Previous work in our laboratory has demonstrated that the addition of recombinant protein ASIP altered mRNA expression of genes related to lipid metabolism and significant increases triglycerides and cholesterol content in bMECs (unpublished data). These studies could imply the involvement of ASIP genes in the synthesis and catabolism of lipids in cattle. To further explore the effect of the ASIP gene on lipid metabolism in bMECs, we successfully constructed the ASIP knockout bMECs using CRISPR/Cas9 technology. The results showed that the expression of ASIP was significantly reduced at the mRNA level in the knockout cell lines.
The content of triglycerides, cholesterol and fatty acids was firstly measured to investigate further the effect of
ASIP knockout on lipid metabolism in bMECs. The results presented that
ASIP knockout affected the composition of medium and long-chain fatty acids, increasing saturated and polyunsaturated fatty acids content and a decreased level of monounsaturated fatty acids in bMECs. The content of saturated fatty acids myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), heptadecanoic acid (C17:0), stearic acid (C18:0), arachidonic acid (C20:0), 21-carbonic acid (C21:0), and ditetradecanoic acid (C24:0) were significantly up-regulated. The significant increase in saturated fatty acids of 14-24 C-atoms, which are substrates for triglycerides production [
23], could provide the fundamental element for the elevation of triglycerides in bMECs after knockout of the
ASIP gene. It has been widely accepted that saturated fatty acids and TG in dairy fat, which may lead to obesity, and high blood pressure, are detrimental to health. Moreover, saturated fatty acids such as myristic (14:0) and palmitic (16:0) acids could enhance the level of low-density lipoprotein (LDL- and high-density lipoprotein (HDL-) cholesterol in the blood [
24]. Despite their inherent ability to inhibit bacteria and viruses, a high intake of these fatty acids could raise blood cholesterol content which could cause heart disease, weight gain, and obesity [
5]. For some polyunsaturated fatty acids, there were significant increases in linoleic acid (C18:2N6), gamma-linoleic acid (C18:3N6), and alpha-linolenic acid (C18:3N3) in bMECs with
ASIP knockout. The elevation of these fatty acids could lower blood lipids and improve vascular health status [
25,
26]. Ni Dan et al. investigated the effect of the complete absence of certain long-chain fatty acids (LCFA) on milk lipid metabolism in bMECs using the univariate principle. It was found that triglycerides synthesis was significantly down-regulated in bMECs with the absence of C18:0, C18:2N6 or C18:3N3 [
27]. This implies that elevated C18:2N6 and C18:3N3 may also have a facilitating effect on triglycerides synthesis. Furthermore, the monounsaturated fatty acid oleic acid (C18:1N9), which accounted for the largest proportion of milk lipid [
28], and other monounsaturated fatty acids such as palmitoleic acid (C16:1N7) and C20:1N9 were significantly down-regulated under
ASIP knockout conditions. It has been indicated that consuming diets containing high amounts of monounsaturated fatty acids effectively reduces plasma lipids, such as cholesterol and triacylglycerol concentrations [
29]. Furthermore, monounsaturated fatty acids are more protective against atherosclerosis than polyunsaturated fatty acids [
30]. Thus, the reduction of
ASIP can alter fatty acid composition in the intracellular bMECs and may further in milk. However, this alteration in milk may be detrimental to human health. It suggested that the lower expression level of
ASIP in mammary epithelial cells was not recommended to breed dairy cows with high milk quality in the future.
