TBS, horseradish peroxidase-
conjugated antibodies, and SuperSignal Enhanced Chemi-
luminescence Reagents were from Pierce Chemical Co
(Rockford, IL). QIAzol Lysis Reagent for RNA extraction
was obtained from Qiagen, Inc (Valencia, CA). The AMV
1st Strand cDNA Synthesis kit, the Universal Probe Library
probes, and Reference gene assays were purchased from
Roche Applied Science (Indianapolis, IN). The Serum
Triglyceride Determination kit, monoclonal anti-ceramide,
and synthetic oligodeoxynucleotides were purchased from
Sigma-Aldrich Co (St. Louis, MO). All other monoclonal
antibodies and immunodetection reagents were purchased
from Abcam (Cambridge, MA), Upstate (Billerica, MA),
CA), or Chemicon (Temecula, CA). Fine chemicals were
purchased from CalBiochem (Carlsbad, CA) or Sigma-
Aldrich (St. Louis, MO).
2.2. Source of Human Liver Tissue. Liver tissue biopsies from
patients with chronic ALD and cirrhosis were obtained from
the Liver Tissue Procurement Distribution System (NIH
Contract no. N01-DK-9-2310). Normal human control liver
biopsy tissue samples were obtained from the Lifespan-
Rhode Island Hospital Tumor Bank (Providence, RI). Con-
trol liver samples represented remnant tissue from surgical
biopsies performed for diagnosis. 10 samples were analyzed
in each group. All specimens were deidentified and their use
in this study was approved by the Lifespan Human Studies
Committee and Investigational Review Board. Snap-frozen
fresh tissues stored at −80◦C were used for RNA, protein,
and lipid studies. Formalin-fixed paraffin-embedded tissue
with Masson’s trichrome for histological assessment.
Oxidative Medicine and Cellular Longevity3
2.3. Quantitative Reverse Transcriptase Polymerase Chain
Reaction (qRT-PCR) Assays. RNA extracted from fresh
frozen liver using the RNeasy Mini Kit was reverse-
transcribed using random oligonucleotide primers and the
AMV 1st Strand cDNA Synthesis Kit. Gene expression
was measured in duplicate reactions with a hydrolysis
probe-based duplex qRT-PCR assay in which hypoxanthine-
guanine phosphoribosyltransferase (HPRT) was included
as a reference gene. Reactions (20μL) contained Taqman
Gene expression master mix, 400nM of gene-specific and
HPRT primers, and 200nM of HPRT (Y555 labeled) and
gene of interest (FAM labeled) probes. Gene-specific primer
sequences and matched probes were determined with the
ProbeFinder Software (Roche, Indianapolis, IN) (Table 1).
PCR amplifications were initiated by a 10-minute, 95◦C
denaturation step and followed by 45–50 2-step cycles of
denaturation (15-seconds at 95◦C) and annealing/extension
(1-minute at 60◦C). The amplifications were performed in
a LightCycler 480 PCR machine (Roche, Indianapolis, IN).
Fluorescence signals corresponding to the genes of interest
were acquired in the FAM Channel (Em: 520–530nm), and
the HPRT signal was acquired in the Y555/HEX channel
(Em: 550–568nm). Results were analyzed using LightCycler
Software 4.0. Alternatively, gene expression was measured in
PCR Mix and gene-specific primer pairs , with results
normalized to 18S rRNA as described .
2.4. Enzyme-Linked Immunosorbent Assay (ELISA). Liver
homogenates were prepared in lysis buffer containing
50mM Tris (pH 7.5), 150mM NaCl, 5mM EDTA (pH
8.0), 50mM NaF, 0.1% Triton X-100, and protease and
phosphatase inhibitors . Direct binding ELISAs were
performed in 96-well MaxiSorp plates. Protein homogenates
(100ng/50μL) were adsorbed to the well bottoms by
overnight incubation at 4◦C, and then blocked for 3 hours
with 1% bovine serum albumin (BSA) in TRIS buffered
saline (TBS). After washing, the samples were incubated
with primary antibody (0.1–0.4μg/mL) for 1 hour at
37◦C. Immunoreactivity was detected with horseradish-
peroxidase-(HRP-) conjugated secondary antibody and
intensity was measured (Ex 565nm/Em 595nm) in a
SpectraMax M5 microplate reader (Molecular Devices,
Sunnyvale, CA). Ceramide immunoreactivity was quantified
by ELISA as previously described .
2.5. Bead-Based Multiplex ELISA. We used bead-based mul-
tiplex ELISAs to assess the integrity of signaling through the
insulin and IGF-1 receptors, insulin receptor substrate, type
1 (IRS-1), and downstream through Akt-related pathways
using Akt Total and Phospho 7-Plex Panels. The Akt Total
7-Plex panel measured immunoreactivity to insulin receptor
(IR), IGF-1 receptor (IGF-1R), IRS-1, Akt, proline-rich Akt
substrate of 40kDa (PRAS40), ribosomal protein S6 kinase
(p70S6K), and glycogen synthase kinase 3β (GSK-3β). The
Akt Phospho 7-Plex panel measured immunoreactivity to
pT246-PRAS40,pTpS421/424-p70S6K, andpS9-GSK3β. Samples
containing 100μg protein were incubated with the beads
according to the manufacturer’s protocol. Captured antigens
were detected with secondary antibodies and phycoerythrin-
conjugated anti-rabbit IgG. Proinflammatory cytokine levels
say (200μg protein/well). Plates were read in a Bio-Plex 200
system (Bio-Rad, Hercules, CA).
