The Journal of Nutrition
Nutrition and Disease
Menhaden Oil Decreases High-Fat Diet–Induced
Markers of Hepatic Damage, Steatosis,
Inflammation, and Fibrosis in Obese
Christopher M. Depner,4Moises Torres-Gonzalez,4,5Sasmita Tripathy,4Ginger Milne,6
and Donald B. Jump4*
4School of Biological and Population Health Sciences and the Linus Pauling Institute, Oregon State University, Corvallis, OR;
5Endocrinology and Cardiology, School of Medicine, University of California–San Diego, La Jolla, CA; and6Eicosanoid Core
Laboratory, Vanderbilt University Medical Center, Division of Clinical Pharmacology, Nashville, TN
The frequency of nonalcoholic fattyliverdisease(NAFLD)andnonalcoholic steatohepatitis (NASH)hasincreasedin parallel
with obesity in the United States. NASH is progressive and characterized by hepatic damage, inflammation, fibrosis, and
oxidative stress. Because C20–22 (n-3) PUFA are established regulators of lipid metabolism and inflammation, we tested
the hypothesis that C20–22 (n-3) PUFA in menhaden oil (MO) prevent high-fat (HF) diet–inducedfatty liver disease in mice.
Wild-type (WT) and Ldlr2/2C57BL/6J mice were fed the following diets for 12 wk: nonpurified (NP), HF with lard (60% of
energy from fat), HF–high-cholesterol with olive oil (HFHC-OO; 54.4% of energy from fat, 0.5% cholesterol), or HFHC-OO
supplemented with MO (HFHC-MO). When compared with the NP diet, the HF and HFHC-OO diets induced
hepatosteatosis and hepatic damage [elevated plasma alanine aminotransferase (ALT) and aspartate aminotransferases]
and elevated hepatic expression of markers of inflammation (monocyte chemoattractant protein-1), fibrosis (procollagen
1a1), and oxidative stress (heme oxygenase-1) (P # 0.05). Hepatic damage (i.e., ALT) correlated (r = 0.74, P , 0.05) with
quantitatively higher (.140%, P , 0.05) hepatic cholesterol in Ldlr2/2mice fed the HFHC-OO diet than WT mice fed the
HF or HFHC-OOdiets. Plasmaand hepatic markersof liver damage,steatosis,inflammation, and fibrosis, but not oxidative
stress, were lower in WT and Ldlr2/2mice fed the HFHC-MO diet compared with the HFHC-OO diet (P , 0.05). In
conclusion, MO [C20–22 (n-3) PUFA at 2% of energy] decreases many, but not all, HF diet–induced markers of fatty liver
disease in mice. J. Nutr. 142: 1495–1503, 2012.
Nonalcoholic fatty liver disease (NAFLD)7has increased in
parallel with central obesity, and its prevalence is anticipated to
continue to increase (1,2). NAFLD is now the most common
cause of liver disease in developed countries (3) and is defined as
excessive lipid accumulation in the liver, i.e., hepatosteatosis
(4,5). The American Liver Foundation estimates that ;25% of
the U.S. population has NAFLD and 75% of obese and 100% of
morbidly obese individuals have NAFLD. NAFLD is the hepatic
manifestation of metabolic syndrome (MetS) (4); MetS risk
factors include obesity, elevated plasma TG and LDL choles-
terol, reduced HDL cholesterol, high blood pressure, and fasting
NAFLD ranges in severity from simple fatty liver (steatosis)
to nonalcoholic steatohepatitis (NASH) (6). Simple steatosis is
relatively benign until it progresses to NASH, which is charac-
terized by hepatic injury (hepatocyte ballooning and cell death),
increased blood levels of hepatic enzymes [alanine aminotrans-
ferase (ALT)], hepatic inflammation, oxidative stress, and
fibrosis (1,2,7). Approximately 30–40% of individuals with
simple steatosis progress to NASH (8), and NASH can progress
to cirrhosis (8), which is a major risk factor for hepatocellular
carcinoma (2). In the “2 hit hypothesis” for NASH (9), the first
hit involves chronic hepatosteatosis as TG and cholesterol (free
1Supported by the USDA, National Institute of Food and Agriculture grant
2009-65200-05846, and NIH grant DK-43220.
2Author disclosures: C. M. Depner, M. Torres-Gonzalez, S. Tripathy, G. Milne,
and D. B. Jump, no conflicts of interest.
3Supplemental Figures 1 and 2 and Supplemental Tables 1–3 are available from
the “Online Supporting Material” link in the online posting of the article and from
the same link in the online table of contents at http://jn.nutrition.org.
