High-fat diet modifies expression of hepatic cellular senescence gene p16(INK4a) through chromatin modifications in adult male rats
Zhang et al. Genes & Nutrition
High-fat diet modifies expression of hepatic cellular senescence gene p16(INK4a) through chromatin modifications in adult male rats
Xiyuan Zhang 2
Guanying Bianca Xu 1
Dan Zhou 4
Yuan-Xiang Pan 0 1 3
0 Division of Nutritional Sciences (DNS), University of Illinois Urbana-Champaign , 461 Bevier Hall, MC-182, 905 South Goodwin Avenue, Urbana, IL 61801 , USA
1 Department of Food Science and Human Nutrition, University of Illinois Urbana-Champaign , 461 Bevier Hall, MC-182, 905 South Goodwin Avenue, Urbana, IL 61801 , USA
2 Pediatric Oncology Branch (POB), National Cancer Institute (NCI), National Institute of Health (NIH) , Bethesda, MD 20892 , USA
3 Illinois Informatics Institute, University of Illinois at Urbana-Champaign , 461 Bevier Hall, MC-182, 905 South Goodwin Avenue, Urbana, IL 61801 , USA
4 Hongqiao International Institute of Medicine, Shanghai Tongren Hospital/Faculty of Public Health, Shanghai Jiao Tong University School of Medicine , Shanghai 200025 , China
Background: Liver is the crucial organ as a hub for metabolic reactions. p16(INK4a) is a well-established cyclindependent kinase (CDK) inhibitor that plays important role in the molecular pathways of senescence, which lead to irreversible cell cycle arrest with secretion of proinflammatory cytokines and mitochondrial dysfunction. This study tested the hypothesis that cellular senescence regulated by p16(INK4a) is associated with high-fat diet in adult male rats. Methods: Sprague Dawley rats were fed a high-fat (HF) diet or a control (C) diet for 9 weeks after weaning. At 12 weeks of age, liver samples of male rats were collected to investigate the key genes and liver physiological status. Results: Both mRNA and protein expression level of cellular senescence marker, p16(INK4a), was increased significantly in HF group when compared to C group. A decrease of tri-methylated histone H3 lysine 27 (H3K27Me3) in the coding region of p16(INK4a) was observed. On the other hand, mRNA and protein expression of another inhibitor of cyclindependent kinase, p21(Cip1), was decreased significantly in HF group; however, no significant chromatin modification was found in this gene. Histological analysis demonstrated hepatic steatosis in HF group as well as severe fat accumulation. Conclusions: Our study demonstrated that HF diet regulated cellular senescence marker p16(INK4a) through chromatin modifications, which may promote hepatic fat accumulation and steatosis.
High-fat diet; p21(Cip1); Fatty liver; Hepatic cellular senescence; Chromatin modification
Dietary factors play pivotal role in modulating liver
function and physiology as well as in the development of
hepatic cells: a high-fat (HF) diet can increase hepatic
lipid content and plasma insulin concentration, causing
insulin resistance in obese animals [
], while maternal
high-fat diet leads to altered regulation of liver
development in offspring through DNA methylation, which may
cause early hepatic dysfunction [
]. Several animal
models have been reported to establish the association
between high-fat diet and the physiological changes in
the liver [
Among the annotated genes using the Gene Oncology
database, cell cycle control genes were the second largest
group of altered genes associated with HF diet-induced
], and this link between HF diet and cell
cycle regulation has also been reported in other studies
using either cell or animal models [
4, 12, 13
p16(INK4a) is a biomarker of cellular senescence and
], and its expression has been linked to
replicative hypofunction in many types of cells, including
hematopoietic progenitors , lymphocytes [
neural stem cells , and pancreatic beta cells [
Pervious study has shown that high-fat diet induced
increasing level of p16(INK4a) transcription rate in an
obese rat model, which was associated with a higher
acetylation levels of histone H4 and lower methylation
level of histone H3 lysine 27 in p16(INK4a) promoter
and coding region [
]. p21(Cip1), on the other hand,
belongs to the family of CDK inhibitor proteins and is a
universal inhibitor of CDKs which include cyclin D-, E-,
and A-dependent kinases [
]. Upregulation of
p21(Cip1) protein was proved to be involved in impaired
regeneration of fatty livers in obese mice . Although
literature indicates that p16(INK4a) and p21(Cip1)
alteration may not be necessary for either exogenous or
endogenous liver carcinogenesis [
], it remains unclear
how high-fat diet affects expressions of these CDK
inhibitors and how the expression changes affect liver
function as well as hepatic physiology.
As briefly mentioned before, the expressions of
p16(INK4a) gene is regulated through epigenetic
modifications. Hypermethylation of DNA in promoter CpG
islands is associated with the transcriptional silencing of
p16(INK4a) gene, and the di-methylation of histone H3
lysine 9 (H3K9) correlates with the DNA methylation
status in colon cancer development [
]. High-fat diet
was proved to alter DNA methylation in genes related to
liver lipid metabolism and hepatic steatosis: for example,
Ndufb9, or NADH dehydrogenase 1 beta subcomplex 9,
was found hyper-methylated in CpG sites in the HFD
group, which downregulates its expression [
However, few studies have been done to investigate the role
of dietary high fat in regulation of p16(INK4a)
expression through epigenetic modifications.
