1 Introduction
With the improvement of living standards, excessive intake of fatty and
luscious foods leads to a rapidly increasing trend of obesity, which is
a serious public healthy issue all over the world. Obesity can also lead
to other diseases, such as inflammation, diabetes, and cardiovascular
diseases.
Current therapies for obesity include the use of weight-loss drugs,
increasing exercises, and reducing fat intake. Alternative measures are
also investigated. Studies have shown that obesity is correlated with
vitamin D (VD) deficiency (Leary et al., 2017), thus, supplementation of
VD could counteract the symptoms of obesity. For example, the Xi-an
Children’s Hospital, China conducted a study on VD intervention in 198
overweight and obese children and found that the intervention moderately
increased the circulating VD content in obese children, but the response
to VD supplementation was weaker than in healthy children (Liu et al.,
2008). VD is a natural steroid hormone, which is involved in a variety
of physiological functions and can promote the release of leptin, thus
promoting mobilization of body fat and reducing insulin resistance (Zhou
et al., 2017), but high levels of body fat can lower absorptivity of VD
in the gut (Liu et al., 2008). Therefore, obesity people may develop VD
deficiency. If so, VD supplementation is necessary in obese people.
Diacylglycerol (DAG) can lower blood lipids, body weight and body fat
deposition compared with triglycerides (TG), as well as has other
important physiological functions in rats (Diao et al., 2014; Han,
2014). The deposit of body fat depends on dynamic changes in fat
synthesis and mobilization, which are regulated by a number of genes.
Fatty acid synthase (FAS) is a fatty acid synthetase that catalyzes the
generation and elongation of fatty acid chains using acetyl-CoA and
malonyl-CoA as the substrates (Menendez et al., 2009). Carnitine
acyltransferase Ⅰ, also known as carnitine palmitoyltransferase I
(CPT1), is a key enzyme in fatty acid oxidation. Carnitine can promote
fatty acid transfer from cytosol to mitochondria where fatty acid
oxidation occurs (Wang et al., 2018). Studies have shown that the
carnitine content in liver tissues in high-fat diet induced obese rats
decreased significantly compared with healthy rats (Kim et al., 2010),
suggesting that the carnitine demand may be increased in obese rats. Wu
et al. (2019) found that the expression of CPT1 in the liver tissue of
rats on a high-fat was down-regulated while the FAS expression was
up-regulated, indicating that fat mobilization could be decreased and
fat synthesis increased in high-fat rats.
Aforementioned, VD could increase body fat mobilization via its effect
on leptin secretion, and DAG may reduce triglycerides in the body. We,
therefore, hypothesized that VD and DAG have synergistic effect, via
molecular and microbial mechanisms, on body fat deposit in obese people
and test this hypothesis in this study. We used a high fat diet to
induce obesity in rats as a model and then applied various DAG and VD
treatments. The corresponding changes in cytokines associated with
lipids metabolism and the microbiota in the caecum were monitored to
assess the effects.
Materials and Methods
2.1 Duck oil DAG preparation and detection
Duck oil DAG was prepared by esterifying glycerol and free fatty acids
decomposed from duck oil using an enzymatic method (Wang et al., 2019).
Purification of duck oil DAG in the preparation was proceeded using
molecular distillation equipment (Liu et al., 2020).
The content of DAG was analyzed using a high-performance liquid
chromatography method using Agilent 1100 Series (USA) HPLC equipped with
a Refractive Index Detector according to the method of Liu et al.
(2020).
VD was provided by Shanghai Yuanye Biotechnology Co., Ltd. (CAS:67-97-0,
China).
2.2 Fatty acid composition analysis
Fatty acid compositions (% of the total fatty acids) in duck oil DAG
preparation and duck oil were analyzed using the gas chromatography
method (GB5009-168-2016), and the results are shown in Table 1.
2.3 Animals and experimental design
A total of 75 four-week-old male Sprague-Dawley (SD) rats were used in
this study. Rats were housed in stainless steel cages (5 rats per cage)
in a temperature and humidity controlled room with a 12:12-h light/dark
cycle, free access to a commercial diet for rodents and water. The use
of the animals and the experimental procedures were approved by the
Animal Ethics Committee of the College of Animal Science and Technology,
Qingdao Agricultural University, in accordance with the China National
Standard – Laboratory Animals – Guideline of Welfare and Ethics
(2016).
The experiment lasted for 28 days. Rats were acclimated for seven days
and fed a commercial diet that contained 7% soybean oil. At the end of
acclimation, rats were allocated into five groups (n = 15, 5 rats per
cage) and randomly assigned to five treatments respectively: non-obesity
(NO), obesity control (OC), DAG: 10 mL per kg body weight (DG10),
VD:12.5 μg per kg body weight (VD12.5), and DAG × VD: 10 mL/d × 12.5 μg
per kg body weight (DG10VD12.5). From day 8 to day 28, NO group was
continuously fed the diet as in the acclimation period, and the other
three groups were fed a high fat (3.3% soybean oil and 30.1% lard).