2.6. Liquid Chromatography, Tandem Mass Spectrometry
(LC-MS-MS) for Quantification of Ceramides in Liver Tissue.
Lipids were extracted for ceramide profiling as described
[40, 41]. In brief, 60–70mg of fresh frozen tissue were
homogenized in 10 volumes of ice cold phosphate-buffered
saline (PBS). 200μL of homogenate were extracted with
isopropanol:water:ethyl acetate (30:10:60 by volume).
The combined extracts were transferred to clean glass
centrifuge tubes and evaporated at room temperature
in a SpeedVac evaporator (Model SPD1010 Speed Vac
System, Thermosavant, Holbrook, NY). The residues
were redissolved in 100μL of methanol, vortex-mixed
and then centrifuged; 5μL were injected in the analytical
column. The system comprised a binary pump and
autosampler (Shimadzu, Kyoto, Japan) coupled to an
API 3200 triple quadruple mass spectrometric detector
(AB Sciex, Toronto, Ontario, Canada), equipped with a
Turbo V source electrospray ionization (ESI) probe. The
chromatographic data were collected and analyzed using
the Analyst package (version 1.4.1., AB Sciex). Multiple
reaction monitoring (MRM) scanning was used for mass
spectral detection and quantification of C14-Ceramide
(N-myristoyl-D-erythro-sphingosine), C16-Ceramide (N-
stearoyl-D-erythro-sphingosine), C20-Ceramide (N-arachy-
ceroyl-D-erythro-sphingosine). C17-Ceramide (Heptade-
canoyl-D-erythro-sphingosine) was used as an internal
Thermo-Hypersil GOLD PFP (50 × 2.1mm, 3.0μm)
analytical columns were used for chromatographic sepa-
ration of the lipids using Buffer A (1.0mmol/L ammo-
nium acetate, 0.5% formic acid, v/v/v) and Buffer B
(1.0mmol/L ammonium acetate, 0.5% formic acid, HPLC-
grade methanol, v/v/v). The mobiles phase was comprised
of solvent mixtures: A:B 50:50 (v/v) at 0.0min; A:B
20:80 (v/v) for 0.01–8.0min; A:B 5:95 (v/v) for 8.1–
15.0min; A:B 5:95 (v/v) for 15.1–20.0min; A:B 5:95
(v/v) for 20.1-20.2min; followed by column equilibration
at A:B 50:50 (v/v) for five minutes, giving an injection
cycle of 25 minutes. The flow rate was 250μL/min. Spe-
cific pairs of precursor/product ions were as follows: the
ion transitions were C14-Ceramide (510.5→492.4), C16-
Ceramide (538.8→520.5), C17-Ceramide (552.6→534.4),
C18-Ceramide (566.4→548.4), C20-Ceramide (594.7→
576.2), and C24-Ceramide (650.6→632.5). The retention
times for C14-Ceramide, C16-Ceramide, C17-Ceramide,
C18-Ceramide, C-20-Ceramide, and C24-Ceramide were
9.4, 10.0, 10.4, 10.7, 11.1, and 12.1 minutes, respectively.
The ionization was set at electrospray positive ion mode
4 Oxidative Medicine and Cellular Longevity
Table 1: List of primers used for qRT-PCR analysis of gene expression.
Sequence (5?→ 3?)
Amplicon size (bp)
UPL probe no.
Abbreviations: CERS: ceramide synthase; SMPD: sphingomyelin phosphodiesterase; UGCG: UDP glucose ceramide glycosyltransferase; SPTLC: serine
palmitoyl transferase; CERD: ceramidase; GM3Syn: GM3-synthase; EDEM: ER degradation-enhancing α-mannosidase-like protein; CHOP: C/EBP
homologous protein; p58IPK: inhibitor of interferon-induced and double stranded RNA activated kinase; GRP78: glucose regulated protein 78; ATF:
activating transcription factor-4; HERP: homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1; WARS: tryptophanyl-
Oxidative Medicine and Cellular Longevity5
and at a temperature of 150◦C utilizing nitrogen for the
drying and collision gas (curtain gas, 10psi; collision gas,
3psi; ionspray voltage, 5500V; ion source gas 1, 25psi;
ion source gas 2, 25psi). Separate stock solutions of all
compounds each containing 1.0mM were prepared in
methanol. Working standard solutions of combined analytes
were prepared by serial dilution in methanol. Calibration
standards were prepared in concentrations ranging from
0.5μM to 500μM (0.1μM, 0.5μM, 1μM, 5μM, 10μM,
50μM, 100μM, 500μM). The internal standard was used at
a final concentration of 20nM.
2.7. Statistical Analysis. Data depicted in box plots reflect
group medians (horizontal bar), 95% confidence interval
limits (upper and lower box limits), and range (whiskers).