This study was designed to investigate the expression
and chromatin modifications of CKIs p16(INK4a) and
p21(Cip1) in adult male rat model induced by high-fat
diet and its effects on hepatic cellular senescence as well
as liver physiology. We hypothesized that high-fat diet
regulates hepatic cellular senescence in male adult rats
by regulating p16(INK4a) and p21(Cip1) expression
through chromatin modifications, which may promote
hepatic fat accumulation and steatosis.
Sprague Dawley rats were fed a high-fat diet (HF) or a
control (C) diet for 9 weeks after weaning. All information
regarding the diets (Research Diets, Inc., New Brunswick,
NJ) is listed in Table 1. Animals were individually housed
in standard polycarbonate cages in a humidity- and
temperature-controlled room on a 12-h light-dark cycle
and had free access to food and water. At 12 weeks of age,
the animals were sacrificed and the left lobe of the liver
was collected, rapidly frozen in liquid nitrogen, and stored
in − 70 °C for future use. We confirm that all applicable
institutional and governmental regulations regarding the
ethical use of animals were followed during this research
(University of Illinois Institutional Animal Care and Use
Committee approval #09112).
RNA isolation and cDNA synthesis
Frozen liver samples (50–100 mg) were ground using
mortar and pestle with liquid nitrogen, and total RNA
was extracted with TRI reagent (Sigma, St. Louis, MO).
RNA samples were incubated with DNase Ι (Roche,
Mannheim, Germany) at room temperature for 20 min,
followed by 10 min in 90 °C heat block to prevent
contamination of genomic DNA. High-capacity cDNA
Reverse Transcription Kit (Applied Biosystems, Foster City,
CA) was used for reverse transcription of 2 μg of total
RNA. Reverse transcription was performed in a 2720
Thermal Cycler (Applied Biosystems) with 20 μL
reaction volume with the following program: heated at 25 °C
for 10 min, incubated at 37 °C for 2 h, and then heated
at 85 °C for 5 s. The 20 μL of final product was diluted
to 400 μL for 5 ng/μL using RNase free water (Fisher,
Fair Lawn, NJ) and stored at − 20 °C.
Real-time quantitative PCR
To measure the relative transcriptional level of target
genes, qPCR was performed in a 96-well plate using a
7300 real-time PCR System (Applied Biosystems). Five
microliters of cDNA, with the final concentration of
5 ng/μL, was mixed with 10 μL SYBR Green master mix
(Quanta Biosciences Inc., Gaithersburg, MD) and 1 μL
of each 5 nmol/L primer (forward and reverse). The
reaction was incubated using the following program: 95 °C
for 10 min, followed by 35 cycles of 95 °C for 15 s and
60 °C for 1 min. High efficiency of the machine and the
presence of a unique product were ensured by checking
that the slope of the standard was in the range of −
3.3 ± 0.3 and the R square to be larger than 0.98. Kinetic
analysis was conducted to detect the exponential phase
of amplification in each well with 25 ng template cDNA.
mRNA level of ribosomal protein L7a was utilized as the
internal control. In order to determine the gene
transcription rate, primers were designed to amplify a region
including both exon and intron of that gene. The relative
expression level of this region can indicate the
premRNA rate of the gene and further present the
transcription rate. Primers used for qPCR were designed
using Vector NTI software (InforMax Inc., Frederick,
MD), and all the primers used for real-time PCR are
listed in Table 3.
Triacylglycerol and non-esterified fatty acid assay in plasma and liver tissues
One hundred milligrams of frozen liver samples were
ground using a mortar and pestle with liquid nitrogen
and mixed with 0.3 mL saline (0.9% w/v NaCl).
Homogenized samples were quickly frozen in liquid nitrogen
and kept in − 70 °C until analysis. The samples were
quickly thawed in 37 °C and diluted five times to
1.5 mL. Twenty microliters of the diluted samples was
incubated with 20 μL 1% deoxycholate in 37 °C for
5 min, and 10 μL of the samples was analyzed via the
Thermo Infinity Triglycerides Liquid Stable Reagent
(Thermo Fisher Scientific, Rockford, IL) following
company protocol and using a commercially available
standard reference kit (Verichem Laboratories, Providence,
RI). Lowry assay was performed to determine the protein
concentration, which was used to normalize the
triacylglycerol (TAG) concentration in the liver. To determine
plasma TAG levels, plasma samples were thawed on ice
and analyzed accordingly following company protocol as
for liver samples. Non-esterified fatty acid concentration
in the liver was determined using a commercially available
kit (HR-2 Series, Wako Diagnostics, Richmond, VA).
