Plasma microRNAs are sensitive indicators of inter-strain differences in the severity of liver injury induced in mice by a choline- and folate-deficient diet
MicroRNAs (miRNAs) are a class of small, conserved, tissue-specific regulatory non-coding RNAs that modulate a variety of biological processes and play a fundamental role in the pathogenesis of major human diseases, including nonalcoholic fatty liver disease (NAFLD). However, the association between inter-individual differences in susceptibility to NAFLD and altered miRNA expression is largely unknown. In view of this, the goals of the present study were (i) to determine whether or not individual differences in the extent of NAFLD-induced liver injury are associated with altered miRNA expression, and (ii) assess if circulating blood miRNAs may be used as potential biomarkers for the noninvasive evaluation of the severity of NAFLD. A panel of seven genetically diverse strains of inbred male mice (A/J, C57BL/6J, C3H/HeJ, 129S/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were fed a choline- and folate-deficient (CFD) diet for 12weeks. This diet induced liver injury in all mouse strains; however, the extent of NAFLD-associated pathomorphological changes in the livers was strain-specific, with A/J, C57BL/6J, and C3H/HeJ mice being the least sensitive and WSB/EiJ mice being the most sensitive. The morphological changes in the livers were accompanied by differences in the levels of hepatic and plasma miRNAs. The levels of circulating miR-34a, miR-122, miR-181a, miR-192, and miR-200b miRNAs were significantly correlated with a severity of NAFLD-specific liver pathomorphological features, with the strongest correlation occurring with miR-34a. These observations suggest that the plasma levels of miRNAs may be used as biomarkers for noninvasive monitoring the extent of NAFLD-associated liver injury and susceptibility to NAFLD.
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Plasma microRNAs are sensitive indicators of inter-strain differences in the severity
of liver injury induced in mice by a choline- and folate-deﬁcient diet☆
Volodymyr P. Tryndyak
, John R. Latendresse
, Beverly Montgomery
, Sharon A. Ross
Frederick A. Beland
, Ivan Rusyn
, Igor P. Pogribny
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA
Toxicologic Pathology Associates, National Center for Toxicological Research, Jefferson, AR 72079, USA
Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892, USA
Department of Environmental Sciences & Engineering, University of North Carolina, Chapel Hill, NC 27599, USA
Received 5 March 2012
Revised 5 April 2012
Accepted 16 April 2012
Available online 24 April 2012
Nonalcoholic fatty liver disease
MicroRNAs (miRNAs) are a class of small, conserved, tissue-speciﬁc regulatory non-coding RNAs that modulate
a variety of biological processes and play a fundamental role in the pathogenesis of major human diseases, in-
cluding nonalcoholic fatty liver disease (NAFLD). However, the association between inter-individual differences
in susceptibility to NAFLD and altered miRNA expression is largely unknown. In view of this, the goals of the pre-
sent study were (i) to determine whether or not individual differences in the extent of NAFLD-induced liver
injury are associated with altered miRNA expression, and (ii) assess if circulating blood miRNAs may be used
as potential biomarkers for the noninvasive evaluation of the severity of NAFLD. A panel of seven genetically di-
verse strains of inbred malemice (A/J, C57BL/6J, C3H/HeJ, 129S/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ)were fed
a choline- and folate-deﬁcient (CFD) diet for 12weeks. This diet induced liver injury in all mouse strains; how-
ever, the extent of NAFLD-associated pathomorphological changes in the livers was strain-speciﬁc, with A/J,
C57BL/6J,and C3H/HeJ mice being the leastsensitive and WSB/EiJ micebeing the most sensitive. The morpholog-
ical changesin the livers were accompaniedby differences in the levels of hepatic and plasma miRNAs. The levels
of circulating miR-34a, miR-122, miR-181a,miR-192, and miR-200b miRNAs were signiﬁcantly correlatedwith a
severity of NAFLD-speciﬁc liver pathomorphological features, with the strongest correlation occurring with miR-
34a. These observations suggest that the plasma levels of miRNAs may be used as biomarkers for noninvasive
monitoring the extent of NAFLD-associated liver injury and susceptibility to NAFLD.
Published by Elsevier Inc.
The incidence of nonalcoholic fatty liver disease (NAFLD) is in-
creasing dramatically in the United States and developed countries
(Bellentani et al., 2010; Tiniakos et al., 2010). It is estimated that 25%–
30% of adults in the United States have NAFLD (Day, 2011; Pascale
et al., 2010) and that NAFLD accounts for 39% of newly diagnosed
cases of chronic liver disease (Pascale et al., 2010). NAFLD is composed
of several related liver disorders ranging from uncomplicated steatosis
to nonalcoholic steatohepatitis (NASH). More importantly, NAFLD is
considered to be a major risk factor for the development of other
chronic liver diseases,such as ﬁbrosis, cirrhosis, and hepatocellular car-
cinoma (Siegel and Zhu, 2009; Starley et al., 2010; Welzel et al., 2011).