Tabulated data reflect means ± SEMs for each group. Data
were analyzed using GraphPad Prism 5 software (GraphPad
Software, Inc., San Diego, CA), and intergroup comparisons
were made using Student’s t-tests. Computer software gener-
mal chord-like architecture and were free of inflammation,
steatosis, fibrosis, and cholestasis. Livers from the 10 chronic
alcoholics had micronodular cirrhosis with steatohepatitis,
apoptotic bodies, disorganization of the normal chord
architecture, bile duct proliferation, and cholestasis. None
of the livers exhibited dysplasia or hepatocellular carcinoma.
Corresponding with the hepatic steatosis, the livers with
ALD had significantly increased levels of neutral lipids as
demonstrated with the Nile Red assay (Control: 9.41 ±
1.23(FLU/g), ethanol: 13.35 ± 1.45(FLU/g), P = 0.03). In
contrast, mean triglyceride content was similar in the ALD
and control livers (Control: 24.89 ± 2.95(μg/mg), ethanol:
24.38 ± 3.21(μg/mg)).
3.2. Cytokine Activation in Chronic ALD (Table 2). We used
a 21-plex bead-based assay to measure proinflammatory
cytokine and chemokine levels in relation to chronic ALD.
Contrary to what might have been expected, chronic ALD
was not associated with significantly increased levels of
cytokines or chemokines in liver. Instead, the mean levels of
proinflammatory cytokines IL-10, SDF-1α, MCP, IL-1β, IL-6
and TRAIL, and pro-fibrogenic cytokines VEGF, PDGF, and
β-FGF were significantly lower in the chronic ALD relative
to control livers. In contrast, the mean level of HGF, which
is progrowth and antifibrogenic was significantly elevated
in chronic ALD relative to control livers. However, qRT-
PCR analysis demonstrated higher levels of IL-1β, IL-6, and
TNF-α in the ethanol group, with significant intergroup
differences obtained with respect to IL-6 (Table 2). A similar
phenomenon was observed in experimental models of
chronic ethanol feeding . The discrepancies between
mRNA and protein could be due to increased release of
3.3. Chronic ALD Is Associated with Impaired Hepatic Insulin
Signaling. The qRT-PCR analyses demonstrated signifi-
of insulin, IGF-1, and IGF-2 receptor mRNA transcripts
in chronic ALD relative to control livers. In contrast, the
mRNA levels of insulin and IGF-1 polypeptide genes were
similar in the two groups (Figure 1). Multiplex ELISAs also
demonstrated significantly higher levels of insulin and IGF-1
receptor expression, and significantly reduced levels of IRS-
1 protein in the ALD group (Figure 2). Although tyrosine
phosphorylated insulin receptor levels were also increased
in the ALD group, the ratio of phosphorylated/total insulin
ALD versus control livers. ALD was also associated with sig-
nificantly increased levels of S312 phosphorylation of IRS-1.
S312 phosphorylation of IRS-1 inhibits IRS-1 signaling .
Additional studies characterized the effects of chronic
ALD on downstream signaling through Akt pathways.
Chronic ALD was associated with significantly increased
levels of total and phosphorylated AKT levels, but similar
levels of phospho/total Akt relative to control. ALD was
associated with significantly reduced levels of total GSK-
3β, but significantly increased Ser9 phospho/total GSK-3β,
reflecting reduced levels of GSK-3β activity. Correspond-
ingly, we detected no significant effects of chronic ALD on
the total, phosphorylated or phospho/total levels of proline-
rich Akt substrate 40kDa (PRAS 40) or p70S6 kinase, which
mediate mTOR signaling (Table 3).
3.4. Chronic ALD Is Associated with Increased Activation
of Proceramide Mechanisms in Liver. Previous studies
in experimentalanimal models
steatohepatitis from various causes, including chronic
ethanol exposure, is associated with increased proceramide
gene expression and ceramide levels in liver [10, 11].
To determine if human chronic ALD exhibits the same
abnormalities, we measured mRNA levels of proceramide
genes corresponding to the de novo synthesis (Ceramide
synthases 1, 2, 4, 5, 6, and the subunits 1 (regulatory) and 2
(catalytic) of serine palmitoyl transferase subunit (SPTLC1
and 2)), catabolic (sphingomyelin phosphodiesterases 1 and
3 (SMPD-1 and SMPD-3) and ceramidases (CERD)), and
to biosynthesis of complex sphingolipids (ganglioside GM3
synthase and UDP-glucose ceramide glucosyltransferase
(UGCG)) pathways by qRT-PCR analysis (Table 4). Chronic
ALD was associated with significantly increased expression
increased ceramide biosynthesis, increased mRNA levels
of SMPD1 and SMPD3, corresponding to increased levels
of sphingomyelin hydrolysis, and decreased expression of
CERD2, an enzyme responsible for deacylation of ceramide
to sphingosine and fatty acids. In addition, chronic ALD
was associated with significantly increased levels of GM3
synthase and UGCG, which mediate formation of complex
sphingolipids from ceramides. We also measured acidic,
neutral, and alkaline sphingomyelinase activities using
commercial assay reagents and found significantly higher
levels of all three in ALD relative to control livers (Figure 3).