Frozen liver samples from offspring were embedded in
O.C.T. compound (Gentaur, Kampenhout, Belgium)
prior to sectioning. All sections were then stained with
hematoxylin and eosin (H&E) or oil red O (ORO,
Newcomer Supply, Middleton, WI) and evaluated for
steatosis, fat accumulation, and inflammation, by a
pathologist blind to the identity of the experimental groups.
Chromatin immunoprecipitation (ChIP)
To determine the specific chromatin modification and
transcription factor binding, ChIP analysis was employed
according to a modified protocol [
]. Two hundred
milligrams of frozen liver samples were ground using a mortar
and pestle with liquid nitrogen and washed with PBS. The
samples were resuspended in PBS and cross-linked in 1%
formaldehyde for 10 min at room temperature. After
centrifugation, the pellet was resuspended in nuclei swelling
buffer (5 mmol/L Pipes [NaOH] pH 8.0, 85 mmol/L KCl,
0.5% NP40) containing protease inhibitor and
phosphorylation inhibitor. The nuclei were lysed in SDS lysis buffer
(50 mol/L Tris-HCl pH 8.1, 10 mmol/L EDTA, 1% SDS)
containing protease inhibitor and phosphorylation
inhibitor. The chromatin was sonicated (Fisher Scientific model
100 Sonic Dismembrator, Pittsburgh, PA) on ice with six
bursts for 40 s at power setting 5 with 2 min cooling
between each burst. After removing the cell debris by
centrifuging the sonicated product at 13,000 rpm in 4 °C for
10 min, sheared chromatin was diluted in ChIP Dilution
Buffer to 10 mL to perform 10 immunoprecipitations
(IPs). One milliliter of the diluted lysate was incubated
overnight on a hematology mixer (Model 346, Fisher
Scientific) with 2 μg of primary antibodies at 4 °C (all the
antibody information is listed in Table 2). Sixty microliters
of pre-blocked salmon sperm DNA/protein G agarose
beads (60 μl, 50% slurry; Upstate Biotechnology, Lake
Placid, NY) was then added to each chromatin sample,
followed by 2 h of incubation at 4 °C. The mixture was
then centrifuged at 2000 rpm for 1 min at 4 °C.
Supernatant of normal rabbit IgG was saved as input control.
The pellets containing immunoprecipitated complexes
were washed sequentially with 1 mL of low-salt solution
(0.1% SDS, 1% triton X-100, 2 mM EDTA, 20 mmol/L
Tris-HCl pH 8.0, 150 mmol/L NaCl), high-salt solution
(0.1% SDS, 1% triton X-100, 2 mM EDTA, 20 mmol/L
Tris-HCl pH 8.0, 500 mmol/L NaCl), and LiCl solution
(0.25 mol/L LiCl, 1% NP40, 1% sodium deoxycholate,
1 mM EDTA, 10 mmol/L Tris-HCl pH 8.0) and twice with
TE (pH 8.0). Antibody/protein/DNA complexes were
eluted from Protein G beads by adding twice 250 μL of
the elution buffer (1% SDS and 50 mmol/L NaHCO3)
followed by shaking at 37 °C for 15 min at 300 rpm and
flash spinning down at room temperature. The combined
supernatants were incubated at 65 °C for 5 h with 20 μL
5 mol/L NaCl and 1 μg of RNase A (Qiagen, Hilden,
Germany) to reverse the formaldehyde cross-linking and
release the DNA fragments. Samples were then treated
with proteinase K (Sigma) at 37 °C for 1 h to remove
protein. DNA was purified with a DNA miniprep system
(Qiagen). Five percent of immunoprecipitated DNA was
used for real-time PCR reaction to detect promoter and
coding regions of the p16(INK4a) and p21(Cip1) genes.
Primers were designed using Vector NTI software
(InforMax Inc., Frederick, MD) and are listed in Table 3.
Unfixed, frozen colon sections were embedded in
Tissue-Tek OCT compound (VWR, Radnor, PA) and cut
to 8-μm-thick sections using Leica CM3050 S cryostat
(Leica Microsystems, Inc., IL) at − 24 °C; sections were
stored in − 80 °C before immunofluorescence staining.
