The effects of a diet with high fat content from lard on the health and adipose-markers’ mRNA expression in mice

Abstract

Pork is one type of the most frequently consumed meat with about 30% globally. Thus, the questions regarding to the health effects of diet with high fat content from lard are raised. Here, we developed a model of mice fed with high fat (HF) from lard to investigate and have more insights on the effects of long-time feeding with HF on health. The results showed that 66 days on HF induced a significant gain in the body weight of mice, and this weight gain was associated to the deposits in the white fat, but not brown fat. The glucose tolerance, not insulin resistance, in mice was decreased by the HF diet, and this was accompanied with significantly higher blood levels of total cholesterol and triglycerides. Furthermore, the weight gains in mice fed with HF seemed to link to increased mRNA levels of adipose biomarkers in lipogenesis, including Acly and Acaca genes, in white fat tissues. Thus, our study shows that a diet with high fat from lard induced the increase in body weight, white fat depots’ expansion, disruption of glucose tolerance, blood dyslipidemia, and seemed to start affecting the mRNA expression of some adipose biomarkers in a murine model.

Full text

The effects of a diet with high
fat content from lard on the
health and adipose-markers’
mRNA expression in mice
Dinh-Toi Chu
1,2
, Hue Vu Thi
1,2
,
Nhat-Le Bui
1,2
and Ngoc-Hoan Le
3
1
Center for Biomedicine and Community Health, International School,
Vietnam National University, Hanoi, Vietnam
2
Faculty of Applied Sciences, International School, Vietnam National
University, Hanoi, Vietnam
3
Faculty of Biology, Hanoi National University of Education, Hanoi,
Vietnam
Abstract
Pork is one type of the most frequently consumed meat with about 30% globally. Thus, the ques-
tions regarding to the health effects of diet with high fat content from lard are raised. Here, we
developed a model of mice fed with high fat (HF) from lard to investigate and have more insights
on the effects of long-time feeding with HF on health. The results showed that 66 days on HF
induced a significant gain in the body weight of mice, and this weight gain was associated to the
deposits in the white fat, but not brown fat. The glucose tolerance, not insulin resistance, in
mice was decreased by the HF diet, and this was accompanied with significantly higher blood levels
of total cholesterol and triglycerides. Furthermore, the weight gains in mice fed with HF seemed
to link to increased mRNA levels of adipose biomarkers in lipogenesis, including Acly and Acaca
genes, in white fat tissues. Thus, our study shows that a diet with high fat from lard induced
the increase in body weight, white fat depots’expansion, disruption of glucose tolerance, blood
dyslipidemia, and seemed to start affecting the mRNA expression of some adipose biomarkers
in a murine model.
Keywords
High fat diet, lard, weight gains, white fat, glucose tolerance, blood dyslipidemia, adipose-markers,
mRNA expression
Corresponding author:
Dinh-Toi Chu, Center for Biomedicine and Community Health, International School, Vietnam National
University, Hanoi, Vietnam; and Faculty of Applied Sciences, International School, Vietnam National University,
Hanoi, Vietnam.
Emails: chudinhtoi.hnue@gmail.com, toicd@vnu.edu.vn
Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative
Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/)
which permits non-commercial use, reproduction and distribution of the work without further permission provided the original
work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage).
Original Research Article SCIENCE PROGRESS
Science Progress
2024, Vol. 107(3) 1–16
© The Author(s) 2024
Article reuse guidelines:
sagepub.com/journals-permissions
DOI: 10.1177/00368504241269431
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Introduction
A high fat (HF) is described as a diet containing lipids that contribute to over 30% of the
total calorie intake, and it may be up to 50%, according to some current research.
1
US
data for the period from 1908 to 1985 showed that the proportion of total energy
intake from carbohydrates fell from 57% to 46%, while the total calorie intake from
fats grew from 32% to 43%.
2
The trends for carbohydrate and fat consumption were
still maintained in the following years, which reached 35% and 45%, respectively.
2
Various physical health consequences are linked to HF such as obesity, diabetes, intes-
tinal diseases, autoimmune disorders, and several types of cancer.
3,4
An HF-induced
imbalance between energy intake and expenditure causes a redundant accumulation of
fat in white adipose tissue (WAT), which may lead to obesity and diabetes.
5
The exces-
sive intake of saturated fats, including a minute amount of omega-3 polyunsaturated fatty
acids (PUFAs) and a considerable amount of omega-6 PUFAs is associated with the
abnormality of metabolism.
2
HF could also induce abnormal liver tissue, diversity loss
of intestinal microbiota and inhibit intestinal development.
6
In terms of autoimmune dis-
orders such as systemic lupus erythematosus, the association between diet and their risk
was suggested around 50 years ago.
7
Moreover, HF aggravates prostate cancer, pancre-
atic cancer and colitis-related carcinogenesis.
8–10
The burden of HF is undeniably immense in mental health, economy and environment.
HF-induced obesity also has a notable association with attention deficit hyperactivity dis-
order, bipolar disorder, depression and substance abuse.
11
Furthermore, weight stigma
was negatively related to self-esteem and positively related to body image dissatisfaction,
anxiety and eating disturbances.
12
Obesity-induced stress caused by HF accelerates hair
thinning by targeting hair follicle stem cells.
13
Obesity also aggravates sebaceous glands
causing acne, increased skin infection incidence and premature hair graying.
14
The
medical costs for treating such diseases in the USA are expected to reach $66 billion
in 2030.
3
Obese patients perform less effectively at work due to their low life expectancy
and physical limitations.
3
Productivity loss caused by hospitalization and low life expect-
ancy was estimated to be $74 million and $444 million in the USA, respectively.
3
The
consequences of HF disease have also been proven for the environment. A study has
shown diets in the Xinjiang Autonomous Region, which includes high carbohydrates
and fats intake were associated with high ecological footprint, high water footprint and
high carbon footprint.
15
Pork is one of the most frequently consumed meats, with about 30% globally. In lard,
we can find about 46% monounsaturated (MUFAs), about 37% saturated (sFA), and
about 17% polyunsaturated (PUFAs). Lard contains extremely high levels of oleic acid
(44%), palmitic acid (27%) and stericand oleic acid (11%).
16
Because of the composition,
lard has been recommended and a commonly used as fat source in metabolic studies on
mice. These diets have been particularly effective in mimicking the metabolic effects of
the human diet due to the significant proportion of lard consumption in human diets in the
world. Notably, Vietnam ranked fifth among the top 10 largest pork consumers in 2021 of
the world.
17
This issue raises the question of how over-eating fat from pork lard affects
health and how we can control this problem in the countries that consume the most pork
2Science Progress 107(3)