6Oxidative Medicine and Cellular Longevity
Table 2: Effects of chronic ethanol exposure on proinflammatory cytokine levels in liver tissue.
58.19 ± 3.02
18.20 ± 1.19
1793.48 ± 559.87
1305.97 ± 299.12
30.14 ± 3.14
79.25 ± 11.76
195.74 ± 12.52
111.52 ± 9.94
2769.95 ± 244.39
39.05 ± 1.29
948.78 ± 81.77
734.19 ± 479.61
2279.33 ± 791.99
365.60 ± 48.50
41.76 ± 4.26
806.30 ± 321.41
6.66 ± 0.37
123.40 ± 44.97
316.64 ± 108.93
12.95 ± 1.03
61.14 ± 13.65
51.27 ± 3.06
13.95 ± 0.97
1123.61 ± 496.75
3887.81 ± 691.31
28.14 ± 1.29
29.75 ± 2.76
120.72 ± 9.74
59.73 ± 4.69
3058.46 ± 219.60
33.54 ± 2.83
546.07 ± 46.79
398.13 ± 96.21
1177.78 ± 142.71
672.60 ± 218.45
48.60 ± 8.14
929.34 ± 185.10
6.93 ± 0.55
34.23 ± 7.75
126.60 ± 91.79
15.93 ± 2.72
25.67 ± 2.84
0.0254 ± 0.0098
0.0670 ± 0.027
0.0253 ± 0.005
0.0341 ± 0.0112
0.2727 ± 0.1033
0.04072 ± 0.010
Liver protein homogenates were used to measure immunoreactivity of the indicated cytokines by multiplex ELISA. Immunoreactivity was normalized to
protein concentration and data are expressed as mean ± SEM of fluorescence light units (arbitrary). mRNA was used to measure gene expression of TNF-α,
IL-1β, and IL-6 via qRT-PCR. Results were normalized to ribosomal 18S levels measured in the same samples. Intergroup comparisons were made using t-test
and significant P values are indicated in the right columns.
3.5. Chronic ALD Results in Increased Ceramide Levels and
Altered Ceramide Profiles in Liver. We measured ceramide
immunoreactivity in liver tissue homogenates by ELISA and
and C24:0 by LC-MS/MS (Figure 3). Livers from patients
with chronic ALD were associated with significantly higher
levels of ceramide immunoreactivity relative to controls.
The LC-MS/MS studies demonstrated the most abundant
ceramide species in control human livers were C18:0,
followed by C16:0, and then C20:0 and the least abundant
was C14:0. In the chronic ALD samples, the mean levels of
C14:0, C16:0, C18:0, and C20:0 were significantly elevated
relative to control, whereas the levels of C24:0 were similar
for the two groups.
3.6. ER Stress in ALD. Steatohepatitis, ceramide accumula-
tion, and insulin resistance can each contribute to the activa-
tion of ER stress pathways, and ER stress can promote oxida-
tive injury and inflammation, which cause ALD to progress.
To determine if chronic ALD in humans is associated with
increased ER stress, we measured mRNA expression of
multiple genes that mediate ER stress at various levels within
the cascade . Chronic ALD was associated with signif-
icant upregulation of the chaperone Bip/GRP78, the tran-
scription factor ATF-4, the homocysteine-responsive endo-
plasmic reticulum-resident ubiquitin-like domain mem-
ber 1 (HERP) and tryptophanyl-tRNA synthetase (WARS)
(Figure 4). ER degradation-enhancing α-mannosidase-like
1 (EDEM) and protein kinase inhibitor p58 (P58IPK)
mRNA transcripts were also elevated in the ALD group,
but the intergroup differences failed to reach statistical
significance due to the large within group variances. Finally,
since sustained UPR activation drives pathological responses
leading to apoptosis, we measured expression of BAX and
the transcription factor CHOP, and both were found to be
significantly elevated in chronic ALD.
ELISAs were used to measure immunoreactivity to
Bip/GRP78, CHOP, IRE-1α, calnexin, the oxidoreductase
ERO1α, protein disulphur isomerase (PDI), PERK, phospho
(Thr980) PERK, eIF2α, and phospho (Ser51) eIF2α. Chronic
ALD was associated with significantly increased levels of
Bip/GRP78, CHOP, IRE-1α, calnexin, ERO-1a, and PDI
Oxidative Medicine and Cellular Longevity7
P = 0.0027
Insulin R/18S (×10−5)0.00005
P < 0.0001
P < 0.0001
P = 0.008
P = 0.04
Figure 1: Effects of chronic ALD on hepatic expression of insulin/IGF ligands and receptors, and IRS genes. RNA extracted from livers of
control and chronic ALD subjects (N = 8/group) was reverse transcribed, and the cDNAs were used to measure gene expression by qPCR
analysis. Graphs depict relative mRNA levels for (a) insulin, (b) insulin-like growth factor (IGF-1), (c) IGF-2, (d) insulin receptor (R), (e)
IGF-1 receptor (IGF-1R), (f) IGF-2R, (g) insulin receptor substrate-1 (IRS-1), (h) IRS-2, and (i) IRS-4. Intergroup comparisons were made
using Student’s t-tests. Significant P values are shown over the graphs.
relative to control livers. In addition, the chronic ALD
livers had significantly higher levels of PERK, p(Thr980)-
PERK, and eIF2α relative control. In contrast, levels of
phosphorylated eIF2α (Ser51) were similar for the two
groups, while the relative level of phospho/total eIF2α was
significantly reduced in the chronic ALD relative to control
group (Figure 5). Further Western blot analyses confirmed
that ER stress pathways through PERK, Bip/GRP78, and
CHOP were upregulated in chronic alcoholic livers (see
online at doi:10.1155/2012/479348).