When conducting an immunofluorescent staining, slides
were first washed in 1× PBS and fixed in 4%
formaldehyde. Then, slides were blocked with blocking buffer (1×
PBS/5% normal goat serum/0.05% Triton X-100) and
incubated with p21 antibody (F-5) Alexa Fluor 488 (1:50,
sc6246 AF488, Santa Cruz Biotechnologies, Dallas, TX)
and anti-CDKN2A/p16INK4a antibody (1:100, ab211542,
Abcam, Cambridge, UK) at 4 °C overnight. After primary
incubation, slides were washed again with 1× PBS and
then incubated with Goat anti-Rabbit IgG (H+L) Highly
Cross-Adsorbed Secondary Antibody, Alexa Fluor 647
(1:100, Thermo Fisher Scientific, Eugene, OR) at room
temperature for 1 h. All slides were then rinsed in 1× PBS
again and incubated with DyLight 554 Phallioidin (1:50,
Cell Signaling Technology, Danvers, MA) and Hoechst
33342 (Thermo Fisher Scientific, Eugene, OR),
respectively, for 15 min in dark at room temperature. After
incubation, slides were washed with 1× PBS, mounted
with Prolong-Gold Antifade Reagent (Molecular Probes
by Life Technologies, Carlsbad, CA), and dried in dark
overnight at 4 °C. Pictures were taken using the Confocal
LSM 700 microscope (Carl Zeiss Microscopy, LLC, United
States) with Zen software (Carl Zeiss AG, Ontario, CA) at
magnification of × 10. To quantify protein expression, five
350 μm × 350 μm captured pictures in each group were
analyzed. ImageJ Software (NIH, Bethesda, MD) was applied
to separate the nucleus spheres and p16 or p21 protein
spheres by channel (blue, red and green) and to measure
sphere areas. Protein expression is calculated by the protein
spheres (μm2) ratio to nucleus sphere area (μm2).
Results are presented as mean ± SEM (animal
numbers of specific experiments are descripted in the
figure legends). Comparison of food intake and body
weight during treatment period and chromatin
modifications at p16 and p21 genes between HF and C
groups were performed by one-way analysis of
variance (ANOVA) using proc GLM program in SAS v.
9.1 (SAS Institute, Cary, NC). Comparison of other
physiological outcomes, mRNA expression, and
protein expression were performed by two-trailed t test
using proc GLM program in SAS v. 9.1 (SAS
Institute, Cary, NC). Significance (*) was set at p < 0.05
Food intake and body weight
The body weight (g) of rats fed HF diet was significantly
higher than those fed C diet after 7 weeks of feeding
(Fig. 1a). Energy intake (kCal) in HF group was
significantly different from that in the C group (Fig. 1b).
Plasma and liver triacylglyceride (TAG) and non-esterified
fatty acid (NEFA)
Plasma TAG levels were not different between the HF
and C groups, while liver TAG significantly increased in
the rats of HF group when compared to the C group.
No significant difference was observed in liver NEFA
level between C and HF groups (Fig. 2a).
Hematoxylin and eosin (H&E) stain and oil-red-O (ORO)
Photomicrographs of liver sections stained with
hematoxylin and eosin are shown in Fig. 2b. As expected,
based on observations made by previous studies [
comparing to liver tissue collected from C group, liver
tissue from HF group showed development of hepatic
steatosis and extensive degenerative ballooning, which was
predominantly microvascular and involved zone 3
(perivenular) hepatocytes [
]. ORO staining suggests that liver
tissue collected from HF group has increased fat
accumulation with larger lipid droplets (Fig. 2c). Together, these
observations indicated that high-fat diet triggered significant
histological changes in the liver, increased hepatic lipid
accumulation, and hence induced fatty liver.
Cellular senescence parameters
mRNA level of p16(INK4a) gene, as a marker of cellular
senescence, increased significantly in livers of rats in HF
group when compared to the C group, while the mRNA
level of p21(Cip1), another inhibitor of cyclin-dependent
kinase, was significantly lower in HF group than in C
group (Fig. 3a). Transcription rate of p16(INK4a) gene
was accordingly significantly increased in HF group,
while the transcription rate of p21(Cip1) gene did not
change between the two groups (Fig. 3b).
Protein level of p16 (INK4a) and p21 (Cip1) by
Immunofluorescent pictures suggest that there is a
difference in protein expression of p16 (INK4a) and
p21 (Cip1) between C and HF groups (Fig. 4a).
Consistent with mRNA expression, protein level of p16
(INK4a) increased significantly in livers of rats in HF
group when compared to the C group, while protein
level of p21 (Cip1) was significantly lower in HF
group than in C group (Fig. 4b, c).
Chromatin modifications in p16(INK4a) and p21(Cip1) genes
ChIP assay was performed to investigate the possible
chromatin modifications occurring in the livers of C and
HF adult male rats that affected gene transcription.
Results showed that methylation level of histone H3 lysine
27 (H3K27Me3) was significantly lowered in the
p16(INK4a) coding region in HF rats compared with C
rats. Lowered methylation level of histone H3 lysine 4
(H3K4Me2) was also observed at promoter region and
in the coding region of p16(INK4a) in HF rats compared
with C rats; however, no significance was detected
On the other hand, as for gene p21(Cip1), the
methylation level of histone H3 lysine 4 (H3K4Me2) was
increased at the promoter region in HF rats compared
with C rats, while decreased in the coding region, with
no significance detected. Meanwhile, the methylation
level of histone H3 lysine 27 (H3K27Me3) was almost
same at the promoter region and in the coding region of
p21(Cip1) in HF rats compared with C rats (Fig. 6).