particularly and globally. To set the light on the effect of HF from pork fat on health, it is
necessary to develop animal models to sufficiently mimic all characteristics of human
disease. Among prevalent animal models, mice are widely applied to explore HF
impacts due to reasonable prices and faster growth with less feed consumption.
18
Therefore, murine HF has been used to model type-2 diabetes mellitus, wound
healing, Alzheimer’s, aging, dysbiosis and prostate disease.
18–22
To investigate and
have more insights on the effects of longtime-feeding with HF on the health, we have
developed a model of mice fed with HF from pork lard in Vietnam. The effect of the
HF has been comprehensively evaluated through mouse and fat tissues weight, glucose
tolerance and insulin resistance, blood lipids and liver enzymes, and adipose biomarkers
of lipogenesis.
Methods
Animal models
The 6-week-old male Swiss mice or other suitable strains were purchased from the
National Institute of Hygiene and Epidemiology and raised at the animal laboratory at
the Center for Biomedicine and Community Health, VNU-International School. The pro-
cedure for building animal models has been described in detail in our previous
studies.
23,24
Six-week-old mice were kept in a cage and adaptably housed at room tem-
perature and fed ad libitum with a standard diet for acclimatization until starting of the
experiment at the 8 weeks of age. Our study adhered to institutional guidelines concern-
ing the care and utilization of laboratory animals.
Then, mice were guaranteed to eat 6 g of food/mouse/day during the protocol. Mice
were divided into two groups through simple randomization method by lottery. Each
group has 18–19 mice and 3–4 mice were raised per cage. One group was fed the standard
diet (STD) with 5.53% kcal of fat, and the other was provided a HF with 60% kcal from
fat. In the STD, 5.53% kcal of fat was included totally in 6 g of standard rodent pellets/
mouse/day purchased from the National Institute of Hygiene and Epidemiology. On the
other hand, the fat in the HF is obtained from pork hard fat. It was washed, boiled,
chopped, and mixed with standard rodent pellets. Specifically, HF group was fed
2.55 g of standard rodent pellets and 3.45 g of lard/mouse/day to ensure a ratio of 60%
kcal of fat. Throughout the protocol, mice were always housed at 25 °C with a 12-h
light/dark cycle. The mouse cage has been cleaned daily by replacing the coop liner
with sterilized and dried sawdust. The mouse’s weight was monitored using the
SF400D scale (minimum division 0.01 g) every two days. When any mouse with abnor-
mal signs was detected, it would be separated into new cages to avoid affecting to other
animals.
The experiment ended on the 66th day of STD and HF feeding. The mice were sacri-
ficed to collect the tissues and blood for further analyses. The inguinal (ING WAT) and
epididymal (eWAT) white fat tissues, and interscapular brown adipose tissue (iBAT)
were collected, kept in 1.5 ml Eppendorf tubes and weighed, then were instantly
frozen in liquid nitrogen and stored at −80 °C until total RNA isolation.
Chu et al. 3