7Abbreviations used: ALT, alanine aminotransferase; AST, aspartate amino-
transferase; ChREBP, carbohydrate response element binding protein; DNL, de
novo lipogenesis; HF, high-fat; HFHC, high-fat, high cholesterol; LF, low-fat; LXR,
liver X receptor; MetS, metabolic syndrome; MLX, max-like protein X; MO,
menhaden oil; OO, olive oil; NAFLD, nonalcoholic fatty liver disease; NASH,
nonalcoholic steatohepatitis; NEFA, nonesterified fatty acid; NP, nonpurified;
ROS, reactive oxygen species; SREBP1, sterol regulatory element binding
protein-1; WT, wild-type.
* To whom correspondence should be addressed. E-mail: donald.jump@oregonstate.
ã 2012 American Society for Nutrition.
Manuscript received January 26, 2012. Initial review completed March 5, 2012. Revision accepted May 3, 2012.
First published online June 27, 2012; doi:10.3945/jn.112.158865.
by guest on November 21, 2015
Supplemental Material can be found at:
cholesterol and cholesterol esters) accumulation. Excessive
hepatic lipid sensitizes hepatocytes to the second hit, which is
manifested by increased inflammation derived from resident
(Kupffer cells) and recruited macrophages, induction of oxida-
tive stress, activation of stellate cells, and fibrosis (2,10).
Although the management of lifestyle (diet and exercise) is
one approach to control the onset and progression of NAFLD,
the best strategy for managing NAFLD has yet to be defined
(11). On the basis of the well-established effects of C20–22 (n-3)
PUFA to regulate hepatic lipid metabolism, dyslipidemia, and
inflammation (12–14), we tested the hypothesis that dietary
(n-3) PUFA prevents high-fat (HF) diet–induced fatty liver
disease in mice. Recent clinical studies have indicated that
dietary (n-3) PUFA have the potential to reduce hepatic lipid
content in children and adults (15–19). Our studies, however, go
beyond the analysis of hepatic lipids and examine the capacity of
(n-3) PUFA to regulate markers of NASH, such as hepatic
damage, inflammation, oxidative stress, and fibrosis. We used
wild-type (WT) and Ldlr2/2mice and 3 HF diets: HF lard (HF
diet; 60% of energy from fat), which induces obesity and insulin
resistance (20,21), HF–high cholesterol with olive oil (HFHC-
OO diet; 54.4% of energy from fat, 0.5% cholesterol), which
induces fatty liver and oxidative stress (22), and the HFHC-OO
diet supplemented with menhaden oil (MO) (HFHC-MO) a rich
source of EPA [20:5(n-3)] and DHA [22:6(n-3)]. EPA and DHA
in the HFHC-MO diet were 2% of total energy, a level com-
parable to that used to treat dyslipidemia (23). Our studies
established that dietary C20–22 (n-3) PUFA have the capacity to
regulate some, but not all, HF diet–induced markers of NASH.
Materials and Methods
Animals and diets. All procedures for the use and care of animals for
laboratory research were approved by the Institutional Animal Care and
Use Committee at Oregon State University. Male WT and Ldlr2/2mice
(on the C57BL/6J background; Jackson Laboratories) at 2 mo of age
consumed one of the following diets ad libitum for 12 wk: 1) Purina
chow 5001 [nonpurified (NP); 13.5% of energy from fat and 58.0% of
energy from carbohydrates]; 2) HF [60% of energy from fat; D12492;
Research Diets]; 3) HFHC-OO [54.4% of energy from fat, 0.5%
cholesterol (weight%); D08010702; Research Diets]; or 4) HFHC-MO
[54.4% of energy from fat, 0.5% cholesterol (weight%); D08010703;
Research Diets] (Supplemental Table 1). The HF, HFHC-OO, and
HFHC-MO diets were described previously (21,22). Fat energy density
in the HFHC-OO and HFHC-MO diets was identical (54.4% energy
from fat). C20–22 (n-3) PUFA in the HFHC-MO diet represented ;2%
of total energy (Supplemental Table 1). All diets were stored frozen
(2208C) until used to feed the mice; diets were replenished every other
day in an effort to reduce the formation of oxidation products.
The study was carried out twice with 8 mice/diet group in each study.
Energy intake was monitored every other day, and body weight was
monitored weekly. At the end of the 12-wk feeding period, all mice were
feed-deprived overnight (1800 to 0800 the next day); then half of the
mice were refed their diets for 4 h (0800 to 1200). Feed-deprived and
refed mice were killed (isoflurane anesthesia and exsanguination) at
0800 and 1200, respectively, for the collection of blood and liver. Blood
was collected in tubes containing EDTA; plasma was collected by
centrifugation. Livers were removed, weighed, and rapidly frozen in
liquid nitrogen. Plasma, blood cells, and liver were stored frozen (2808C)
until used for specific assays.