The purpose of the present study was to determine the
mRNA expressions of CKIs p16(INK4a) and p21(Cip1)
genes and to investigate the chromatin modification in
these two genes induced by HF diet in livers of adult male
rats and their effects on inducing hepatic physiological
changes. Here, we reported that significantly increased
mRNA expression of p16(INK4a) and significantly
decreased p21(Cip1) mRNA expression were observed in
livers of adult rats fed HF diet, comparing to rats fed C
diet. The protein levels of p16 (INK4a) and p21 (Cip1)
were confirmed by immunofluorescence, and the results
were consistent with mRNA expression. We further
investigated and discovered that expression of p16(INK4a) was
regulated by chromatin modifications in the coding
region. Also, we reported that HF diet induced physiological
outcomes in adult rats including significantly increased
energy intake and body weight, as well as significantly
increased liver TAG concentration, which induced fatty liver
in adult rats.
Lipid accumulation has been confirmed to be associated
with fatty liver development and prevention [
previous studies, an increased liver TAG concentration in
animal models fed HF diet or in their offspring [
observed, as well as increased lipid accumulation
(indicated by larger lipid droplets from ORO staining in
animals fed HF diet) and extensive ballooning degeneration
], which is consistent with our results, indicating
that HF diet induced fatty liver in rats in this current
We also found mRNA and protein expression of both
p16(INK4a) and p21(Cip1), two well-established cell
cycle inhibitors, biomarkers of aging and cellular
] have been altered significantly by HF diet
in livers of adult rats, however, in opposite direction.
The increase of p16(INK4a) mRNA can be partially
explained by the decrease of tri-methylated histone H3
lysine 27 (H3K27Me3) in the coding region of p16(INK4a)
that was observed in this present study, as H3K27Me3
has been considered as a suppressor of gene
]. On the other hand, a significantly
decreased mRNA expression of p21(Cip1) was observed in
HF rats; however, there was no significant difference on
transcription rate of this gene between two diet groups.
Further, an increased methylation level of histone H3
lysine 4 (H3K4Me2) in promoter region of p21 (Cip1) in
HF rats was observed, as well as a decreased methylation
level in the coding region, although no significance was
detected. Consistent with our results, a declined amount
of p21(Cip1)1 with an elevated expression of p16(INK4a)
were observed in human diploid fibroblasts after cell
achieve senescence by an early research [
]. The same
research also demonstrated that p16(INK4a) and
p21(Cip1) have very different age-related accumulation
patterns, and the downregulation of p21(Cip1) might be
a necessary part for putative differentiation program in
late senescent cells. Thus, the decrease of p21(Cip1)
mRNA might be explained by the differential role it
plays in the mechanisms of senescence in cells. Histone
H3 lysine 4 methylation (H3K4me) may also contribute
to p21(Cip1) transcription [
]. Another study suggests
that the absence of H3K4me2 in coding region induces
increased of H3K4ac, while the degree of H3K4ac
enrichment is proportional to the rate of transcription
]. The two studies together may be used to explain
the results gained from current study: in the liver of
adult rats fed HF diet, a higher level of H3K4me2 in
promoter region of p21(Cip1) repressed its expression;
meanwhile, a lower level of H3K4me2 in coding region
maintained its transcription rate, keeping it at almost
the same level as in C group rats.
The regulatory mechanisms of p16(INK4a) and
p21(Cip1) on cellular senescence remain unclear at this
point. Previous study demonstrated that p16(INK4a)
regulates cell senescence through both CDK4/6-dependent
and CDK4/6-independent mechanisms. Under
CDK4/6dependent mechanism, enhanced p16(INK4a) expression
disassociates and releases p21(Cip1) from cyclinD-CDK4/
6 complexes, inhibiting CDK4/6 activity, and therefore
promotes cell senescence and decreases regenerative
capacity. However, the study also suggests that mechanism
through which p16(INK4a) induces senescence varies,
depending on aging condition, cell type and species [
Meanwhile, it is reported that lowered p21(Cip1)
expression causes lower serum adiponectin, a protein that
inhibits proliferation of liver cancer cells, and therefore
impairs apoptosis and/or induces cell cycle progression in
the liver [
]. On the other hand, an enhanced
p21(Cip1) expression in the liver is usually linked to
accelerated hepatocyte senescence [
Overall, our study demonstrates that, in adult rats fed
HF diet, it activates cellular senescence through interplay
between p16(INK4a) and p21(Cip1) in the liver. This is
accompanied by elevated TAG accumulation. The increased
expression of p16(INK4a) was associated with histone
modifications, in particular, the trimethylation of histone
3 lysine 27 and the demethylation of histone 3 lysine 4,
happened in promoter and coding regions respectively.
C: Control; CDK: Cyclin-dependent kinase; ChIP: Chromatin
immunoprecipitation; CKI: CDK inhibitor; H&E: Hematoxylin and eosin;
H3K27Me3: Trimethylation histone H3 lysine 27; H3K4: Histone H3 lysine 4;
H3K4Me2: Demethylation histone H3 lysine 4; H3K9: Histone H3 lysine 9;
HF: High fat; HFD: High-fat diet; NEFA: Non-esterified fatty acid; ORO: Oil red
O; qPCR: Quantitative real-time polymerase chain reaction;
Rb: Retinoblastoma; TAG: Triacylglycerol; TSA: Trichostatin A
This project was supported by the United States Department of Agriculture
Cooperative State Research, Education, and Extension Service, Hatch project
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on a reasonable request.