Glucose tolerance test (GTT) and insulin tolerance tests (ITT)
GTT and ITT were performed after the mice had fasted for 12 h, as described.
24
For
GTT, we diluted 30% glucose solution to 20% glucose solution with distilled water,
then intraperitoneal injection of 20% glucose solution was used following the 2 g/kg
body weight ratio. After GTT 2 days, mice were conducted ITT, the 100 IU/ml
insulin solution was diluted 1000 times to form a 0.1 IU/ml insulin solution, then
mice were injected with intraperitoneal insulin at 0.75 IU/kg of body weight. Blood
glucose levels were measured using an On Call Plus II Blood Glucose Meter (ACON
Laboratories, Inc., USA) with five-time points: before injection, 15 min, 30 min, 90 min,
and 120 min after injection.
Measurement of blood lipids and liver enzymes
Fasting blood samples from the heart of each mouse were collected. We cut the artery
of the mouse’s heart and used a 5 ml syringe to suck the blood and put it in a Heparin
tube. Plasma was obtained by centrifuging a blood sample immediately after collec-
tion. It was then stored at −80 °C until blood lipids and liver enzymes were tested.
Triglyceride (TRI), Total Cholesterol (CHO), High-Density Lipoprotein-cholesterol
(HDL), and Low-Density Lipoprotein-cholesterol (LDL) were determined to assess.
Liver function has been evaluated through indicators such as glutamic oxaloacetic
transaminase (GOT) and glutamic pyruvic transaminase (GPT). All the above analyses
were performed using an automated blood analyzer (Type Architect C8000; Abbott
Ltd, USA) at the Genome Sciences Technology And Services Company Limited,
Hanoi, Vietnam.
RNA isolation and QRT-PCR
Total RNA was isolated from frozen fat tissues by the QIAamp viral RNA mini Kit
(Qiagen, 52906). The total RNA’s absorbance qualities and purification levels were
assessed using a NanoDrop spectrophotometer. The ratio of absorbance at 260 nm
versus 280 nm (260/280), and 260 nm versus 230 nm (260/230), as well as the total
RNA concentration (ng/μl) were recorded. Total RNA was used to perform
Quantitative reverse transcription-PCR (qRT-PCR). We used the TaqMan probe for
Acly and Acaca genes available from previous studies upon request. Acly and Acaca
can be considered as biomarkers of adipose tissue because it directly involved in
fatty acid synthesis, an important process in adipose tissue.
23,25,26
All mRNA expres-
sion data has been normalized by using the cyclophilin as the reference gene.
Data analysis
The data was analyzed by STATA 14 (StataCorp LLC, Texas, USA) and GraphPad Prism
8.0. It was expressed as mean ±SEM. Student’st-test and two-way ANOVA tests were
used for single comparisons and multiple comparisons, respectively. Significance was
accepted at p< 0.05 and not significant (n.s.) as p≥0.05.
4Science Progress 107(3)

Results
Effect of the high fat diet on mouse body weight
Eight-week-old mice were fed with HF or STD in 66 days to check the effect of HF on the
health and mRNA expression of some adipose biomarkers of lipogenesis. The amount of
food consumed by the two groups did not differ during the experiment. The results
showed a significant increase in mouse body weight in the HF group but almost no
change in the STD group. In particular, the mice’s body weight increased rapidly in
the first 30 days in response to HF, then continued to increase slightly until the 66th
day. On the opposite side, the body weight was slightly decreased but not statistically
in the group of mice fed with STD. When comparing the body weight of mice in the
two groups (Figure 1(a)), our data indicated that body weight did not significantly
differ between the two groups at the start of the experiment (Day 0). Thereafter, body
weight was significantly higher at HF than STD on test days 10, 30, and 66 at 6.45(g),
9.97(g), and 12.79(g) body weight, respectively. Obviously, we calculated the changes
in body weight of mice by the diets at the experimental time points versus the day of start-
ing (Day 0) (Figure 1(b)), the data clearly shows that the mice body weight was signifi-
cantly changed by HF, up to the peak of 9.27 (g) at the day 30, then still high and reached
10.23 (g) at the end of the experiment, but the STD did not seem to make the significantly
changes in animal bodyweight. Thus, the current data show that after 66 days fed with HF
in our laboratory condition, mice’s weight was significantly elevated compared to those
fed with STD, and also versus the beginning of experiment.
Effect of the high fat diet on the weight of fat tissues
One of the most lipid-accumulative tissues in animals is white adipose tissue. We have
weighed the fat tissues of mice fed by STD and HF (Figure 2). The current data
showed that the HF affected the weight accumulation of the WAT, but not BAT.
Particularly, the ING WAT and eWAT were significantly increased by HF compared
to STD by about 15% and 30%, respectively. The increase in ING WAT and eWAT
seems to reflect the increase in the body weight of animals by HF. On the other hand,
the data clearly indicate that the HF did not significantly change the weight of interscapular
brown adipose tissue (iBAT). Therefore, this result proved that the HF made the accumu-
lation in the body weight when a significant increase in white adipose tissue appears.
Effect of high fat diet on glucose tolerance and insulin resistance
One of the health effects of the longtime eating of high fat-contained-food is the induction
of the disruption of the lipid and glucose metabolism with the initial signs of dysfunction
of glucose tolerance and insulin resistance. We have conducted GTT and ITT for the two
groups of animals fed with STD or HF for 66 days (Figure 3). As expected, the HF sig-
nificantly affected the glucose tolerance of the experimental mice, that is the HF-fed-mice
had the higher blood glucose levels in 15, 30, and 90 min compared to the STD group
after GTT test. After that, blood glucose levels cannot return to normal levels as seen
Chu et al. 5