Our studies used WT and Ldlr2/2mice fed the NP diet as controls.
Because there was no significant difference (Student’s t test) in any
variable measured between WT and Ldlr2/2mice fed the NP diet, NP-
fed Ldlr2/2mice served as the control group for all studies described
below. Thefollowing6groupdesignations areusedto describethestudy:
Ldlr2/2mice fed NP (NP), WT mice fed HF (WT-HF), WT mice fed
HFHC-OO (WT-HFHC-OO), WT mice fed HFHC-MO (WT-HFHC-
MO), Ldlr2/2mice fed HFHC-OO (Ldlr2/2HFHC-OO), and Ldlr2/2
mice fed HFHC-MO (Ldlr2/2HFHC-MO). During the course of our
studies, we found that markers of NASH were induced to higher levels in
Ldlr2/2mice compared with in WT mice. As such, much of the data
presented below describe the capacity of MO to regulate NASH markers
in Ldlr2/2mice. A comparison of the effects of the 4 diets on
inflammation and fibrosis markers in WT and Ldlr2/2mice is shown
in Supplemental Table 3.
Measurement of plasma markers. Plasma glucose (Autokit Glucose),
TG (L-type TG H triglyceride), nonesterified fatty acids (NEFA; NEFA-
C), and cholesterol (Cholesterol E) were measured with the use of kits
from Wako. Plasma b-hydroxybutyrate was measured with the use of a
kit (b-hydroxybutyrate Liquicolor) from Stanbio. Plasma apo B (ApoB
K-Assay) and apo CIII (ApoCIII K-Assay) were measured by immuno-
turbidimetric assay from Kamiya Biomedical. Alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) were measured by using
kits from Thermo Electron.
Measurement of urinary isoprostanes. After 11 wk of being fed the
NP or HFHC diets, Ldlr2/2mice were placed in metaboliccages for 24-h
urine collection. Collected urine was stored at ,808C until extracted for
F2- and F3-isoprostanes and F4-neuroprostanes analysis. Results were
normalized to urinary creatinine as described (22,24).
Lipid extraction and analyses. Totallipidswereextractedfromliverand
plasma; FAME were prepared andquantified bygas chromatography(20).
To measure hepatic total TG and cholesterol, extracted hepatic lipids were
dried, dissolved in 10% Triton X-100 (Fisher Scientific), and assayed for
TG and cholesterol content using the L-type TG H triglyceride and total
cholesterol assay kits from Wako as described above (20). Hepatic protein
was measured by using the Quick Start Bradford Reagent (Bio-Rad) and
bovine serum albumin (Sigma) as the standard (20).
RNA extraction and qRT-PCR. Total RNAwas extracted from liver, and
specific mRNA were quantified by qRT-PCR (20,25). Specific primers for
each gene were described previously (20,25) or are listed in Supplemental
Table 2. Cyclophilin was used as the internal control for all genes.
Immunoblotting. Hepatic nuclear protein extracts were prepared
by using both protease (Roche Diagnostics) and phosphatase inhibitors
(1 mmol/L b-glycerol phosphate, 2.5 mmol/L Na-pyrophosphate,
1 mmol/L Na3VO4) (20). Proteins (25–100 mg) were separated electro-
phoretically and transferred to nitrocellulose for immunoblotting. TBP
(TATA-binding protein) served as a loading control for all immunoblot
studies. Antibodies used in these studies were described previously (20);
antibodies for NF-kB-p50 and NF-kB-p65 were purchased from Santa
Cruz Biotechnology and Cell Signaling, respectively.
Statistical analysis. We used 1- and 2-way ANOVA and Tukey’s
honestly significant difference post hoc analysis to establish significant
differences. One-way ANOVA was used to detect dietary effects when
only Ldlr2/2mice were included in the analysis. Two-way ANOVAwas
used to detect diet-gene interactions, when both WT and Ldlr2/2mice
were includedin theanalysis andfor the analysisof refeedingeffects(Fig.
1A). Data were analyzed for homogeneous variances by using the Levene
test. If unequal variances were detected, the data were log-transformed.
ANOVA was performed on both transformed and untransformed data.
Untransformed data are presented for interpretation purposes. The
Student’s t test was used when only 2 groups were compared. P , 0.05
was considered different. The correlation analysis used linear regression
analysis. Statistical analysis was performed with VassarStats (http://
vassarstats.net/) and Statgraphics (StatPoint Technologies, Inc.). All
values reported are means 6 SD.
Body weight and plasma markers. After 12 wk of being fed
the NP and test diets, the body weights of WT mice fed the HF or
HFHC diets and Ldlr2/2mice fed the HFHC diets were .39%
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