The underlying molecular mechanisms involved in the etiology and
pathogenesis of NAFLD are only partially understood and pharmaco-
therapy options are limited (Cohen et al., 2001). Supportive treatment
strategies for NAFLD are focused on managing the metabolic syndrome
and chronic inﬂammation in the liver; thus, early detection of the dis-
ease is critical for improved prognosis and prevention of more serious
liver illnesses. A recent report by Paie et al. in a small cohort of patients
with NAFLD demonstratedthat isolated steatosis does progress to NASH
accompanied by a worsening of the metabolic syndrome (Pais et al.,
2011), and suggested the crucial need for continued hepatic monitoring
in patients diagnosed with uncomplicated NAFLD. Unfortunately, at the
present time, a liver biopsy is the only method for the diagnosis and as-
sessment of disease progression (Sanyal et al., 2011).
Even though the incidence of NAFLD in the general population is
appreciable, it is estimated that only 10% of those diagnosed will pro-
gress to liver ﬁbrosis or cirrhosis (Siegel and Zhu, 2009). In addition, the
incidence of NAFLD varies greatly among racial groups (Browning et al.,
2004) and NAFLD is a highly heritable trait (Schwimmer et al., 2011).
Toxicology and Applied Pharmacology 262 (2012) 52–59
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase;
LDH, lactate dehydrogenase; miRNAs, microRNAs; NAFLD, nonalcoholic fatty liver dis-
ease; NASH, nonalcoholic steatohepatitis; qRT-PCR, quantitative reverse transcription
☆The views expressed in this manuscript do not necessarily representthose of the U.S.
Food and Drug Administration.
⁎Corresponding author at: Division of Biochemical Toxicology, NCTR, 3900 NCTR
Rd., Jefferson, AR 72079, USA. Fax: +1 870 543 7720.
E-mail address: firstname.lastname@example.org (I.P. Pogribny).
0041-008X/$ –see front matter. Published by Elsevier Inc.
Contents lists available at SciVerse ScienceDirect
Toxicology and Applied Pharmacology
journal homepage: www.elsevier.com/locate/ytaap
Author's personal copy
A recent genome-wide association study (GWAS) of the subjects with
histopathologically-conﬁrmed NAFLD identiﬁed several single nucleo-
tide polymorphisms as potential genetic modiﬁers of the NAFLD activity
score, ﬁbrosis, inﬂammation, and serum alanine aminotransferase (ALT)
levels (Chalasani et al., 2010). Because the pathogenesis of NAFLD is
complex and involves dysregulation of several interdependent physio-
logical processes, including lipid metabolism, insulin resistance, im-
mune response, inﬂamation, oxidative stress, and apoptosis (Larter
et al., 2010; Marra et al., 2008), additional studies in human populations
or animal models of the human population (Rusyn et al., 2010)are
needed to elucidate the genetic determinants of NAFLD.
Investigating the molecular basis of how genetic factors inﬂuence
the susceptibility to NAFLD in humans is frequently impractical and
always very complex, whereas using relevant animal models may
substantially overcome many limitations of human-only studies. Several
studies have demonstrated an inter-strain variability in the suscepti-
bility of mice to NAFLD, NASH, and hepatocarcinogenesis induced by
either high-fat diet, or methionine- and choline-deﬁcient diet (Hill-
Baskin et al., 2010; Pogribny et al., 2009; Yamazaki et al., 2008). Each
of these dietary models has some advantages and limitations. For in-
stance, feeding high-energy diets compromises the metabolic status
and induces obesity but does not uniformly cause liver injury (Maher,
2011). In contrast, feeding either a methionine- and choline-deﬁcient
or choline-deﬁcient diet to rats or mice causes liver injury similar to
NAFLD but does not attain the compromised metabolic status observed
Previous reports have convincingly demonstrated that dysregulation
of miRNA expression is an important early event in the pathogenesis of
NAFLD in humans (Cheung et al., 2008) and mice (Wang et al., 2009a).
However, the association between inter-individual differences in sus-
ceptibility to NAFLD and altered miRNA expression is largely unknown.
To explore further the potential mechanisms of inter-individual
differences in susceptibility and severity of NAFLD-related liver injury,
we fed a panel of seven genetically diverse inbred mouse strains that
are parental lines in the Collaborative Cross (Aylor et al., 2011)the
choline- and folate-deﬁcient (CFD) diet that consistently induces fat-
related liver injury resembling pathomorphological features of human
NAFLD (Maher, 2011). We determined the inter-strain variability in
severity of NAFLD induced by the CFD diet and its association with aber-
rations in miRNA expression. We also assessed whether or not circulat-
ing blood miRNAs may be used as potential biomarkers for noninvasive
evaluation of liver injury in NAFLD. Because miRNA expression in mouse
liver varies little among inbred strains (Gatti et al., 2011) the establish-
ment of circulatory miRNA biomarkers as noninvasivediagnostics of the
severity of NAFLD may ﬁll a critical gap in clinical practice.