in STDs at the end of the GTT test (120 min) (Figure 3(a) and (b)). But unexpectedly, we
did not see any difference between HD and STD in the glucose levels in the mouse blood
after insulin injection during the ITT test (Figure 3(c) and (d)), as we found in previous
work when we fed B6 mice with a HF diet for 154 days (22 weeks).
24
In that report,
Figure 1. Effect of the high fat diet on the body weight of mice. Adult mice at eight weeks of age
were fed with HF or STD for 66 days. A. Body weight of mice was measured every two
days. B. The mouse body weight was compared between HF and STD groups at the selective time
points (0, 10, 20, 20, 40, 50, and 66 days from starting day of the experiment –Day 0). C. The
changes in mouse body weight every two days compared to Day 0 in each mouse group. D. The
heat map representing the changes in mouse body weight in HF and STD groups at each time point
during the course of experiment. Student’s t-test was performed as the statistical test. (n =18–19
mice per group).
6Science Progress 107(3)

154 days on HF increased the blood glucose levels in the ITT test compared to the
STD-fed-mice group.
24
So this result means that mice had reduced glucose tolerance
for 66 days in HF but were not induced with insulin resistance.
Effects of the high fat diet on the blood lipids and liver enzymes
Furthermore, we looked more closely at the effects of high fat feeding on the lipid metab-
olism of animals by checking profiles of the lipids and liver enzymes in the blood of
experimental mice in two groups (Figure 4). In 66 days of eating the food with high
content of fat, the levels of blood lipids were changed selectively (Figure 4(a)).
Specifically, we saw that CHO and TRI levels were significantly elevated by the high
fat feeding, which was clearly higher than those in the standard diet group (P< 0.01).
However, the 66 days in HF only slightly increased the blood levels of HDL and LDL,
and this increase was not statistical significance. Furthermore, the plasma levels of GOT
and GPT were not different between mice in HF versus STD groups (Figure 4(b)).
Effects of the high fat diet on adipose biomarkers of lipogenesis
In this study, we deeply investigated if the longtime-eating-high fat affected the mRNA
expression of the adipose markers related to the lipid metabolism, and we selected to
Figure 2. Effect of the high fat diet on the weight of fat tissues. The mice were sacrificed at the
end of the experiment –day 66. The ING, eWAT, and iBAT were collected from every mouse in
both STD and HF groups, then the tissues were quickly weighed to compare the effects of diet.
Student’s t-test was performed as the statistical test. (n =18–19 mice per group).
Chu et al. 7

check the mRNA levels of two important lipogenic genes including ATP Citrate Lyase
(Acly) and Acetyl-CoA Carboxylase Alpha (Acaca) in white and brown fat tissues of
the mice in two groups. We found that the effects of HF on mRNA levels of Acly and
Acaca genes in fat tissues (Figure 5) seems to reflect those effects on the changes on
the fat tissues and animals’body weight (Figure 2). That is, except the mRNA level of
Acaca in eWAT, the high fat feeding significantly increased the mRNA levels of the
Acly and Acaca genes in the white adipose tissues including ING WAT (Figure 5(b))
and eWAT (Figure 5(c)) (p< 0.05). While the p-value indicates no statistical significance,
Figure 3. Effect of the high fat diet on glucose and insulin tolerance. Before finishing experiments
4 and 2 days, we have done the GTT and ITT. A. The glucose levels in mouse blood in two diet
groups were measured at 0, 15, 30, 90, and 120 min after glucose injection. B. The comparison of
blood glucose levels in HF versus STD group at the indicated time points in GTT. C. The blood
glucose levels of mice in two diet groups were measured at 0, 15, 30, 90, and 120 min after insulin
injection in ITT. D. Blood glucose levels in HF-fed mice were compared to those of the STD group
at the indicated time points in ITT. Student’s t-test was performed as the statistical test. (n =18–19
mice per group).
8Science Progress 107(3)

Figure 4. Effects of the high fat diet on the blood lipids and on liver enzymes. The mouse blood
was collected from each mouse in both groups at the end of the experiment to check plasma lipids
and liver enzyme levels. A. The levels of 4 types of most important blood lipids, including CHO and
TRI, HDL and LDL in mice fed with HF versus STD. B. The levels of plasma liver enzymes –GOT
and GPT in HF versus STD group. Student’s t-test was performed as the statistical test. (n =18–19
mice per group).
Figure 5. Effects of the high fat diet on adipose biomarkers of lipogenesis. The mRNA levels of
selective adipose biomarkers functioning on the lipogenesis were quantitated by RT-PCR in the fat
tissues collected from mice at the end of experiment. A. mRNA levels of Acly and Acaca in iBATof
HF versus STD group. B. mRNA levels of Acly and Acaca in the ING of HF versus STD group. And
C. mRNA levels of Acly and Acaca in eWAT of HF versus STD group. Student’s t-test was
performed as the statistical test. (n =7 mice per group).
Chu et al. 9

