Short/branched chain acyl-CoA dehydrogenase (ACADSB) is a member of the acyl-CoA dehydrogenase family of enzymes that catalyze the dehydrogenation of acyl-CoA derivatives in the metabolism of fatty acids. Our previous transcriptome analysis in dairy cattle showed that ACADSB was differentially expressed and was associated with milk fat metabolism. The aim of this study was to elucidate the background of this differential expression and to evaluate the role of ACADSB as a candidate for fat metabolism in dairy cattle. After analysis of ACADSB mRNA abundance by qRT-PCR and Western blot, overexpression and RNA interference (RNAi) vectors of ACADSB gene were constructed and then transfected into bovine mammary epithelial cells (bMECs) to examine the effects of ACADSB on milk fat synthesis. The results showed that the ACADSB was differentially expressed in mammary tissue of low and high milk fat dairy cattle. Overexpression of ACADSB gene could significantly increase the level of intracellular triglyceride (TG), while ACADSB gene knockdown could significantly reduce the TG synthesis in bMECs. This study suggested that the ACADSB was important in TG synthesis in bMECs, and it could be a candidate gene to regulate the metabolism of milk fat in dairy cattle.
ACADSB gene is a short/branched chain acyl-coenzyme a dehydrogenase,
which is a member of the ACADS family that is indispensable in fatty
acid and amino acid metabolism in mitochondria (Ikeda et al., 1985;
Alfardan et al., 2010). ACADS also catalyzes
Partial data about the differentially expressed genes of transcriptome analysis.
Note: description of genes, ABCA1, uncharacterized protein; ACSM1, Acyl-coenzyme A synthetase ACSM1 mitochondrial; ADAMTS1, ADAMTS1 protein; C1QTNF5, C1QTNF5 protein; ACADSB, short/branched chain-specific acyl-CoA dehydrogenase, mitochondrial; ADAM8, uncharacterized protein.
Animal experiments were performed in strict accordance with the guide for the care and use of laboratory animals by the Jilin University animal care and use committee (permit number: SYXK (Ji) 2012-0010/0011).
In this study, 3 high milk fat cattle and 3 low milk fat cattle were
chosen by a milk ingredient analyzer (Lactoscan SP, Milkatronic
Ltd., Nova Zagora, Bulgaria) from 15 healthy dairy cattle, which were
in the same environment and the same lactation period. Dairy cattle of
different experimental groups were slaughtered at Haoyue
slaughterhouse in Changchun, and then the fresh breast tissues were
collected from the carcass of dairy cattle with different milk fat
percentage and frozen in liquid nitrogen for subsequent
experiments. The total RNA was extracted from mammary tissue using
TRIzol Reagent (Tiangen, Beijing, China) and reverse transcribed into
cDNA using a RT cDNA synthesis kit (TOYOBO, Japan) and following the
manufacturer's protocols. ACADSB gene expression in mammary tissues
from cattle with different milk fat percentages was analyzed by
quantitative real-time fluorescent PCR (qRT-PCR). The qRT-PCR was
carried out using SYBR
A 21-mer siRNA oligonucleotide targeting the coding sequence of ACADSB
and the complementary oligonucleotide were designed according to the
design principles of shRNA. The target sequence on ACADSB gene is
GCTGGGTATAAAGGAA TTACC and the sequences of the primer pair were as follows:
(5
Negative control plasmid was constructed by inserting annealed scrambled siRNA oligonucleotides into pGPU6/GFP/Neo.
The cDNA for the predicted mature coding region of ACADSB in cow was
amplified by PCR with sense primer
5
The bovine mammary epithelial cells in this study were established by
our laboratory (Lu et al., 2012). Cells were cultured in 10
BMECs transfected with ACADSB RNAi vector and overexpression vector
were harvested after transfection for 24 h, and total RNA was
extracted using the PREP RNA mini kit (Analytik Jena, Germany). Total
RNA was checked with agarose gel electrophoresis, and the concentration
was measured using a spectrophotometer. The cDNA was synthesized using
a RT cDNA synthesis kit (ToYoBo, Japan) following the manufacturer's
protocols. The qRT-PCR was carried out using SYBR
The cells were collected after transfection for 48 h and washed
two times with PBS (phosphate buffered saline). Cells were then resuspended in RIPA buffer (RIPA
splitting buffer, BOSTER, China) with protease inhibitor. Cell lysates
were cleared by centrifugation at 12 000
The TG contents were determined using TG kit
(Applygen Technologies, Beijing, China) according to the
manufacturer's protocols. BME cells were plated at a concentration of
0.6
Experimental data are shown as mean
Cattle short/branched chain acyl-CoA dehydrogenase gene is located on
bovine (BTA) chromosome 26 (
The ACADSB gene expression in mammary tissues from the three high milk fat
cattle and three low milk fat cattle was determined by quantitative
real-time PCR. The expression of ACADSB gene in mammary tissues of
high milk fat cattle is significantly higher than that in the low milk
fat cattle (
The ACADSB gene expression levels in mammary tissue of high milk fat and low milk fat cattle.
Construction of ACADSB gene RNA interference and
overexpression vector and transfection into bovine mammary
epithelial cells.
The milk fat percentage data were obtained from a total of 15 Chinese Holstein dairy cattle 10 days continuously.