Materials and methods
Animals and experimental design. Male A/J, C57BL/6J, C3H/HeJ,
129S1/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ mice (6 weeks of age)
were obtained from the Jackson Laboratory (Bar Harbor, ME). These
strains were selected because they provide an excellent representation
of the broad genetic diversity and their genomes have been fully se-
quenced (Yang et al., 2011). The mice were housed in sterilized cages
in a temperature-controlled room (24 °C) with a 12 h light/dark cycle,
and given ad libitum access to puriﬁed water and NIH-31 pelleted diet.
At 8 weeks of age, mice from each strain were allocated randomly into
control and experimental groups. Mice in the experimental groups
were maintained on the CFD diet, a diet lacking choline and folic acid
(Diet #519541, choline and folate deﬁcient, iron supplemented, and L-
amino acid deﬁned diet; Dyets, Inc., Bethlehem, PA) for 12 weeks.
Mice in the control groups received the same diet supplemented with
0.4% methionine, 0.3% choline bitartrate, and 2 mg/kg folic acid. Diets
were stored at 4 °C before use and given ad libitum with replacement
twice a week. Body weights of the mice were recorded weekly. Five ex-
perimental and ﬁve control mice from each strain were euthanized by
exsanguination following deep isoﬂurane anesthesia 12 weeks after
diet initiation. Blood was collected into BD vacutainer EDTA containing
blood collection tubes (BD Biosciences, Franklin Lakes, NJ) by direct
puncture of the heart. Plasma was isolated by centrifugation at
3000 rpm for 10 min at 4 °C according to the manufacturer's instruc-
tions and stored at −80 °C. The livers were excised and a slice of the
median lobe was ﬁxed in neutral buffered formalin for 48 h for histo-
pathological examination. The remaining liverwas snap-frozen immedi-
ately in liquid nitrogen and stored at −80 °C for subsequent analyses.
All experimental procedures were reviewed and approved by the Na-
tional Center for Toxicological Research Animal Care and Use
Biochemical analyses. Serum triglyceride, ALT, aspartate amino-
transferase (AST), lactate dehydrogenase (LDH), total cholesterol,
and glucose levels were measured using an ACE Alera® Clinical Chem-
istry System (Alfa Wassermann Inc., West Caldwell, NJ) according to the
Tissue processing, histological analysis and criteria for pathology
assessment. After 48 h, a slice of the median lobe of the liver that
was ﬁxed in 10% neutral buffered formalin was trimmed, processed,
and embedded in inﬁltrating media (Surgipath Formula R®, Leica Bio-
systems, Richmond, IL), sectioned at approximately 5 μm, mounted
on a glass slide, and stained with hematoxylin and eosin. The liver
sections were examined histopathologically for NAFLD-speciﬁc le-
sions, including cytoplasmic alteration, steatosis, hepatocellular de-
generation, inﬂammation, hepatocellular karyocytomegaly, and oval
cell proliferation, and graded using a severity score system for each
of the morphological parameters as follows: grade 0, absent; grade
1, minimal; grade 2, mild; grade 3, moderate; and grade 4, severe
changes. Total liver pathology scores were calculated as the mean se-
verity for all of the NAFLD-speciﬁc lesions detected in the livers of the
RNA extraction and miRNA expression analysis by quantitative reverse
transcription real-time PCR (qRT-PCR). Total RNA was extracted
from liver tissue using miRNAeasy Mini kits (Qiagen, Valencia, CA)
according to the manufacturer's instructions. Total RNA (100 ng) was
used for qRT-PCRs of miR-122, miR-192, miR-34a, miR-200b, miR-
221, and miR-181a using TaqMan miRNA assays (Applied Biosystems,
Foster City, CA), according to the manufacturer's instructions.
SnoRNA202 was used as an endogenous control. The relative amount
of each miRNA was measured using the 2
and Livak, 2008). All qRT-PCR reactions were conducted in triplicate
and repeated twice.
Total plasma RNA, including miRNA, was isolated using QIAzol re-
agent (Qiagen) according to the manufacturer's instructions with minor
modiﬁcations. In brief, 700 μl of QIAzol reagent containing 1.2 μgofcar-
rier MS2 RNA (Roche Diagnostics Corporation, Indianapolis, IN) was
added to 100 μl of each plasma sample. The sample was mixed in a
tube, spiked with 4 μlof0.5μM cel-miR-54 miRNA (Qiagen), and
140 μl of chloroform was added. After mixing vigorously for 15 s, the
sample was then centrifuged at 12,000 gfor 15 min. The upper aqueous
phase was carefully transferred to a new collection tube, and precipi-
tated with 2 volumes of isopropanol, centrifuged at 18,000 gfor 15 min,
and washed with 75% ethanol. After air drying, the RNA was dissolved
in 30 μl RNase-free water. The quality and quantity of the RNA was
evaluated using a NanoDrop 200c spectrophotometer (Thermo Fisher
Scientiﬁc, Waltham, MA). The efﬁciency of small RNA isolation was
monitored by determining the amount of spiked miRNA recovered by
using TaqMan miRNA assays (Applied Biosystems). Total RNA (1.5 μl
per reaction) was used for qRT-PCRs of miR-122, miR-34a, miR-200b,
miR-192, miR-221, and miR-181a using TaqMan miRNA assays (Applied
Biosystems), according to the manufacturer's instructions. The relative
53V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 52–59
Author's personal copy
amount of each miRNA was measured using the 2
normalized to mmu-miR-16, a ubiquitous non-liver-speciﬁcmiRNA.