the mean differences observed suggest a potentially meaningful biological effects in
mRNA levels in the classical brown fat tissues (iBAT) (n.s.) (Figure 5(a)). This result
suggests that the longtime on HF effects not only on the changes in body weight,
white fat depots’expansion, the ability of glucose tolerance, the blood lipids’levels,
but also affect the mRNA expression of the adipose biomarkers.
Discussion
Nowadays, the prevalence of metabolic disorders is increasing day by day.
27
Obesity has
been considered a global health problem and a major risk factor for chronic diseases and
cancer.
28
Thus, scientists have been trying to generate mouse models to elucidate the
influence of HF on mouse development, especially on the physiological mechanisms
of obesity. Although this disease is related to genetic factors, it is largely caused by
diet and exercise habits.
28
A series of tests have consistently shown that there was a dif-
ference in weight between HF-fed mice and standard-fed mice after follow-up time-
lines.
29–31
The increase in body weight in mice with HF was thought to be between
10% and 20% of standard-feeding mice. However, the change in weight between each
group depends on the fat composition of each diet.
32,33
The researchers also demonstrated
that weight changes were associated with different strains of mice when it was fed by
HF.
33
On the contrary, some scientists have asserted that HFs do not cause weight
gain if they do not exceed the threshold of total daily calories, even if those mice had
up to 90% of their total calories coming from HF.
34
The reason for this mechanism is
due to the homeostasis and pleasure system of the brain. In our laboratory condition,
we found that the 66 days fed with HF the weight of mice was significantly higher in com-
parison to the STD, and the weight gain of mice in HF group was reached at around 10 (g)
versus the beginning of the experiment.
Based on the function, adipose tissues are classified into white and thermogenic fat
tissues. The white adipose tissues (WAT) function to store energy as lipids and locates
along the body especially under the skin such as the inguinal white adipose tissues
(ING WAT), and around or in visceral such epididymal white adipose tissue (eWAT),
perirenal white adipose tissue (prWAT), retroperitoneal white adipose tissue (rWAT),
and mesenteric white adipose tissue (mWAT).
35
But the thermogenic fat tissues function
to burn lipids into heat, and they include interscapular brown adipose tissue (iBAT), and
brite/beige adipose tissues inside the white fat deports.
36
Thus, in this work we examined
the effect of longtime-feeding by a HF from lards on the weight of the ING WAT, eWAT
and iBAT in mice, because the ING WAT and eWAT are typical white fat tissues, and
iBAT is typical thermogenic fat tissues, and we found that the HF diet significantly
increased the expansion of ING WAT and eWAT, but not iBAT, this is continent with
the increase in the bodyweight of mice induced by the HF (Figure 2).
Moreover, we further found that the significant increase in the body weight of mice by
HF was accompanied with the dysfunction glucose tolerances, and the elevation in the
weight of white fat tissues but not classical brown fat. Lipid metabolism involves the bio-
synthesis of lipid particles like fatty acids, cholesterol, or triglycerides as well as their
degradation. HDL is known for its function in removing excess cholesterol, and LDL
is related to plaque formation. CHO, HDL, and LDL are commonly utilized to assess
10 Science Progress 107(3)

overall lipid metabolism and cardiovascular risk, while TRI is a key indicator of lipid
metabolism. Thus, these four indexes are the most important and popular indicators to
predict the detrimental effects of HF on lipid metabolism and health
37,38
According to
Li et al. (2020), mice feeding with HF had dramatically higher levels of serum TRI,
CHO, and LDL, while HDL was substantially lower than those in the STD group.
39
Consistent results from similar studies were indicated that dyslipidemia was often accom-
panied by HF and obesity.
40
HF also accelerates the excessive accumulation of lipid dro-
plets and oxidase stress by breaking the lipid synthesis and decomposition balance. We
observed similar results with Li et al. (2020), in which longtime fed with HF from pork
hard fat remarkably increased TRI and CHO levels, suggesting dyslipidemic induction.
Moreover, the elevated TRI is associated with a higher risk of atherosclerosis, occurring
when fatty deposits build up in the walls of arteries, and restrict blood stream.
41
The ele-
vated CHO contributes to plaque formation in arteries, increasing the risk of coronary
heart disease.
42
Thus, a long intake of saturated fats, like pork hard fat, which is one
of the main worldwide food sources, may lead to a higher risk of several cardiovascular
consequences and metabolic syndromes. However, we saw only slight changes in the
blood levels of HDL and LDL, this may be due to the differences in experimental con-
ditions and times of feeding HF, and origins of fat content between laboratories.
Another hypothesis is that an adaptive metabolic response may occur and moderate
the changes in LDL and HDL.
It has been reported that HF promotes liver dysfunction by regulating the plasma
level and activity of liver enzymes. HF negatively affected liver function in C3 h mice
treated with dietondiethylnitrosamine, as indicated by increased alkaline phosphatase
(ALP), GPT, and GOT plasma levels and increased γ-glutamyltransferase activity.
43
Matsumoto M. et al. stated that HF increased serum GPT in male A/J mice and
C57BL/6J mice.
44
Hamsters fed by HF also had significantly higher GOT and GPT
plasma levels.
45
Since GOT and GPT are present in hepatic cells, the increased plasma
level of GOT and GPT is one of the earliest signals of hepatic injury. However, unexpect-
edly, the plasma levels of GOT and GPT were not affected by 66 days of feeding a diet
with high fat content from lard in our experiment. This finding can be explain by HF can
affect liver function, however this change may not occur immediately. Since the 66-day
duration of HF may not be long enough, the liver is thought to be temporarily able to
adapt to changes in fat metabolism without causing any significant changes in liver
enzyme levels.
The changes in the expression level of lipogenesis genes are thought to be directly
related to diet-induced obese mice. Acaca is a paramount gene in controlling fatty acid
synthesis, while Acly has been shown to highly affect histone acetylation and adipocyte
differentiation.
46
In our HF-fed-animals, we found that the mRNA levels of Acly and
Acaca genes in the white adipose tissues, including ING WAT and eWAT were elevated
compared to the STD group. Voigt et al. (2013) indicated that HF could elevate the
expression of both Acaca and Acly in eWAT, which was consistent with our results.
47
Another similar study suggested that age and sex could be responsible for the difference
in gene expression level of adipogenesis gene, in which Acaca expression level in HF-fed
females was significantly higher than in males, thus leading to higher metabolic activity
and lower risk of obesity.
48
However, controversial results were still reported. When
Chu et al. 11