This study was designed to gain a better understanding of
the molecular pathogenesis of chronic progressive ALD in
humans by determining if the abnormalities detected were
similar to those previously identified in experimental animal
models. Therefore, we examined several interrelated aspects
of liver injury including (1) histopathology; (2) integrity
of insulin/IGF signaling through upstream and Akt down-
stream pathways; (3) activation of proinflammatory medi-
ators; (4) ceramide production, accumulation, and profiles;
(5) ER stress activation. Together, these approaches enabled
us to characterize the nature and extent of hepatocellular
dysfunction in human chronic progressive ALD. Moreover,
the findings suggest that multipronged therapeutic strategies
are needed to prevent or reduce ALD progression.
4.1. ALD Features. The histopathological studies demon-
strated that all cases of chronic ALD included in this study
had severe steatohepatitis with cirrhosis. As the livers were
obtained from patients scheduled for liver transplantation,
their clinical disease status was regarded as advanced.
The presence of active steatohepatitis with conspicuous
foci of apoptosis and necrosis indicates that the disease
process was still progressing at the time of tissue sampling,
thereby enabling us to determine the molecular and patho-
physiological processes contributing to the ongoing liver
injury and degeneration. The finding of reduced levels of
8Oxidative Medicine and Cellular Longevity
Insulin R (FLU)
P = 0.06
IGF-1 R (FLU)
P = 0.0003
P = 0.036
P = 0.0005
P = 0.0008
P = 0.023
Figure 2: Effects of chronic ALD on insulin signaling mechanisms. Protein homogenates of liver were used in bead-based multiplex ELISAs
to measure immunoreactivity corresponding to (a) insulin receptor (InR), (b) IGF-1 receptor (IGF-1R), (c) IRS-1, (d)pYpY1162/1163InR,
(e)pYpY1135/1136IGF-1R, and (f)pS312IRS-1. Calculated phospho/total ratios of (g) InR, (h) IGF-1R, and (i) IRS-1 reflect relative levels of
phosphorylation. Intergroup comparisons were made using Student’s t-tests and significant P values are shown over the graphs.
Table 3: Effects of chronic ALD on the integrity of the Akt signaling pathway.
Protein (Phospho/Total) ∗ 100
18599.50 ± 744.87
2295.38 ± 250.09
462.13 ± 31.84
1204.38 ± 130.96
22377.37 ± 478.47
1604.090 ± 208.06
438.298 ± 15.13
1258.14 ± 170.01
273.47 ± 720.05
612.40 ± 155.72
65.65 ± 5.42
184.44 ± 23.30
252.78 ± 33.53
636.30 ± 46.82
78.07 ± 7.37
114.32 ± 19.48
1.43 ± 0.32
28.42 ± 5.12
14.29 ± 2.26
13.91 ± 4.14
1.12 ± 0.14
44.02 ± 8.15
17.65 ± 1.46
9.01 ± 0.97
Liver homogenates were used to measure immunoreactivity by multiplex ELISA. Phospho/total signaling protein levels were calculated. Immunoreactivity
was normalized to protein concentration and data are expressed as mean ± SEM of fluorescence light units (arbitrary). Intergroup comparisons were made
using t-tests. Significant P values are indicated in the right column.
Oxidative Medicine and Cellular Longevity9
Table 4: Effects of chronic ethanol consumption on hepatic expression of proceramide genes-biosynthetic pathways.
0.542 ± 0.162
3634 ± 509.2
160.2 ± 36.36
1.62 ± 0.22
15.70 ± 3.59
121.4 ± 9.29
2615 ± 444.8
685.7 ± 133.9
765.4 ± 140.5
6.524 ± 2.439
5.583 ± 1.458
282.6 ± 68.27
56.21 ± 7.80
1.145 ± 0.228
3982 ± 450
172.7 ± 22.86
3.62 ± 0.27
33.52 ± 5.48
253.3 ± 26.26
3187 ± 309.5
1214 ± 244.8
1353 ± 216.6
1.887 ± 0.3439
6.896 ± 1.853
609.9 ± 174.5
72.32 ± 6.981
RNA extracted from normal or chronic alcoholic livers (N = 8/group) was reverse transcribed, and the cDNAs were used to measure gene expression by
qPCR analysis with gene-specific primer pairs in a duplex qPCR reaction, in which the genes of interest were co-amplified with HPRT for normalization (see
Section 2 and Table 1). Table represents relative levels of gene expression for Ceramide synthases (CERS), UDP glucose ceramide glycosyltransferase (UGCG),
ceramidases (CERD), GM3-synthase, serine palmitoyl transferase subunits (SPTLC), sphingomyelinases (SMPD). Intergroup comparisons were made using
Student’s t-tests and significant P values are shown over the graphs.