The experiments were performed in the laboratory of Y-XP at the University
of Illinois at Urbana-Champaign. Y-XP, XZ, and DZ were responsible for the
conception and design of the experiments. XZ and DZ conducted the
experiments and collected the data. XZ and GX analyzed the data, performed the
statistical analysis, and wrote the manuscript. Y-XP, XZ, and GX had the
primary responsibility for the final content. All authors read and approved the
We certify that all applicable institutional and governmental regulations
regarding the ethical use of animals were followed during this research
(University of Illinois Institutional Animal Care and Use Committee approval
Consent for publication
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1. Vilar L , Oliveira CP , Faintuch J , Mello ES , Nogueira MA , Santos TE , Alves VA , Carrilho FJ . High-fat diet: a trigger of non-alcoholic steatohepatitis? Preliminary findings in obese subjects . Nutrition . 2008 ; 24 : 1097 - 102 .
2. Jornayvaz FR , Jurczak MJ , Lee HY , Birkenfeld AL , Frederick DW , Zhang D , Zhang XM , Samuel VT , Shulman GI. A high-fat, ketogenic diet causes hepatic insulin resistance in mice, despite increasing energy expenditure and preventing weight gain . Am J Physiol Endocrinol Metab . 2010 ; 299 : E808 - 15 .
3. Liu Z , Patil IY , Jiang T , Sancheti H , Walsh JP , Stiles BL , Yin F , Cadenas E. Highfat diet induces hepatic insulin resistance and impairment of synaptic plasticity . PLoS One . 2015 ; 10 : e0128274 .
4. Dudley KJ , Sloboda DM , Connor KL , Beltrand J , Vickers MH . Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation . PLoS One . 2011 ; 6 : e21662 .
5. Gao M , Ma Y , Liu D . High-fat diet-induced adiposity, adipose inflammation, hepatic steatosis and hyperinsulinemia in outbred CD-1 mice . PLoS One . 2015 ; 10 : e0119784 .
6. Nakamura A , Terauchi Y. Lessons from mouse models of high-fat dietinduced NAFLD . Int J Mol Sci . 2013 ; 14 : 21240 - 57 .
7. Shearn CT , Mercer KE , Orlicky DJ , Hennings L , Smathers-McCullough RL , Stiles BL , Ronis MJ , Petersen DR . Short term feeding of a high fat diet exerts an additive effect on hepatocellular damage and steatosis in liver-specific PTEN knockout mice . PLoS One . 2014 ; 9 : e96553 .
8. Ishimoto T , Lanaspa MA , Rivard CJ , Roncal-Jimenez CA , Orlicky DJ , Cicerchi C , McMahan RH , Abdelmalek MF , Rosen HR , Jackman MR , et al. High-fat and high-sucrose (western) diet induces steatohepatitis that is dependent on fructokinase . Hepatology . 2013 ; 58 : 1632 - 43 .
9. Ganz M , Csak T , Szabo G . High fat diet feeding results in gender specific steatohepatitis and inflammasome activation . World J Gastroenterol . 2014 ; 20 : 8525 - 34 .
10. Tan X , Xie G , Sun X , Li Q , Zhong W , Qiao P , Sun X , Jia W , Zhou Z. High fat diet feeding exaggerates perfluorooctanoic acid-induced liver injury in mice via modulating multiple metabolic pathways . PLoS One . 2013 ; 8 : e61409 .
11. Kirpich IA , Gobejishvili LN , Bon HM , Waigel S , Cave M , Arteel G , Barve SS , McClain CJ , Deaciuc IV . Integrated hepatic transcriptome and proteome analysis of mice with high-fat diet-induced nonalcoholic fatty liver disease . J Nutr Biochem . 2011 ; 22 : 38 - 45 .
12. Wu Y , Zhang Z , Liao X , Wang Z. High fat diet triggers cell cycle arrest and excessive apoptosis of granulosa cells during the follicular development . Biochem Biophys Res Commun . 2015 ; 466 : 599 - 605 .
13. Zhu QC , Gao RY , Wu W , Guo BM , Peng JY , Qin HL . Effect of a high-fat diet in development of colonic adenoma in an animal model . World J Gastroenterol . 2014 ; 20 : 8119 - 29 .
14. Krishnamurthy J , Torrice C , Ramsey MR , Kovalev GI , Al-Regaiey K , Su L , Sharpless NE . Ink4a/Arf expression is a biomarker of aging . J Clin Invest . 2004 ; 114 : 1299 - 307 .
15. Campisi J , d'Adda di Fagagna F . Cellular senescence: when bad things happen to good cells . Nat Rev Mol Cell Biol . 2007 ; 8 : 729 - 40 .