investigating the mRNA expression levels of several genes related to fatty acid synthesis
like Acaca and Scd1 among HF mice, scientists found that the gene expression of Scd1
was even significantly higher in their offspring compared to those of STD mice while this
trend was not found in Acaca.
49
Besides, results from Jian Dong et al. (2024) indicated
that both mRNA and protein expression of Acaca in hepatic cells were increased in
response to the HF diet, right after 2 weeks of fat intake. The authors also reported
that the lipid accumulation process, especially the plasma level of TG and CHO, and
mitochondrial function were declined by inhibiting Acaca via in vitro model. Thus,
Acaca was considered as a potential target for therapeutic development in obesity and
non-alcoholic fatty liver disease.
50
On the other hand, we found that there was no statis-
tically significant difference in gene expression in iBAT, but the mean differences
observed suggest a potentially meaningful biological effect. Future studies with larger
sample sizes are suggested to shed more light on this finding.
Although our study has shed light demonstrate that a high-fat diet containing lard leads
to an increase in body weight, expansion of white fat depots, disruption of glucose toler-
ance, blood dyslipidemia, and appears to initiate alterations in the mRNA expression of
certain adipose biomarkers in a murine model. However, our study still has limitations
that need to be considered. Firstly, we selected the number of mice without using a spe-
cific sample size calculation formula. However, we chose the sample size based on build-
ing animal models in the previous study.
23,24
Secondly, the experiment lasted for 66 days.
Although this period allows for the observation of certain metabolic changes, extended
studies might be required to comprehensively understand the long-term effects of
dietary interventions on metabolism and health outcomes.
Conclusion
In this study, we performed a mice model to clarify the effect of a high-fat diet form lard
on several indexes. The current data suggest that the 66 days exposed to a HF diet induced
the increase in body weight, white fat depots’expansion, disruption of glucose tolerance,
blood dyslipidemia with the total cholesterol and triglycerides, and seemed to start affect-
ing the mRNA expression of some adipose biomarkers in a mouse model. However, we
observed no significant changes in iBAT, insulin resistance, other blood lipid indexes
like HDL and LDL, and hepatic function. Future researches should deeply delve into
explaining the underlying mechanisms of these above observations. Longitudinal
studies with extended dietary interventions and larger sample sizes may unveil
nuanced metabolic changes and shed light on the temporal progression of metabolic
dysfunction. In conclusion, a long-term intake of high fat from lard had a detrimental
effect on health and a potential induction of the changes in the expression levels of lipo-
genesis genes in mice.
Acknowledgments
We would like to thank Ms. Mai Vu Ngoc Suong, Thuy Duong Vu, and other members at Center
for Biomedicine and Community Health members for contributing to some experiments, collecting
some parts of data, and checking to improve the manuscript.
12 Science Progress 107(3)

Consent for publication
Not applicable.
Credit authorship contribution statement
Dinh Toi Chu: Conceptualization, Methodology, Analysis, Investigation, Validation, Visualization,
Supervision, Project administration, Funding acquisition, Writing –review & editing; Hue Vu Thi:
Methodology, Analysis, Investigation, Validation, Visualization, Writing –review & editing; Nhat
Le Bui: Hue Vu Thi: Methodology, Analysis, Investigation, Validation, Visualization, Writing –
review & editing; Ngoc Hoan Le: Methodology, Analysis, Investigation, Validation, Visualization,
Supervision, Writing –review & editing.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship,
and/or publication of this article.
Ethical approval
This study was approved by the ethics committees of the VAST Institute of Genome Research
(No. 02-2020/NCHG-HĐĐĐ).
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or
publication of this article: This research is funded by Vietnam National Foundation for Science and
Technology Development (NAFOSTED) under grant number 106.02-2019.314.
ORCID iD
Dinh-Toi Chu https://orcid.org/0000-0002-4596-2022
References
1. Qiao J, Wu Y and Ren Y. The impact of a high fat diet on bones: potential mechanisms. Food
Funct 2021; 12: 963–975.
2. Malesza IJ, Malesza M, Walkowiak J, et al. High-fat, western-style diet, systemic inflamma-
tion, and gut microbiota: a narrative review. Cells 2021; 10. doi:10.3390/cells10113164.
3. Chu DT, Minh Nguyet NT, Dinh TC, et al. An update on physical health and economic
consequences of overweight and obesity. Diabetes Metab Syndr 2018; 12: 1095–1100.
4. Chu DT, Nguyet NTM, Nga VT, et al. An update on obesity: mental consequences and psy-
chological interventions. Diabetes Metab Syndr 2019; 13: 155–160.
5. Seo DC, Choe S and Torabi MR. Is waist circumference >/=102/88 cm better than body mass
index >/=30 to predict hypertension and diabetes development regardless of gender, age group,
and race/ethnicity? Meta-analysis. Prev Med 2017; 97: 100–108.
6. Wang J, Cheng R, Luo Y, et al. High-fat diet induces metabolic syndrome in mice and its influ-
ence on intestinal development, liver function and intestinal microbiota. Wei Sheng Yan Jiu
2021; 50: 93–99.
7. Kono M, Nagafuchi Y, Shoda H, et al. The impact of obesity and a high-fat diet on clinical and
immunological features in systemic lupus erythematosus. Nutrients 2021; 13. doi:10.3390/
nu13020504.
Chu et al. 13