Statistical analyses. Results are presented as mean±S.D. Clinical
chemistry values, body weights, and miRNA levels were analyzed by
two-way analysis of variance (ANOVA), with pair-wise comparisons
being made by the Student–Newman–Keuls method. When necessary,
the data were natural log transformed before conducting the analyses
to maintain a more equal variance or normal data distribution. Histopa-
thology scores were evaluated by one-way ANOVA, using strain as the
ﬁxed factor. Pearson product–moment correlation coefﬁcients were
used to determine the relationship between miRNA levels and histopa-
thology scores. P-values b0.05 were considered signiﬁcant.
Inter-strain differences in liver pathology elicited by a choline- and
Over the course of this 12 week study, all mice fed the CFD diet
gained weight, with the 129S1/SvImJ mice gaining signiﬁcantly more
than the methyl-sufﬁcient control mice, and the WSB/EiJ mice gaining
signiﬁcantly less than the methyl-sufﬁcient control mice (Table 1).
These results are in contrast to some of the previous reports that found
a substantial loss of the body weight, often between 20 and 40%, as a re-
sult of feeding mice various formulations of “lipogenic methyl-deﬁcient
diets”, e.g., methionine- and choline-deﬁcient, or choline-deﬁcient diets
(Ariz et al., 2010; Hebbard and George, 2011; Larter and Yeh, 2008).
This is considered as one of the major disadvantages of the mouse
model of NAFLD induced by these diets (Ariz et al., 2010; Hebbard and
George, 2011; Larter and Yeh, 2008). A signiﬁcant increase, 1.5–3-fold,
in relative liver weight was observed in six of the seven strains fed the
CFD diet (Table 1).
Serum triglycerides, cholesterol, and glucose levels were affected
by the methyl donor-deﬁcient diet in a strain-dependent manner
(Table 1). A decrease in triglycerides was signiﬁcant in ﬁve of the
seven strains tested and a greater than 50% reduction was observed
in CAST/EiJ mice. The magnitude of change in serum cholesterol and
glucose levels was less pronounced. Only two strains, PWK/PhJ, and
WSB/EiJ, exhibited a greater than 50% signiﬁcant reduction in choles-
terol and a signiﬁcant decrease in serum glucose concentrations was
found in WSB/EiJ mice only.
Feeding the CFD diet resulted in a signiﬁcant increase in the activity
of serum enzyme markersof liver injury, ALT and AST,in C57BL/6J, C3H/
HeJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ strains (Table 1),which was signif-
icantly correlated with the degree of liver injury(Supplementary Fig. 1).
Serum activity of LDH was increased signiﬁcantly in CAST/EiJ, PWK/PhJ,
and WSB/EiJ strains by 1.9, 2.4, and 2.6-fold, respectively.
Histopathological evaluation of liver injury revealed the presence of
NAFLD-associated pathomorphological features, including cytoplasmic
alteration, steatosis, necrosis, inﬂammation, oval cell hyperplasia, and
karyomegaly in all mice fed the CFD diet (Table 2). Interestingly, the
magnitude of the liver injury varied greatly among strains with the ex-
tent of total liver injury increasing in the following order according to
the total liver pathology scores: A/J≈C57BL/6J≈C3H/ HeJ b129S1/
SvImJ ≈CAST/EiJbPWK/PhJbWSB/EiJ. Lipid accumulation in hepato-
cytes was characterized by microvesicular, macrovesicular, and most
commonly mixed (microvesicular and macrovesicular) patterns of fat
deposition. Hepatocytes with microvesicularfat accumulation appeared
foamy, with the cytoplasm partially or completely ﬁlled with numer-
ous small lipid vacuoles, which did not displace the nucleus to the pe-
riphery. This pattern of steatosis was most pronounced in A/J mice
(Fig. 1C). Macrovesicular fatty change was morphologically character-
ized by hepatocytes mostly containing a moderately large to large and
well-deﬁned single rounded vacuole within each cell.The nucleus and
cytoplasm were displaced to the periphery in such cells. This pattern
was commonly observed in strains exhibiting the greatest degree of
liver injury, such as WSB/EiJ mice (Fig. 1D).