16. Janzen V , Forkert R , Fleming HE , Saito Y , Waring MT , Dombkowski DM , Cheng T , DePinho RA , Sharpless NE , Scadden DT . Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a . Nature . 2006 ; 443 : 421 - 6 .
17. Signer RA , Montecino-Rodriguez E , Witte ON , Dorshkind K. Aging and cancer resistance in lymphoid progenitors are linked processes conferred by p16Ink4a and Arf . Genes Dev . 2008 ; 22 : 3115 - 20 .
18. Liu Y , Johnson SM , Fedoriw Y , Rogers AB , Yuan H , Krishnamurthy J , Sharpless NE . Expression of p16(INK4a) prevents cancer and promotes aging in lymphocytes . Blood . 2011 ; 117 : 3257 - 67 .
19. Molofsky AV , Slutsky SG , Joseph NM , He S , Pardal R , Krishnamurthy J , Sharpless NE , Morrison SJ . Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing . Nature . 2006 ; 443 : 448 - 52 .
20. Krishnamurthy J , Ramsey MR , Ligon KL , Torrice C , Koh A , Bonner-Weir S , Sharpless NE . p16INK4a induces an age-dependent decline in islet regenerative potential . Nature . 2006 ; 443 : 453 - 7 .
21. Zhang X , Zhou D , Strakovsky R , Zhang Y , Pan YX . Hepatic cellular senescence pathway genes are induced through histone modifications in a diet-induced obese rat model . Am J Physiol Gastrointest Liver Physiol . 2012 ; 302 : G558 - 64 .
22. Sherr CJ , Roberts JM . CDK inhibitors: positive and negative regulators of G1-phase progression . Genes Dev . 1999 ; 13 : 1501 - 12 .
23. Xiong Y , Hannon GJ , Zhang H , Casso D , Kobayashi R , Beach D. p21 is a universal inhibitor of cyclin kinases . Nature . 1993 ; 366 : 701 - 4 .
24. Sasaki Y , Tsujiuchi T , Murata N , Kubozoe T , Tsutsumi M , Konishi Y. Absence of p16, p21 and p53 gene alterations in hepatocellular carcinomas induced by N-nitrosodiethylamine or a choline-deficient L-amino acid-defined diet in rats . Cancer Lett . 2000 ; 152 : 71 - 7 .
25. Yoruker EE , Mert U , Bugra D , Yamaner S , Dalay N. Promoter and histone methylation and p16(INK4A) gene expression in colon cancer . Exp Ther Med . 2012 ; 4 : 865 - 70 .
26. Yoon A , Tammen SA , Park S , Han SN , Choi SW . Genome-wide hepatic DNA methylation changes in high-fat diet-induced obese mice . Nutr Res Pract . 2017 ; 11 : 105 - 13 .
27. Chen H , Pan YX , Dudenhausen EE , Kilberg MS . Amino acid deprivation induces the transcription rate of the human asparagine synthetase gene through a timed program of expression and promoter binding of nutrientresponsive basic region/leucine zipper transcription factors as well as localized histone acetylation . J Biol Chem . 2004 ; 279 : 50829 - 39 .
28. Chao J , Huo TI , Cheng HY , Tsai JC , Liao JW , Lee MS , Qin XM , Hsieh MT , Pao LH , Peng WH . Gallic acid ameliorated impaired glucose and lipid homeostasis in high fat diet-induced NAFLD mice . PLoS One . 2014 ; 9 : e96969 .
29. Sasidharan SR , Joseph JA , Anandakumar S , Venkatesan V , Madhavan CN , Agarwal A . Ameliorative potential of Tamarindus indica on high fat diet induced nonalcoholic fatty liver disease in rats . ScientificWorldJournal . 2014 ; 2014 : 507197 .
30. Ragab SM , Abd Elghaffar S , El-Metwally TH , Badr G , Mahmoud MH , Omar HM . Effect of a high fat, high sucrose diet on the promotion of nonalcoholic fatty liver disease in male rats: the ameliorative role of three natural compounds . Lipids Health Dis . 2015 ; 14 : 83 .
31. Wu J , Zhang H , Zheng H , Jiang Y. Hepatic inflammation scores correlate with common carotid intima-media thickness in rats with NAFLD induced by a high-fat diet . BMC Vet Res . 2014 ; 10 : 162 .
32. Deminice R , da Silva RP , Lamarre SG , Kelly KB , Jacobs RL , Brosnan ME , Brosnan JT . Betaine supplementation prevents fatty liver induced by a highfat diet: effects on one-carbon metabolism . Amino Acids . 2015 ; 47 : 839 - 46 .
33. Benatti RO , Melo AM , Borges FO , Ignacio-Souza LM , Simino LA , Milanski M , Velloso LA , Torsoni MA , Torsoni AS . Maternal high-fat diet consumption modulates hepatic lipid metabolism and microRNA- 122 (miR-122) and microRNA- 370 (miR-370) expression in offspring. Br J Nutr . 2014 ; 111 : 2112 - 22 .