8. Matsushita M, Fujita K, Hatano K, et al. High-fat diet promotes prostate cancer growth through
histamine signaling. Int J Cancer 2022; 151: 623–636.
9. Zhang X, Li W, Ma Y, et al. High-fat diet aggravates colitis-associated carcinogenesis by
evading ferroptosis in the ER stress-mediated pathway. Free Radic Biol Med 2021; 177:
156–166.
10. Garcia DI, Hurst KE, Bradshaw A, et al. High-fat diet drives an aggressive pancreatic cancer
phenotype. J Surg Res 2021; 264: 163–172.
11. Avila C, Holloway AC, Hahn MK, et al. An overview of links between obesity and mental
health. Curr Obes Rep 2015; 4: 303–310.
12. Wu YK and Berry DC. Impact of weight stigma on physiological and psychological health out-
comes for overweight and obese adults: a systematic review. J Adv Nurs 2018; 74: 1030–1042.
13. Morinaga H, Mohri Y, Grachtchouk M, et al. Obesity accelerates hair thinning by stem cell-
centric converging mechanisms. Nature 2021; 595: 266–271.
14. Hirt PA, Castillo DE, Yosipovitch G, et al. Skin changes in the obese patient. J Am Acad
Dermatol 2019; 81: 1037–1057.
15. Yin J, Yang D, Zhang X, et al. Diet shift: considering environment, health and food culture. Sci
Total Environ 2020; 719: 137484.
16. Rendina-Ruedy E and Smith B. Methodological considerations when studying the skeletal
response to glucose intolerance using the diet-induced obesity model. Bonekey Rep 2016; 5.
doi:10.1038/bonekey.2016.71.
17. Estimated Pork Consumption around the World for 2021 and 2022, https://www.pig333.com/
latest_swine_news/estimated-pork-consumption-around-the-world-for-2021-and-2022_18160/
(accessed 1 January 2023).
18. Li J, Wu H, Liu Y, et al. High fat diet induced obesity model using four strains of mice:
Kunming, C57BL/6, BALB/c and ICR. Exp Anim 2020; 69: 326–335.
19. Buettner R, Parhofer KG, Woenckhaus M, et al. Defining high-fat-diet rat models: metabolic
and molecular effects of different fat types. J Mol Endocrinol 2006; 36: 485–501.
20. Li W, Zhang K and Yang H. Pectin alleviates high fat (lard) diet-induced nonalcoholic fatty
liver disease in mice: possible role of short-chain fatty acids and gut Microbiota regulated
by pectin. J Agric Food Chem 2018; 66: 8015–8025.
21. Catta-Preta M, Martins MA, Cunha Brunini TM, et al. Modulation of cytokines, resisting, and
distribution of adipose tissue in C57BL/6 mice by different high-fat diets. Nutrition 2012; 28:
212–219.
22. Heydemann A. An overview of murine high fat diet as a model for type 2 diabetes Mellitus.
J Diabetes Res 2016; 2016: 2902351.
23. Chu DT, Truong DT, Thi HV, et al. Adipogenesis of ear mesenchymal stem cells (EMSCs):
adipose biomarker-based assessment of genetic variation, adipocyte function, and brown/
brite differentiation. Mol Cell Biochem 2022; 477: 1053–1063.
24. Chu D-T, Malinowska E, Jura M, et al. C57BL/6J mice as a polygenic developmental model of
diet-induced obesity. Physiol Rep 2017; 5: 20.
25. Nikonova L, Koza Ra Fau-Mendoza T, Mendoza T, et al. Mesoderm-specific transcript is asso-
ciated with fat mass expansion in response to a positive energy balance. FASEB J 2008; 22:
3925–3937.
26. Chu DT, Malinowska E, Gawronska-Kozak B, et al. Expression of adipocyte biomarkers in
a primary cell culture models reflects preweaning adipobiology. JBiolChem2014; 289:
18478–18488.
27. Tanaka M and Itoh H. Hypertension as a metabolic disorder and the novel role of the gut. Curr
Hypertens Rep 2019; 21: 63.
14 Science Progress 107(3)