` a-vis the histopathological features of chronic active ALD.
However, these findings correspond with results in previous
experimental models of chronic ethanol feeding [11, 44].
Similarly, given the extensive degree of fibrosis, the finding
but it may reflect a host compensatory effort to restore liver
4.2. Role of Insulin Resistance/Impaired Insulin Signaling.
Previous in vitro and experimental animal model studies
demonstrated that short-term ethanol exposure or chronic
ethanol feeding impairs insulin and IGF-1 signaling by
inhibiting tyrosine phosphorylation of the insulin and IGF-
1 receptors, and IRS-1 [45–50]. Impaired signaling through
insulin and IGF-1 receptors was shown to be associated
with reduced ligand-receptor binding and compensatory
upregulation of the receptors, both at mRNA and protein
levels [46, 48]. These effects correlated with inhibition of
downstream signaling through Ras/Raf/Erk MAPK [45, 47,
49, 50], reduced DNA synthesis, and impaired capacity for
the liver to regenerate following partial hepatectomy [48,
51]. In addition, the associated reduced transmission of
apoptosis, DNA damage, mitochondrial dysfunction, and
oxidative stress [46, 48, 52, 53].
The significantly increased levels of the insulin, IGF-1
and IGF-2 receptor expression in human cases of chronic
ALD reflect insulin/IGF resistance. Correspondingly, the
ratio of phosphorylated/total IGF-1 receptor was signifi-
cantly reduced, confirming that chronic ALD impairs signal-
ing through hepatic IGF-1 receptors in humans. Moreover,
the significantly reduced expression of IGF-2 polypeptide
in chronic ALD indicates that IGF-2 signaling was also
impaired, but due to the combined effects of trophic
factor deficiency and receptor resistance. The finding that
IRS-1 and IRS-4 expressions were significantly reduced in
chronic ALD indicates that insulin/IGF receptor signaling
was further impaired by reduced capacity to transmit signals
downstream through IRS docking proteins. An additional
abnormality contributing to hepatic insulin/IGF resistance
in human chronic ALD was the significantly increased levels
of S312 phosphorylation of IRS-1; S312 phosphorylation
inhibits IRS-1 signaling . This is the first study to
demonstrate impairments in insulin/IGF/IRS signaling in
human chronic ALD. However, earlier studies in which
mainly peripheral blood and leukocytes were available for
study showed that patients with alcoholic cirrhosis had
peripheral insulin resistance [54–59] with binding and
postbinding defects in insulin target organ cells [54, 55,
59]. Moreover, analysis of brains from human alcoholics
demonstrated chronic insulin resistance in structural targets
of alcohol-induced neurodegeneration . Therefore, these
earlier studies provide evidence that ethanol-induced insulin
resistance is a multisystem disease that is somehow linked to
To examine the effects of impaired insulin/IGF/IRS
signaling through the downstream Akt pathway, we used
multiplex ELISAs to measure total and phosphorylated levels
of Akt, GSK-3β, PRAS40, and p70S6K. In contrast to the
findings in experimental animals in which impairments
in insulin/IGF/IRS signaling were associated with reduced
AKT and increased GSK-3β activation , human chronic
ALD was not associated with significant impairments in
signaling through survival or metabolic pathways. Instead,
the findings of increased Akt and phospho-Akt, but similar
phospho/total Akt relative control, and reduced total GSK-
3β, phospho-Ser9/GSK-3β, and phospho-Ser9/total GSK-3β
indicate that Akt activation was preserved while GSK-3β
was relatively inhibited. Therefore, survival and metabolic
10 Oxidative Medicine and Cellular Longevity
NSmase (mU/mg protein)
P < 0.0001
pg CerC14/mg protein 1000
P = 0.007
pg CerC20/mg protein60
P = 0.0048
ASmase (mU/mg protein)
P = 0.0056
pg CerC16/mg protein 1000
P = 0.0205
pg CerC24/mg protein
Alkaline SMase/mg protein50000
P = 0.0063
pg CerC18/mg protein
P = 0.01
Figure 3: Effects of chronic ALD on proceramide pathway activation in liver. Using a fluorogenic assay, (a) neutral, (b) acid, and (c) alkaline
sphingomyelinase activities were measured in homogenates of liver from control or chronic ALD subjects. Hepatic ceramide profiles were
measured in lipid extracts by LC/MS/MS. (d) CerC14:0, (e) CerC16:0, (f) CerC18:0, (g) CerC20:0, and (h) CerC24:0 levels are expressed
as pg/mg protein. (i) Total ceramide immunoreactivity was measured by direct-binding ELISA. Intergroup comparisons were made using
Student’s t-tests and significant P values are shown over the graphs.
signaling through the Akt pathway were found to be
intact in human chronic ALD. Similarly, activation of
the mTOR networks via p70S6k and PRAS40 was intact.
The discrepancy between the impairments in upstream
signaling through insulin/IGF/IRS and intact downstream
signaling could be explained by the fact that other signaling
factor or epidermal growth factor, can also activate Akt
pathways independent of IRS-dependent networks [60, 61].