Inﬂammatory foci (Figs. 1C and D, marked by arrowheads) were
composed of a mixed population of polymorphonuclear and mononu-
clear cells, mainly neutrophils, macrophages, and lymphocytes. Single-
cell necrosis was one of the most common histopathological observa-
tions in both of the PWK/PhJ and WSB/EiJ mouse strains, but was most
severe in the WSB/EiJ mice. Karyomegaly, characterized as 2–4times
larger than typical hepatocyte nuclei, was found in CFD diet-fed mice
from PWK/PhJ and WSB/EiJ strains (Fig. 1; compare larger sized nuclei
in Figs. 1C and D). Hyperplasia of oval cells, stem cells of bile ductular
epithelium that appeared as single or double rows of oval- or round-
shaped cells organized in linear arrays in liver sinusoids, was also
observed in choline- and folate-deﬁcient PWK/PhJ and WSB/EiJ mice.
Effect of a choline- and folate-deﬁcient diet on hepatic microRNA
Dysregulation of hepatic expression of miR-34a, miR-122, miR-
181a, miR-192, miR-200b, and miR-221 was previously reported to be
a common feature in human (Cheung et al., 2008)andmouseNAFLD
(Pogribny et al., 2010; Wang et al., 2009a). Because an inter-strain
Inter-strain differences in pathology endpoints in mice fed the choline- and folate-deﬁcient diet.
Strain Group Body
A/J Control 25.4±1.51 6.62±1.26 4.16±0.25 162.8±85.0 203.2± 16.9 1180± 280 175.2 ± 11.7 94.0± 4.5 214.4 ±17.2
MCFD diet 23.2± 2.41 5.02± 1.02 4.76 ±0.45 118.4 ±7.7 204.8 ±32.3 1287±231 94.0±7.1* 116.4 ±15.7 194.8 ±16.5
C57BL/6J Control 30.8±1.22 11.88 ±1.02 4.17 ±0.22 49.6 ±8.3 135.6 ±26.6 1148± 475 188.0± 38.8 102.4 ±21.5 203.6 ± 39.2
MCFD diet 30.7± 2.98 11.90± 3.40 6.39± 0.95* 194.8± 50.6* 209.2± 36.1†1430 ±293 110.8 ±13.8†114.8±14.1 274.4 ±31.3
C3H/HeJ Control 32.4±1.80 11.50±1.92 4.19± 0.14 57.6± 13.0 129.6 ±21.6 1149 ±438 166.4± 23.0 153.2± 4.8 197.6± 9.8
MCFD diet 34.8± 4.33 13.40± 3.40 7.44± 0.47* 266.4± 54.6* 259.2± 54.5* 1472 ±336 126.0 ±10.7* 149.6 ±8.9 202.0 ± 23.5
129S1/SvImJ Control 27.2±2.22 7.88±1.26 3.42±0.19 191.2±92.4 368.0± 99.1 1480± 235 171.2 ± 18.6 138.4± 7.6 248.0± 12.7
MCFD diet 27.7± 1.30 10.14± 1.21* 7.60± 2.05* 336.4 ±68.6 455.6± 50.4 2415 ±631 101.6 ±19.9* 122.6 ±11.6 219.0± 19.4
CAST/EiJ Control 16.9± 0.29 4.46± 0.51 4.16 ± 0.44 184.8±66.9 255.8± 65.4 1227± 198 233.2 ± 22.5 83.6± 5.9 278.8 ±25.9
MCFD diet 17.2± 0.23 4.75± 0.68 7.92 ±0.24* 375.5 ±60.1* 374.5 ±60.7 †2316± 574* 104.5± 11.8* 57.0 ±24.3 329.0 ±36.5 †
PWK/PhJ Control 18.6± 1.27 2.86± 1.49 4.75 ±0.83 89.6± 28.2 197.2 ± 62.4 1066± 199 162.4 ±12.1 84.4 ±5.4 199.6±22.7
MCFD diet 20.1± 1.18 1.96± 0.73 13.2 ±0.72* 760.8 ±74.4* 576.0 ±66.0* 2529±313* 106.0 ±8.4* 33.4 ±8.4* 170.0 ±4.5
WSB/EiJ Control 21.1 ±0.80 6.84 ± 0.51 4.26 ± 0.05 103.6 ± 32.1 329.2 ± 73.5 1184± 343 108.4 ± 5.5 114.4 ± 3.0 195.6 ± 15.5
MCFD diet 19.3± 0.85 4.54± 0.57†7.60 ±0.34* 745.2 ±42.2* 617.6 ±71.0* 3090±301* 113.2 ± 11.7 30.0 ±3.0* 153.6± 6.4*
Bold —Signiﬁcantly different from age-matched control mice;
*—Signiﬁcantly different from age-matched control mice (p-value ≤0.01);
†—Signiﬁcantly different from age-matched control mice (p-value ≤0.05).