28. Wolfenden L, Ezzati M, Larijani B, et al. The challenge for global health systems in preventing
and managing obesity. Obes Rev 2019; 20(S2): 185–193.
29. Han J, Nepal P, Odelade A, et al. High-fat diet-induced weight gain, behavioral deficits, and
dopamine changes in young C57BL/6J mice. Front Nutr 2021; 7. doi:10.3389/fnut.2020.
591161.
30. Guerra-Cantera S, Frago LM, Collado-Pérez R, et al. Sex differences in metabolic recuperation
after weight loss in high fat diet-induced obese mice. Front Endocrinol 2021; 12. doi:10.3389/
fendo.2021.796661.
31. Sougiannis AT, VanderVeen BN, Cranford TL, et al. Impact of weight loss and partial weight
regain on immune cell and inflammatory markers in adipose tissue in male mice. J Appl Physiol
(1985) 2020; 129: 909–919.
32. Lang P, Hasselwander S, Li H, et al. Effects of different diets used in diet-induced obesity models
on insulin resistance and vascular dysfunction in C57BL/6 mice. Sci Rep 2019; 9: 19556.
33. Enerback S, Jacobsson A, Simpson EM, et al. Mice lacking mitochondrial uncoupling protein
are cold-sensitive but not obese. 10.1038/387090a0. Nature 1997; 387: 90–94.
34. Licholai JA, Nguyen KP, Fobbs WC, et al. Why do mice overeat high-fat diets? How high-fat
diet alters the regulation of daily caloric intake in mice. Obesity 2018; 26: 1026–1033.
35. Luong Q, Huang J and Lee KY. Deciphering white adipose tissue heterogeneity. Biology
(Basel) 2019; 8. doi:10.3390/biology8020023.
36. Chu D-T and Tao Y. Human thermogenic adipocytes: a reflection on types of adipocyte, devel-
opmental origin, and potential application. Journal article. J Physiol Biochem 2017; 73: 1–4.
37. Athyros VG, Doumas M, Imprialos KP, et al. Diabetes and lipid metabolism. Hormones 2018;
17: 61–67.
38. Hernáez Á, Soria-Florido MT, Schröder H, et al. Role of HDL function and LDL atherogeni-
city on cardiovascular risk: a comprehensive examination. PLOS ONE 2019; 14: e0218533.
39. Li H, Liu F, Lu J, et al. Probiotic mixture of lactobacillus plantarum strains improves lipid
metabolism and gut microbiota structure in high fat diet-fed mice. Original Research. Front
Microbiol 2020; 11.
40. Gu W, Wang Y, Zeng L, et al. Polysaccharides from Polygonatum kingianum improve glucose
and lipid metabolism in rats fed a high fat diet. Biomed Pharmacother 2020; 125: 109910.
41. Talayero BG and Sacks FM. The role of triglycerides in atherosclerosis. Curr Cardiol Rep
2011; 13: 544–552.
42. Peters SAE, Singhateh Y, Mackay D, et al. Total cholesterol as a risk factor for coronary heart
disease and stroke in women compared with men: a systematic review and meta-analysis.
Atherosclerosis 2016; 248: 123–131.
43. Fu H, Tang B, Lang J, et al. High-Fat diet promotes macrophage-mediated hepatic inflamma-
tion and aggravates diethylnitrosamine-induced hepatocarcinogenesis in mice. Front Nutr
2020; 7: 585306.
44. Matsumoto M, Hada N, Sakamaki Y, et al. An improved mouse model that rapidly develops
fibrosis in non-alcoholic steatohepatitis. Int J Exp Pathol 2013; 94: 93–103.
45. Liao CC, Lin YL and Kuo CF. Effect of high-fat diet on hepatic proteomics of hamsters.
J Agric Food Chem 2015; 63: 1869–1881.
46. Carrer A, Parris JLD, Trefely S, et al. Impact of a high-fat diet on tissue acyl-CoA and histone
acetylation levels *. J Biol Chem 2017; 292: 3312–3322.
47. Voigt A, Agnew K, van Schothorst EM, et al. Short-term, high fat feeding-induced changes in
white adipose tissue gene expression are highly predictive for long-term changes. Mol Nutr
Food Res 2013; 57: 1423–1434.
48. Kadota Y, Kawakami T, Takasaki S, et al. Gene expression related to lipid and glucose metab-
olism in white adipose tissue. Obes Res Clin Pract 2016; 10: 85–93.
Chu et al. 15

49. Aizawa S, Tochihara A and Yamamuro Y. Paternal high-fat diet alters triglyceride metabolism-
related gene expression in liver and white adipose tissue of male mouse offspring. Biochem
Biophys Rep 2022; 31: 101330.
50. Dong J, Li M, Peng R, et al. ACACA Reduces lipid accumulation through dual regulation of
lipid metabolism and mitochondrial function via AMPK- PPARα- CPT1A axis. J Transl Med
2024; 22: 196.
16 Science Progress 107(3)