Future studies will characterize the mechanisms by which
prosurvival and prometabolic signaling are maintained vis-
` a-vis chronic ALD-induced inhibition of insulin/IGF/IRS
4.3. Role of Ceramides in Chronic ALD. Although hepatic
steatosis is generally regarded as benign and reversible,
persistent injury and inflammation can cause steatohepatitis
to become chronic and progressive. Two factors that prob-
ably contribute to ALD progression include (1) aberrant
shifts in membrane lipid composition leading to disrupted
intracellular signaling [29, 62, 63]; (2) the accumulation of
toxic lipids that mediate cellular pathology via increased
oxidative stress, ROS generation, adduct formation, and
ER stress. Growth in our understanding of how abnor-
mal accumulation and composition of lipids contribute to
chronic insulin resistance diseases has been furthered by
modern lipidomic approaches. For example, besides the
histopathological differences, nonalcoholic steatohepatitis
(NAFLD) based on the composition of lipids accumulated in
Diacylglycerol (DAG), free fatty acids, free cholesterol,
and ceramides can all have lipotoxic effects in hepatocytes
contributions of ceramides in chronic ALD because (1)
ceramides have demonstrated roles in the pathogenesis of
steatohepatitis in experimental models of chronic ethanol or
high-fat diet feeding [10, 11]; (2) ceramides can promote
Oxidative Medicine and Cellular Longevity 11
P = 0.0003
P = 0.013
P = 0.0006
P < 0.0001
HERP/HPRT ( ×10−2)
P = 0.024
P = 0.044
Figure 4: Effects of chronic ALD on hepatic ER stress gene activation. RNA extracted from livers of control and chronic ALD subjects (N =
8/group) was reverse transcribed, and the cDNAs were used to measure gene expression by duplex qPCR assays in which the gene of interest
wascoamplifiedwithhypoxanthine-guaninephosphoribosyltransferase(HPRT)ascontrol(seeSection 2andTable 1).Graphsdepictrelative
levels of gene expression for (a) BAX, (b) ER degradation-enhancing α-mannosidase-like protein (EDEM), (c) C/EBP homologous protein
(f) activating transcription factor-4 (ATF-4), (g) homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member
1 (HERP), and (h) tryptophanyl-tRNA synthetase (WARS). Intergroup comparisons were made using Student’s t-tests. Significant P values
are shown over the graphs.
12 Oxidative Medicine and Cellular Longevity
BIP/GRP 78 (FLU)
P = 0.01
P < 0.0001
P = 0.0001
P = 0.023
P = 0.002
P = 0.002
P = 0.0005
P = 0.016
P = 0.01
Figure 5: Chronic ALD results in increased hepatic ER stress. ER stress protein expression and phosphorylation were measured by ELISA
using HRP-conjugated secondary antibody and Amplex UltraRed soluble fluorophore. Graphs depict immunoreactivity corresponding to
(a) Bip/GRP78, (b) CHOP, (c) inositol requiring enzyme-1 (IRE-1), (d) calnexin, (e) endoplasmic oxidoreductin-1 (ERO-1), (f) protein
disulphideisomerase (PDI),(g)PERK,(h) phosphoPERK(Thr980),(i) eIF2-α,and (j) phosphoeIF2-α(Ser51). Fluorescence was measured
(Ex 530nm/Em 590nm) in a Spectramax M5 microplate reader (FLU = fluorescence light units). Intergroup comparisons were made by
Student’s t-test analysis. Significant P values are shown over the graphs.
Oxidative Medicine and Cellular Longevity 13
insulin resistance, inflammation, and oxidative stress in vari-
diabetes and obesity [70–72]; (3) ceramides impair Akt/PKB
signaling through activation of protein phosphatase 2A
(PP2A) which dephosphorylates Akt [18, 73] and inhibition
of protein-kinase-C-ζ- (PKC-ζ-) dependent translocation
of Akt to the plasma membrane, which is required for its
detected in experimental models, it is possible that the
abnormality does occur in the earlier and less severe stages of
ALD, but compensatory mechanisms restore these functions
via activation of other signaling networks, for example, those
driven by EGF or HGF. As demonstrated with the multiplex
array analysis, HGF expression was strikingly increased in
chronic ALD relative to control livers.
Corresponding with the findings in experimental mod-
els of alcohol- and high-fat-diet-induced steatohepatitis,
human livers with severe chronic ALD had significantly
increased expression of multiple proceramide genes as
well as increased levels of ceramide immunoreactivity. The
robustness and significance of this response are underscored
by the constitutive upregulation of proceramide genes in
the biosynthetic, catabolic, and salvage pathways, as well as
Mechanistically, insulin resistance promotes lipolysis, and
increased lipolysis results in the breakdown (hydrolysis)
of complex lipids, thereby promoting increased levels of
ceramide. In addition, ceramides can be generated via
the salvage pathway due to increased levels of precursor
lipids. Conceivably, the pathogenic signaling that activates
lipolysis vis-` a-vis chronic steatohepatitis may also initiate
proceramide cascades by increasing gene expression and
enzymatic activity. The increased levels of ceramide synthase
gene expression could be explained by increased fatty
acid load that characteristically occurs with steatohepatitis,
irrespective of etiology [10, 11, 74].