34. Heden TD , Morris EM , Kearney ML , Liu TW , Park YM , Kanaley JA , Thyfault JP . Differential effects of low-fat and high-fat diets on fed-state hepatic triacylglycerol secretion, hepatic fatty acid profiles, and DGAT-1 protein expression in obese-prone Sprague-Dawley rats . Appl Physiol Nutr Metab . 2014 ; 39 : 472 - 9 .
35. Seet EL , Yee JK , Jellyman JK , Han G , Ross MG , Desai M. Maternal high-fat-diet programs rat offspring liver fatty acid metabolism . Lipids . 2015 ; 50 : 565 - 73 .
36. Jung CH , Cho I , Ahn J , Jeon TI , Ha TY . Quercetin reduces high-fat dietinduced fat accumulation in the liver by regulating lipid metabolism genes . Phytother Res . 2013 ; 27 : 139 - 43 .
37. Wang Y , Ziogas DC , Biddinger S , Kokkotou E. You deserve what you eat: lessons learned from the study of the melanin-concentrating hormone (MCH)-deficient mice . Gut . 2010 ; 59 : 1625 - 34 .
38. Coppe JP , Rodier F , Patil CK , Freund A , Desprez PY , Campisi J . Tumor suppressor and aging biomarker p16(INK4a) induces cellular senescence without the associated inflammatory secretory phenotype . J Biol Chem . 2011 ; 286 : 36396 - 403 .
39. Capparelli C , Chiavarina B , Whitaker-Menezes D , Pestell TG , Pestell RG , Hulit J , Ando S , Howell A , Martinez-Outschoorn UE , Sotgia F , Lisanti MP . CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancerassociated fibroblasts, "fueling" tumor growth via paracrine interactions, without an increase in neo-angiogenesis . Cell Cycle . 2012 ; 11 : 3599 - 610 .
40. Bernardes de Jesus B , Blasco MA . Assessing cell and organ senescence biomarkers . Circ Res . 2012 ; 111 : 97 - 109 .
41. Di Croce L , Helin K. Transcriptional regulation by Polycomb group proteins . Nat Struct Mol Biol . 2013 ; 20 : 1147 - 55 .
42. Simon JA , Kingston RE . Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put . Mol Cell . 2013 ; 49 : 808 - 24 .
43. Hon GC , Hawkins RD , Ren B . Predictive chromatin signatures in the mammalian genome . Hum Mol Genet . 2009 ; 18 : R195 - 201 .
44. Stein GH , Drullinger LF , Soulard A , Dulic V . Differential roles for cyclindependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts . Mol Cell Biol . 1999 ; 19 : 2109 - 17 .
45. Pinskaya M , Morillon A . Histone H3 lysine 4 di-methylation: a novel mark for transcriptional fidelity? Epigenetics . 2009 ; 4 : 302 - 6 .
46. Guillemette B , Drogaris P , Lin HH , Armstrong H , Hiragami-Hamada K , Imhof A , Bonneil E , Thibault P , Verreault A , Festenstein RJ . H3 lysine 4 is acetylated at active gene promoters and is regulated by H3 lysine 4 methylation . PLoS Genet . 2011 ; 7 : e1001354 .
47. LaPak KM , Burd CE . The molecular balancing act of p16(INK4a) in cancer and aging . Mol Cancer Res . 2014 ; 12 : 167 - 83 .
48. Healy ME , Chow JD , Byrne FL , Breen DS , Leitinger N , Li C , Lackner C , Caldwell SH , Hoehn KL . Dietary effects on liver tumor burden in mice treated with the hepatocellular carcinogen diethylnitrosamine . J Hepatol . 2015 ; 62 : 599 - 606 .
49. Saxena NK , Fu PP , Nagalingam A , Wang J , Handy J , Cohen C , Tighiouart M , Sharma D , Anania FA . Adiponectin modulates C-Jun N-terminal kinase and mammalian target of rapamycin and inhibits hepatocellular carcinoma . Gastroenterology . 2010 ; 139 : 1762 - 73 . 1773 e1761- 1765
50. Serra MP , Marongiu F , Sini M , Marongiu M , Contini A , Wolff H , Rave-Frank M , Krause P , Laconi E , Koenig S. Hepatocyte senescence induced by radiation and partial hepatectomy in rat liver . Int J Radiat Biol . 2014 ; 90 : 876 - 83 .
51. Aini W , Miyagawa-Hayashino A , Ozeki M , Adeeb S , Hirata M , Tamaki K , Uemoto S , Haga H . Accelerated telomere reduction and hepatocyte senescence in tolerated human liver allografts . Transpl Immunol . 2014 ; 31 : 55 - 9 .
52. Toshima T , Shirabe K , Fukuhara T , Ikegami T , Yoshizumi T , Soejima Y , Ikeda T , Okano S , Maehara Y. Suppression of autophagy during liver regeneration impairs energy charge and hepatocyte senescence in mice . Hepatology . 2014 ; 60 : 290 - 300 .