54 V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 52–59
Author's personal copy
difference in liver injury phenotype elicited by the CFD diet was ob-
served in this study, we investigated whether or not hepatic expression
of these miRNAs may reﬂect variability in the degree of NAFLD or may
be associated with strain-speciﬁc susceptibility to liver injury. The ex-
pression of miR-122 was down-regulated by 25 to 60% in the livers of
A/J, C57BL/6J, PWK/PhJ, and WSB/EiJ mice fed the CFD diet (Fig. 2). No
change was observed in C3H/HeJ, 129S1/SvlmJ, or CAST/EiJ mice. MiR-
192 was down-regulated by 50% only in C57BL/6J mice and slightly
up-regulated in CAST/EiJ mice.
The expression of miR-34a, miR-200b, and miR-181a was signiﬁ-
cantly increased in CFD diet-fed mice of a majority of strains in a
strain-dependent manner, with the maximum response occurring in
WSB/EiJ mice (Fig. 2). Up-regulation of hepatic expression of miR-221
was most pronounced (>10-fold) in PWK/PhJ mice, with a smaller,
but signiﬁcant, increases being observed in C3H/HeJ, 129S1/SvlmJ,
CAST/EiJ, and WSB/EiJ mice. In contrast, feeding the CFD diet did not af-
fect the expression of a ubiquitous non-liver-speciﬁc miRNAlet-7c in all
but WSB/EiJ mice (Supplementary Fig. 2).
Effect of a choline- and folate-deﬁcient diet on plasma microRNA
Plasma levels of miRNAs have been shown to be correlated with
the degree of liver injury in response to drug treatment (Laterza et
al., 2009; Wang et al., 2009b). We evaluated the differences in pat-
terns of miRNA changes in plasma of mice fed control or CFD diet.
Summary of the type and extent of hepatic lesions in mice fed the choline- and folate-deﬁcient diet for 12 weeks.
Strain Group Lesion
Karyomegaly Steatosis Necrosis, hepatocyte Inﬂammation Oval cell hyperplasia Cytoplasmic alteration
A/J Control 0/5
0/5 0/5 1/5 (0.2±0.5)
0/5 5/5 (1.6 ±0.6)
MCFD diet 0/5 5/5 (1.4 ±0.6) 0/5 5/5 (1.0 ±0.0) 1/5 (0.2± 0.5) 4/5 (1.2±0.8)
C57BL/6J Control 0/5 0/5 1/5 (0.2±0.5) 1/5 (0.2 ±0.5) 0/5 2/5 (0.8 ±1.1)
MCFD diet 0/5 5/5 (1.8 ±0.5) 0/5 5/5 (1.4 ±0.6) 3/5 (0.6± 0.6) 3/5 (0.6±0.6)
C3H/HeJ Control 0/5 0/5 0/5 0/5 0/5 0/5
MCFD diet 0/5 5/5 (2.6 ±0.6) 0/5 5/5 (1.0 ±0.0) 0/5 0/5
129S1/SvImJ Control 0/5 0/5 0/5 1/5 (0.2± 0.5) 0/5 5/5 (1.8 ±0.5)
MCFD diet 0/5 5/5 (3.8 ±0.5) 0/5 5/5 (1.4 ±0.6) 1/5 (0.2 ±0.5) 0/5
CAST/EiJ Control 0/5 0/5 0/5 0/5 0/5 2/5 (0.8± 1.1)
MCFD diet 4/4 (1.0 ±0.0) 4/4 (3.5 ±0.6) 0/4 3/4 (0.8± 0.5) 1/4 (0.3 ±0.5) 3/4 (1.0±0.8)
PWK/PhJ Control 0/5 0/5 0/5 1/5 (0.2± 0.5) 0/5 1/5 (0.2 ±0.5)
MCFD diet 5/5 (1.4 ±0.6) 5/5 (3.8 ±0.5) 5/5 (1.0± 0.0) 5/5 (2.0± 0.0) 5/5 (1.2 ±0.5) 5/5 (4.0 ±0.0)
WSB/EiJ Control 0/5 0/5 0/5 1/5 (0.2±0.5) 0/5 4/5 (1.0±0.7)
MCFD diet 5/5 (2.0 ±0.0) 5/5 (4.0 ±0.0) 5/5 (3.8± 0.5) 5/5 (3.8± 0.5) 5/5 (3.6 ±0.6) 5/5 (2.2 ±0.5)
Fig. 1. Representative hematoxylin and eosin staining of liver tissues from control A/J and WSB/EiJ mice (A,B) mice fed the choline- and folate-deﬁcient diet (C,D).(A) Representa-
tive liver section from control A/J mice. (B) Representative liver section from control WSB/EiJ mice. (C) Representative liver section from A/J mice fed the CFD diet for 12 weeks.
Minimal steatosis and inﬂammation (arrows) in the livers. (D) Representative liver section from WSB/EiJ mice fed the CFD for 12 weeks. Severe steatosis, inﬂammation (arrow-
heads) and necrosis (arrows) of hepatocytes in the livers.