We extended our analyses by characterizing the ceramide
profiles in liver tissue using LC-MS/MS. The rationale was
that (1) ceramides associated with specific fatty acids can
have diverse biological functions and can be generated
in response to diverse stimuli [75, 76]; (2) intracellular
mediating insulin resistance and lipotoxicity . Coupling
liquid chromatography with tandem mass spectrometry
enabled us to identify the different ceramide species in liver
based on retention time and specific mass transition using
synthetic reference standards .
The LC/MS/MS studies demonstrated that the ceramide
profiles in chronic ALD differed significantly from those in
control livers due to the relatively higher levels of C14:0,
C16:0, C18:0, and C20:0. Previously it was shown that
increased levels of C16:0, and also C24:0 ceramides could
promote cell death [79, 80], while C18:0 ceramide inhibits
cell growth . Therefore, the ongoing cell death associated
with chronic progressive ALD in humans could be mediated
in part by increased levels of C16:0 ceramide. Mechanisti-
cally, CERS1 has a high specificity for C18:0-CoA generating
C18:0-ceramide, and CERS2 and CERS4 mainly synthesize
C20:0-, C22:0-, C24:1-, C24:0-, C26:1-, and C26:0-ceramide.
In contrast, shorter chain ceramides like C14:0, C16:0 are
primarily generated by CerS5 and CerS6 . The qRT-
PCR analyses demonstrated significantly higher levels of
CERS1, CERS5, and CERS6 in human chronic ALD, which
could partly account for the associated shifts in hepatic
ceramide profiles. As the field of lipidomics grows, our
ability to interpret these types of results in relation to disease
pathogenesis, including insulin resistance, lipotoxicity, and
inflammation will improve. In addition, we may arrive at
the point where targeting specific proceramide mediators
4.4. ER Stress in Progressive ALD. Insulin resistance, inflam-
mation, and ceramide accumulation can promote ER stress,
and ER stress exacerbates insulin resistance, inflammation,
oxidative stress, and ceramide accumulation [35, 82–88].
Moreover, ethanol exposure in experimental animal models
leads to increased activation of ER stress responses in
liver [36, 89, 90]. The studies herein demonstrate that
in human chronic progressive ALD, ER stress signaling is
strikingly upregulated via multiple mechanisms including
(1) Bip/GRP78, which is an important chaperone protein;
(2) IRE-1α, which activates JNK; (3) PERK, which promotes
global arrest of protein synthesis, translation of ATF4, and
upregulation of CHOP; (4) CHOP, which activates caspases
and inhibits Bcl2 prosurvival function by activating BAX.
The increased levels of ATF-4 in chronic ALD correspond
with the significantly higher levels of chaperones and other
components of the folding and ERAD machinery, that is,
PDI and GRP78. In addition, chronic ALD was found to be
associated with significantly increased expression of calnexin
and ERO-1a. Calnexin binds glycosylated proteins after they
pass into the ER, while ERO1 and BAX are activated by
CHOP. Therefore, consequences of constitutive activation of
the UPR in chronic ALD progression include (1) increased
apoptosis via CHOP ; (2) disruption of calcium and
redox homeostasis ; (3) further dysregulation of lipid
homeostasis  leading to enhanced production of choles-
terol and triglycerides via activation of the transcription
Human chronic progressive ALD is associated with impaired
insulin and IGF signaling through insulin/IGF receptors,
IRS-1, and IRS-4. However, downstream signaling through
Akt pathways were found intact, probably due to com-
pensatory adaptive responses such as signaling through
other receptors such as EGF or HGF. In contrast to the
histopathological findings, chronic ALD was not associated
with increased activation of proinflammatory cytokines or
chemokines or profibrogenic markers. The most striking
abnormalities in human livers with advanced chronic ALD
were prominent activation of proceramide mechanisms with
ceramide accumulation and altered ceramide profiles, and
increased activation of the UPR via several pathways and
14Oxidative Medicine and Cellular Longevity
at multiple levels of the network. The aberrant shifts in
ceramide profiles could have contributed to the insulin
At the late stages of disease examined, it is not possible
to designate any single mechanism as the cause of ALD
progression. However, the findings in experimental animal
models suggest that one of the earliest abnormalities in
chronic steatohepatitis is insulin resistance. We hypothe-
size that once steatohepatitis becomes sufficiently severe
and long-standing, hepatic insulin resistance mechanisms
with activation of mal-signaling cascades become initiated
whereby lipolysis leads to increased ceramide generation
and accumulation in membranes including the ER and
plasma membrane. Ceramide accumulation in the ER pro-
motes ER stress, and ceramide incorporation into lipid
rafts exacerbates insulin resistance, inflammation, oxidative
stress, and proapoptosis signaling. Increased oxidative stress
leads to ROS formation, lipid peroxidation, DNA damage,
and cell death. Altogether, the findings herein suggest that
therapeutic prevention or reduction of ALD progression
requires multipronged strategies to reverse or stabilize the
consequences of hepatic insulin resistance and dysregulated
L. Longato, K. Ripp, and M. Setshedi contributed equally to
This work is supported by AA-11431, AA-12908, and AA-
16126 from the National Institutes of Health.
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