Key genes of the fatty acid metabolic pathway were detected to explore the molecular mechanisms of fatty acid alterations in bMECs after
ASIP knockout. As an enzyme directly related to lipid synthesis and fatty acid transport and degradation, ACSL1 can significantly influence fatty acid metabolism. By overexpressing
ACSL1 in bovine adipocytes, Zhao et al. found a significant increase in saturated and polyunsaturated fatty acid (PUFA) content, particularly C16:0 and C18:0. Overexpression of ACSL1 further increased the proportion of eicosapentaenoic acid (EPA) [
31]. In the present study,
ACSL1 expression was significantly up-regulated in
ASIP knockout bMEC, which may lead to SFA accumulation such as palmitic acid (C16:0) and C18:0 content as well as an increase in PUFA and C20:5N3. Moreover, the primary function of
FABP4 is to transport fatty acids across the membrane, and overexpression increases FA transport to enhance energy and lipid metabolism [
32]. The deletion of
FABP4 results in an impaired fatty acid efflux, leading to an increase in fatty acids in FABP4-deficient cells. It has been shown that FABP4 maintains eicosanoid homeostasis in macrophages in mice [
33]. The FASN gene is a multifunctional peptidase for producing saturated fatty acids. It is responsible for all steps in the ab initio synthesis of palmitic acid (C16:0) from acetyl-coenzyme an (acetyl coenzyme a) and malonyl coenzyme a (malonyl coenzyme a) [
34]. The specific knockout of
FASN in mouse mammary glands significantly reduces the content of medium and total long-chain fatty acids in milk [
35]. This may indicate the down-regulation of
FASN may lead to a decreased synthesis rate of C16:0 and finally lower the level of C16:0 in bMEC with
ASIP knockout. Furthermore,
SCD is the rate-limiting enzyme that catalyses the synthesis of monounsaturated fatty acids (MUFAs) from saturated fatty acids (SFAs). The major substrates of
SCD are C16:0 and C18:0 FA, which can be converted to C16:1 cis9 and C18:1 N9 [
36]. Association analysis of SNP loci of
SCD with milk lipid ty acid traits in 297 Holstein cows was conducted by Mele et al. They found that cows with the AA genotype had higher levels of cis-9C18:1, total monounsaturated fatty acid levels and C14:1/C14:0 ratios in milk compared to cows with the VV genotype [
37]. In this study, there was a significant down-regulation of
SCD expression in
ASIP knockout bMEC, leading to SFA accumulation, such as palmitic acid (C16:0) and C18:0 content and the reduction of MUFA, like C16:1N7 and C18:1N9. For C16:0, both
FASN and
SCD could influence its level in bMEC. As C16:0 content was lower in the control group, it may imply that the consumption rate of C16:0 by
SCD could be lower than the synthesis rate by
FASN in
ASIP knockout cells. Furthermore, Junjvlieke et al. used adenovirus to overexpress the
ELOVL6 gene, a rate-limiting enzyme in the long-chain fatty acid elongation reaction, in bovine adipocytes and found a significant increase in the proportion of C18:0 and C20:4N6 fatty acids [
38]. Our results suggested that the mRNA expression level of
ELOVL6 was significantly increased in bMEC with
ASIP knockout which could contribute to the upregulation of C18:0. Additionally, the expression of
HACD4 mRNA was detected in this study, as it is an important enzyme at the third step of fatty acid extension. Even though there was a significant elevation in 18:2N6 and 18:3N3 fatty acids after
ASIP knockout in bMECs, the higher mRNA expression of
HACD4 was not detected. It may indicate that the enhancement of 18:2N6 and 18:3N3 fatty acids in
ASIP knockout of bMECs was not mainly regulated by
HACD4. Moreover, we found that the knockout of the
ASIP gene in bMECs resulted in significant downregulation of the peroxisome proliferator-activated receptor (PPARγ), which has a key role in lipid metabolism, promoting free fatty acid uptake and accumulation of triacylglycerols in adipose tissue and liver [
39]. The down-regulation of
PPARγ may lead to triglycerides down-regulation. Garin-Shkolnik et al. found that high expression of fatty acid-binding protein 4 (FABP4, also known as aP2) could down-regulate
PPARγ expression [
40]. Tingting Li et al. found that in human hepatocytes overexpressing
ASCL1 caused a significant decrease in fatty acid synthesis pathway-related genes such as
FASN and
SCD1 by suppressing
PPARγ expression to an increase in triglycerides levels [
41]. This is consistent with the results of gene and triglycerides changes in our experiments. In another human hepatocyte line (L02 cells) overexpressing
PPARγ, it was found that up-regulation of PPARγ could affect cholesterol efflux and thus lower cholesterol levels through multiple pathways [
42]. Our cholesterol assay results indicate a trend of up-regulation, but one not significant in bMECs after knockout of
ASIP, but its molecular mechanism needs to be further investigated. These results suggest that the knockout of
ASIP can influence the expression of key genes in the fatty acid metabolism pathways, thus affecting the intracellular lipid metabolism, especially the composition of fatty acids. It has been shown that
ASIP affects the cellular content of cAMP by competitive binding to MC1R [
43,
44]. However, whether
ASIP affects the expression of key genes in lipid metabolism in bMECs by regulating cAMP content still needs further investigation.