55V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 52–59
Author's personal copy
Fig. 2. Expression changes of miR-122, miR-34a, miR-200b, miR-192, miR-221, and miR-181a miRNAs in the livers of control mice and mice fed the choline- and folate-deﬁcient
diet.The miRNA expression data are presented as fold change of each miRNA normalized to that of snoRNA202 in the livers of mice fed the CFD diet compared to control mice
(n=5, mean ±SED). Gray bars —control groups; black bars —CFD diet groups.* —Signiﬁcantly different from age-matched control mice.
Fig. 3. Levels of miR-122, miR-34a, miR-200b, miR-192, miR-221, and miR-181a miRNAs in plasma of control mice and mice fed the choline- and folate-deﬁcient diet. The miRNA
expression data are presented as fold change of each miRNA normalized to that of normalized to mmu-miR-16, a ubiquitous non-liver-speciﬁc miRNA in plasma of mice fed the CFD
diet compared to control mice. (n =5, mean ±SED). Gray bars —control groups; black bars —CFD diet groups.* —Signiﬁcantly different from age-matched control mice.
56 V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 52–59
Author's personal copy
Fig. 3 demonstrates that there were strain-speciﬁc differences in the
levels of miRNAs in the mice fed the CFD diet. Notably, in A/J mice,
one of the strains with the lowest extent of methyl donor-deﬁcient
diet-induced liver injury, none of the miRNAs was increased by feeding
the CFD diet. In contrast, in plasma of PWK/PhJ and WSB/EiJ mice,
strains that exhibited the greatest degree of liver injury, each of miRNAs,
with the exception of miR-200b was signiﬁcantly increased by choline
and folate deﬁciency.
Because strain-dependent effects of treatment on miRNA levels
in liver and plasma were observed, we tested whether the inter-
strain difference in the degree of liver injury correlated with miRNA
expression. Fig. 4 shows that the induction of miR-34a, miR-181a,
miR-200b and miR-221 in the livers is signiﬁcantly correlated with
the histopathology score in CFD diet-fed mice, with the strongest cor-
relations being observed with miR-181a and miR-200b. Interestingly,
expression of miR-122 and miR-192 in the livers, two of the most
abundant hepatic miRNAs (Gatti et al., 2011), did not correlate with
the extent of liver injury.
Fig. 5 shows that plasma levels of each of the miRNAs were signif-
icantly correlated with the degree of liver injury induced by the CFD
diet, with strongest correlation occurring with miR-34a, whereas
level of circulating miRNA let-7c did not differ (Supplementary
The results of the present study demonstrate that feeding a mul-
tistrain panel of inbred mice the CFD diet for 12 weeks resulted in
strain-speciﬁc changes in biochemical indicators of liver injury in plasma
(Table 1) and accumulation of microscopically observed lesions in
the livers similar to human NAFLD (Table 2,Fig. 1). The previous
comprehensive studies have established that dietary-based models of
NAFLD induced by methionine- and choline-deﬁcient or choline-
deﬁcient diets are the best models to study mechanisms of liver injury
in NAFLD, despite the fact that some of the features do not resemble
pathogenesis of NAFLD in humans (Ariz et al., 2010; Hebbard and
George, 2011; Larter and Yeh, 2008; Maher, 2011). One of the major dis-
advantage of the mouse NAFLD model induced by methionine- and
choline-deﬁcient or choline-deﬁcient diets is associated with signiﬁcant
loss of body weight (Ariz et al., 2010; Hebbard and George, 2011; Larter
and Yeh, 2008). The results of the present study showing that feeding
the methionine-containing CFD diet induced liver injury without the
loss of body weight, overcome this shortcoming of the commonly used
methionine- and choline-deﬁcient or choline-deﬁcient diets.
The magnitude of biochemical indicators of liver injury in plasma
and the extent of histomorphological changes in the livers ranged,
with the order being: A/J≈C57BL/6J≈C3H/HeJb129S1/SvImJ≈CAST/
EiJbPWK/PhJbWSB/EiJ. More importantly, the pathomorphological
alterations in the livers were accompanied by different expression pat-
terns of hepatic miR-122, miR-181a, miR-192, miR-34a, miR-200b, and
miRNA-122 is the most abundant and highly liver-speciﬁc miRNA,
accounting for 70% of all miRNAs in the adult liver (Girard et al., 2008;
Lewis and Jopling, 2010). The expression of hepatic miR-122 has been
reported to decrease signiﬁcantly in individuals with viral- and alcohol-
induced liver injuries and NASH (Cheung et al., 2008; Morita et al.,
2011) and to be inversely correlated with the severity of liver histopa-
thology. Similar down-regulation of miR-122 has been reported in ex-
perimental animals with NAFLD (Wang et al., 2009b; Pogribny et al.,
2010). However, the results of present study demonstrate clearly the
existence of inter-strain variability in expression of hepatic miR-122
in response to feeding the CFD diet. Speciﬁcally, down-regulation of
Fig. 4. Correlation plots of total liver pathology scores and induction of miRNAs in the livers of mice fed the choline- and folate-deﬁcient diet.Total liver pathology scores represent
the mean severity for all of the lesions detected in the liver of mouse. Each symbol represents individual animal in the CFD diet group in respected mouse strain.