In order to reveal the effect of ACADSB gene on the synthesis of
TGs, overexpression vector and RNAi vector of
bovine ACADSB gene were successfully constructed (Fig. 2b). Bovine
mammary epithelial cells were transiently transfected with plasmids of
pGPU6/GFP/Neo-ACADSB and pBI-CMV3-ACADSB. The expression of green
fluorescence could be observed from both overexpression vector
(pBI-CMV3-ACADSB) transfected group and RNAi vector
(pGPU6/GFP/Neo-ACADSB) transfected group after 24
To investigate the effect of pBI-CMV3-ACADSB and pGPU6/GFP/Neo-ACADSB
vector on cellular ACADSB mRNA level, total RNA was extracted from
cells which transfected with various plasmids and analyzed by
qRT-PCR. The results showed that ACADSB mRNA expression levels in RNAi
transfected cells were significantly decreased compared with those in
the negative control vector transfected cells (
To investigate the effect of pBI-CMV3-ACADSB and pGPU6/GFP/Neo-ACADSB
plasmid on cellular ACADSB protein level, total protein was isolated
from cells that transfected with various plasmids of overexpression
vector and RNAi vector. The Western blot analysis results revealed
that ACADSB protein expression in RNAi transfected cells was
significantly down-regulated compared to those in the negative
control vector transfected cells (
To examine the effect of ACADSB gene on intracellular TG
synthesis, TG content was detected by TG kit. The result
showed that TG content in bMEC transfected with
pGPU6/GFP/Neo-ACADSB was significantly lower than that in the control
group and pBI-CMV3-ACADSB transfected group (
Comparison of the TG content of bMECs which transfected with
RNAi vector or overexpression vector.
ACADSB is a member of the acyl-CoA dehydrogenase family, which can catalyze the dehydrogenation of acyl-CoA derivatives in the metabolism of fatty acid (Willard et al., 1996; Arden et al., 1995). At least nine members of this family have been described (Ensenauer et al., 2005; Ikeda et al., 1985). Substrate specificity is the most important characteristic to define the members of the ACADS family. Studies have shown that SCAD, ACADSB and IBD can all utilize butyryl-CoA as a substrate, whereas IVD, ACADSB and IBD are most active with short/branched chain acyl-CoAs as a substrate (Zhang et al., 2002; He et al., 2003). ACADSB encodes for a mitochondrial precursor protein with a molecular weight of 47.1 kDa, which has the active activity toward the short/branched chained acyl-CoA derivative (Rozen et al., 1994; Alfardan et al., 2010). ACADSB gene is located on chromosome 10 in human at 10q26.13 (Andresen et al., 2000). ACADSB is differentially expressed among different tissues (Gibson et al., 2000). Studies in human dehydrogenase deficiency have indicated the involvement of ACADSB gene in the degradation of valine, leucine and isoleucine and the metabolism of fatty-acid metabolism (Chaudhari et al., 2016; Cheng et al., 2016; Wang et al., 2015), suggesting that ACADSB gene maybe a candidate gene of fatty-acid metabolism.
Since the pathway of glycerolipid biosynthesis was illuminated in the 1950s, more studies have been gained about the mechanism of lipid biosynthetic and triacylglycerol biosynthesis (Coleman and Lee, 2004). TG has a critical effect on energy store and a repository of essential and nonessential fatty acids and also was the precursor of phospholipid biosynthesis. Fatty acids are packaged in very low-density lipoprotein and chylomicra as TG for distribution to other tissues (Coleman and Lee, 2004). Fatty-acid composition of TGs affects the absorption and the distribution in the organism (Rodríguez et al., 2012). A published study showed that dietary TG may influence lipid metabolism in humans, and it also found enhanced absorption of fatty acid in the sn-2 position of dietary TGs (Hunter, 2001). Lipogenesis resulted in cellular lipid accumulation, via the uptake of lipogenic substrate. Then the endogenous fatty acid was synthesis and mainly stored in form of TGs (Desvergne et al., 2006). TG is also an essential component of milk (Coleman and Lee, 2004), suggesting that ACADSB gene maybe a candidate gene of milk fat metabolism.
In this study, we have examined the milk fat rate in 15 Chinese Holstein cattle with the same lactation period and compared ACADSB gene expression levels in mammary tissue by quantitative RT-PCR between dairy cattle groups of high fat rates and low milk fat percentage. In order to further validate the function of ACADSB gene, we cloned ACADSB gene from bovine tissue by RT-PCR and constructed the silencing vector and overexpression vector of ACADSB, which were then transfected into bMEC. As a result of our experiment, the ACADSB can significantly affect the synthesis of TGs in the fatty-acid metabolic pathway in bMEC.
The pBI-CMV3 vector was selected to construct the overexpression vector, which is bidirectional mammalian expression vector and is designed to constitutively express a protein of interest and the green fluorescent protein ZsGreen1, and it also can be transfected into mammalian cells using any standard transfection methods (Yu et al., 2017). Therefore, it greatly improves the test process and also ensures the well-off completion of the study. In cell transfection, we recommend the following: when the bMEC confluence is above 80 %, the use of FuGENE transfection reagent could improve transfection efficiency.
In conclusion, our previous transcriptome analysis and current study results identified ACADSB as a differentially expressed gene in mammary tissue between low and high milk fat cattle. Our results showed that ACADSB expression level was positively correlated with cellular TGs content, and ACADSB gene was actively involved in fatty-acid metabolism pathway in bMEC and milk fat synthesis of dairy cattle.
Data are available from the corresponding author upon request.
The authors declare that they have no conflict of interest.
This work was supported by the National Major Special Project on New Varieties Cultivation for Transgenic Organisms (2016ZX08009003006), the National Natural Science Foundation of China (no. 31672389, 31772562) and the Jilin Scientific and Technological Development Program (no. 20170519014JH, 20180101275JC). Edited by: Steffen Maak Reviewed by: Hong Guo and one anonymous referee