Rats in the NO and OC groups was orally drenched once with 10 mL/d
water, rats in the DAG group drenched with 10 mL DAG per kg body weight,
and rats in the VD group drenched with 12.5 μg VD per kg body weight,
and rats in the DAG × VD group drenched with 10 mL DAG plus 2.5 μg VD
per kg body weight. The drench was administered by oral gavage, once a
day. Those treatments lasted for 21 days.
2.4 Sample collection
Blood sample (each about 5 mL) was taken from rat retro-orbital eye vein
into a centrifuge tube, and centrifuged at 2,800×g 4°C for 15 min. Serum
was harvested, and stored at -40℃ until analysis.
Once blood sample was taken, rats were immediately dissected, the liver,
testicles and visceral fat (around the kidneys, intestinal fat and
epididymal fat) were collected, and weighed. The caecum was removed, the
content was collected and sampled. All samples were stored at -40℃ until
analysis.
2.5 Body weight and body fat in rats
The weight of rats was weighed weekly using an electronic balance. The
perirenal fat, periepididymal fat and mesenteric fat in rats were
removed and weighed using an electronic balance. The fat to the body
weight percentage was calculated as followings:
The fat ratio = total fat weight (g)/body weight (g) × 100%
2.6 Serum lipids assays
The concentrations of TG, total cholesterol (TC), high density
lipoprotein (HDL), and low density lipoprotein (LDL) in serum were
measured using commercial kits (Jiancheng, Nanjing, China) according to
the manufacturer’s instructions. The absorbance was measured at 450 nm.
2.7 Assays for activity of metabolic enzymes in liver
The sample preparation was briefed as follows: the liver was weighed,
cut into tiny pieces in normal saline solution, then homogenized. The
homogenate was centrifuged at 500×g at 4°C for 15 min, the supernatant
was collected. The aspartate aminotransferase (AST) and alanine
transaminase (ALT) activity in the liver was determined using commercial
kits
(Jiancheng, Nanjing, China) according to the manufacturer’s
instructions.
2.8 FAS and CPT 1 mRNA expression in liver
The liver sample was weighed, cut into tiny pieces in normal saline
solution, then homogenized. Total RNA in the liver sample was extracted
using a RNAiso Kit (Takara Bio, China) according to the manufacturer’s
instructions. RNA was reverse-transcribed into cDNA using the 037 A
Reverse Transcription Kit (Takara Bio, China). cDNA synthesis conditions
were set as follows: 37°C for 15 min, 85°C for 5 s. PCR amplification
was performed in a total volume of 20 μL, which contained: 2 μL cDNA
templet, 4 μL forward primer (10μM), 4 μL reverse primer (10μM), 10 μL
SYBR GreenⅠenzyme-mix, and 7.2 μL ddH2O. Thermal cycling
conditions were set as follows: initial denaturation at 95°C for 30 s,
followed by 40 cycles at 95°C for 30 s, 95°C for 3 s and 60°C for 30 s.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal
reference gene. FAS mRNA (forward primer: 5’-TGGACGCCCACCACAAGA-3’;
reverse primer: 5’-AGAGGAGACAGGTCCAGAGT-3’) and CPT1 mRNA (forward
primer: 5’-CCACAAGTGCCTGTCCGTC-3’; reverse primer:
5’-TCAGGTAGGCTTCGTGGATTC-3’) gene expression was detected by SYBR Green
I chimeric fluorescence method according to the instructions of SYBR
Premix Ex Taq II kit (Takara Bio, China).
2.9 Microbial diversity in caecum
Three rats in each group were randomly selected and sacrificed by
dispositioning the neck. The caecum was immediated cut off, and the
content was collected into a sterile centrifuge tube. Total bacterial
DNA in the caecal content sample was extracted using the Power Soil DNA
Isolation Kit (MO BIO Laboratories) according to the manufacturer’s
protocol. DNA quality and quantity were assessed by the ratio of 260
nm/280 nm. Then the extracted DNA was stored at -80°C until the further
processes. The V3-V4 region of the bacterial 16S rRNA gene was amplified
with the common primer pair (forward primer, 5’- ACTCCTACGGGAGGCAGCA-3’;
reverse primer, 5’-GGACTACHVGGGTWTCTAAT-3’) combined with adapter
sequences and barcode sequences. PCR amplification was performed in a
total volume of 50 μL, which contained 10 μL buffer, 0.2 μL Q5
high-fidelity DNA polymerase, 10 μL high GC enhancer, 1 μL dNTP, 10 μM
of each primer and 60 ng genome DNA. PCR conditions were set as follows:
initial denaturation at 95°C for 5 min, followed by 15 cycles at 95°C
for 30 s, 50°C for 30 s, 72°C for 40
s. High-throughput sequencing analysis of bacterial rRNA genes was
performed on the purified, pooled sample using the Illumina Hiseq 2500
platform (2 × 250 paired ends) at Biomarker Technologies Corporation,
Beijing, China.
2.10 Statistics analysis
One-way ANOVA was used for all data analysis. Duncan method was used for
multiple comparisons between the means. All analyses were performed
using SPSS 17.0 software. P value < 0.05 was considered as
statistically significant.