57V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 52–59
Author's personal copy
miR-122 was found only in the livers of A/J, C57BL/6J, PWK/PhJ, and
WSB/EiJ mice only. More importantly, while changes in miR-122 ex-
pression in the livers may be reﬂective of liver injury, the down-
regulation of this miRNA was not associated with severity of liver injury.
This was evidenced by the fact that changes in hepatic miR-122 expres-
sion did not correlate with the magnitude of pathomorphological
changes in the livers of choline- and folate-deﬁcient mice (Fig. 4). Like-
wise, the expression of another highly abundant and liver-speciﬁc
miRNAs, miR-192, was not associated with the severity of liver dam-
age induced by a CFD diet. In contrast, expression of miR-34a, miR-
200b, miR-221 and miR-181a miRNAs increased across strains, with
the greatest magnitude being found in sensitive PWK/PhJ, and WSB/
EiJ strains. More importantly, up-regulation of miR-34a, miR-181a,
miR-200b, and miR-221 in the livers of CFD diet-fed mice strongly
correlated with a severity of NAFLD-related pathomorphological
changes in the livers.
The most notable ﬁnding in this study is that plasma levels of each
of the miRNAs were signiﬁcantly correlated with the severity of liver
injury induced by the CFD diet, with strongest correlation occurring
It has been suggested that plasma miRNAs may be potential bio-
markers to diagnose and monitor diseases (Cortez and Calin, 2009), in-
cluding several types of liver pathologies, ranging from drug-induced
liver injury to chronic viral hepatitis and NAFLD (Wang et al., 2009b;
Laterza et al.,2009; Bihrer et al., 2011; Cermelli et al., 2011). Speciﬁcally,
the increased levels of plasma miR-122 alone or the combination of
miR-122 and miR-192 have been used to detect liver injury induced
by traditional liver toxicants, including trichlorobromomethane, carbon
tetrachloride, and acetaminophen in rats and mice (Wang et al., 2009b;
Laterza et al., 2009). Importantly, several recent clinical studies have
demonstrated that the levels of circulating miR-122, miR-192, and
miR-34a are signiﬁcantly higher in patients with acetoaminophen-
induced acute liver injury, NAFLD, and chronic hepatitis C infection
suggesting that plasma miRNAs may be useful biomarkers to monitor on-
going damage of hepatocytes and the magnitude of necroinﬂammation
(Cermelli et al., 2011; Starkey Lewis et al., 2011).Theresultsofthepresent
study showing a high and signiﬁcant correlation between the levels
of miR-122, miR-181a, miR-192, miR-34a, miR-200b, and miR-221 in
plasma and a severity of NAFLD-speciﬁc pathomorphological changes
in the livers of mice fed the CFD diet reinforce this suggestion. More
importantly, the lack of changes in the plasma level of ubiquitous
non-liver-speciﬁc miRNA let-7c indicates that these circulating miRNAs
may be potential liver-speciﬁc biomarkers for noninvasive evaluation of
liver injury in NAFLD. In addition, a strong correspondence between
correlation of the levels on miRNAs and the activity of ALT and AST in
plasma with the extent of liver injury suggests that miRNAs may serve
as independent robust biomarkers of liver injury (Fig. 5 and Supple-
mentary Fig. 1).
In conclusion, the results of our study demonstrate that develop-
ment of NAFLD in mice induced by a choline- and folate-deﬁcient
diet is associated with altered expression of hepatic miRNAs and
their subsequent elevation in plasma. More importantly, the severity
of NAFLD is strongly associated with levels of circulating miR-122,
miR-181a, miR-192, miR-34a, miR-200b, and miR-221 that mirror
the magnitude of NAFLD-associated liver injury. These results suggest
that circulating miRNAs may be applied in human population-based
studies as sensitive genetic background-independent indicators for
noninvasive monitoring the development and extent of NAFLD-
associated liver injury. Additionally, they can be used as potential
noninvasive indicators of susceptibility to NAFLD liver injury.
Supplementary data related to this article can be found online at
Fig. 5. Correlation plots of total liver pathology scores and induction of miRNA levels in plasma of mice fed the choline- and folate-deﬁcient diet.Total liver pathology scores rep-
resent the mean severity for all of the lesions detected in the liver of mouse. Each symbol represents individual animal in the CFD diet group in respected mouse strain.
58 V.P. Tryndyak et al. / Toxicology and Applied Pharmacology 262 (2012) 52–59
Author's personal copy
Conﬂict of interest statement
The authors declare that there are no conﬂicts of interest.
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