Milk fat which contains a high proportion of one or more unsaturated fatty acids (UFAs; examples are oleic and linolenic acids) is a valuable component in the dairy industry as a result of putative health benefits (Massaro et al., Reference Massaro, Carluccio and De Caterina1999; Shingfield et al., Reference Shingfield, Chilliard, Toivonen, Kairenius and Givens2008). In contrast, some specific saturated fatty acids (SFAs) in milk fat, mainly lauric, myristic and palmitic acids, have been found to have a negative effect on human health when consumed in excess by increasing plasma total and LDL cholesterol concentrations (Williams, Reference Williams2000). In the mammary gland of ruminants, short- and medium-chain fatty acids (SMFAs) and a portion of C16 : 0 fatty acids from acetate and, to a lesser extent, b-hydroxybutyrate must be obtained by de novo synthesis, while long-chain fatty acids (LCFAs) and the remaining C16 : 0 fatty acids are obtained by the exogenous uptake of preformed fatty acids (FAs) from the circulation (Harvatine et al., Reference Harvatine, Boisclair and Bauman2009). A gene network analysis study in bovine mammary tissue showed that peroxisome proliferator-activated receptor gamma (PPARG), a nuclear receptor transcription factor for which acetyl-coenzyme A carboxylase alpha (ACACA), fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) are putative target genes, regulates milk fat synthesis during lactation (Bionaz and Loor, Reference Bionaz and Loor2008). In addition, lipoprotein lipase (LPL) and acyl-CoA synthetase long-chain family member 1 (ACSL1), genes involved in FA transport, were reported to be target genes of PPARG (Desvergne et al., Reference Desvergne, Michalik and Wahli2006). The enzymes ACACA and FASN participate in the metabolic de novo FA synthesis pathway in mammary tissue, whereas the enzyme LPL is involved in the uptake of plasma FAs. Kadegowda et al. (Reference Kadegowda, Bionaz, Piperova, Erdman and Loor2009) demonstrated the regulatory roles of PPARG and exogenous or mammary-synthesized LCFAs in milk fat synthesis in vitro. In addition, Harvatine and Bauman (Reference Harvatine and Bauman2006) suggested that sterol regulatory element binding transcription factor 1 (SREBF-1) is pivotal in the regulation of milk fat synthesis in cows.
Exogenous supplemental LCFAs are crucial for milk fat synthesis in bovine mammary epithelial cells (BMECs). Studies have indicated that LCFAs significantly inhibited de novo synthesis of SMFAs (Hansen and Knudsen, Reference Hansen and Knudsen1987; Warntjes et al., Reference Warntjes, Robinson, Galo, DePeters and Howes2008) as well as the mRNA expression of ACACA and FASN in BMECs (Bauman et al., Reference Bauman, Perfield, Harvatine and Baumgard2008; Kadegowda et al., Reference Kadegowda, Bionaz, Piperova, Erdman and Loor2009). In in vitro studies, the exogenous LCFAs oleate, linoleate, or linolenic acid increased TAG accumulation and altered the mRNA expression of genes related to FA synthesis in BMECs (Yonezawa et al., Reference Yonezawa, Yonekura, Kobayashi, Hagino, Katoh and Obara2004; Qi et al., Reference Qi, Yan, Sheng, Zhao and Guo2014; Sheng et al., Reference Sheng, Yan, Qi and Zhao2015). Furthermore, LCFAs are not only required for milk fat biosynthesis as precursors but also play a role in regulating milk FA composition.
Studies showed that supplementation with a single UFA affected TAG accumulation and lipogenic enzymes in BMECs (Kadegowda et al., Reference Kadegowda, Piperova and Erdman2008; Yonezawa et al., Reference Yonezawa, Sanosaka, Haga, Kobayashi, Katoh and Obara2008). However, few studies regarding the regulatory effect of combination UFA treatment on milk fat synthesis have been reported. In animals, FAs are not present alone, and FAs in the diet also coexist with endogenous FAs. Therefore, an in vitro study was undertaken in BMECs to test the hypothesis that an optimum combination of preformed UFAs consisting of oleic, linoleic, and linolenic acids would regulate cytosolic TAG accumulation and the expression of genes encoding lipogenic enzymes to some extent. To meet this objective, BMECs were cultured with C18-UFAs, following which TAG accumulation, the mRNA expression of lipogenic genes, and the intracellular FA composition were measured.
Materials and methods
Cell culture
BMECs were isolated from the mammary glands of 6 Chinese Holstein dairy cows from a local slaughterhouse during their lactation period. Mammary tissues for cell culture were obtained from the deep layer of the mammary gland and soaked in ice-cold PBS before cell culture. BMECs were cultured according to the methods described by Hu et al. (Reference Hu, Wang, Bu, Wei, Zhou, Li and Loor2009) with modifications by the use of type II collagenase (Gibco BRL, Grand Island, NY, USA). Specific detailed procedures are presented in the online Supplementary Materials and Methods. Cells were seeded at 1 × 104 cells/cm2 in culture flasks and then incubated in basal culture medium for 48 h before stimulation by C18-UFAs treatment and growth in FA-free bovine serum albumin (BSA, Equitech-Bio, Kerrville, TX, USA) medium for an additional 48 h. The FA-free BSA culture medium was prepared as described above for the basal culture medium, except that 10% fetal bovine serum was replaced with 1 g/l FA-free BSA. The control treatment contained only FA-free BSA without UFAs.
Cell proliferation assay
Cell proliferation was determined by MTT assay according to Qi et al. (Reference Qi, Yan, Sheng, Zhao and Guo2014) with modifications (online Supplementary Materials and Methods). The results revealed that cell viability was promoted by each LCFA at a low concentration and then inhibited when the concentration increased (online Supplementary Figure).
TAG accumulation
Based on the MTT assay, three different concentrations for which each C18 UFA did not inhibit cell viability were selected and used to determine the synthesis of cellular TAG utilizing L 9 (33) orthogonal design to finally confirm the effects of C18 UFAs treatment (online Supplementary Table S1). The selected concentrations were 100, 200 and 400 μmol/l oleic acid; 50, 75 and 100 μmol/l linoleic acid; and 1, 1.5 and 2 μmol/l linolenic acid. Synthesized TAG was determined as reported in the online Supplementary Materials and Methods.
RNA extraction and real-time quantitative PCR
Specific details of these procedures are presented in online Supplementary Materials and Methods and online Supplementary Table S2 (primer sequences). RT-PCR analysis was performed with the 2−ΔΔCT method (Livak and Schmittgen, Reference Livak and Schmittgen2001), with the reference genes β-actin, GAPDH and RPS9. The primers were designed according to Sheng et al. (Reference Sheng, Yan, Qi and Zhao2015) and synthesized by Shanghai Sangon Biological Engineering and Technology Service Co., Ltd. (Shanghai, China). Specific gene amplification was confirmed by sequencing.
Measurement of the cellular FA composition
Intracellular FAs were extracted according to a previously described procedure (Zhang et al., Reference Zhang, Ao, Khas-Erdene and Zhang2015) with modifications (online Supplementary Materials and Methods).
Statistical analysis
Treatments in culture wells were replicated at least 3 times, and PCR was performed in triplicate. Data were analyzed using one-way ANOVA with SAS software (SAS Version 9.0; SAS Institute Inc., Cary, NC). RT-PCR data analysis was performed with 2−ΔΔCt values. Differences were considered significant when P < 0.05.
Results
Tag content in the cytosol
The results of the orthogonal experiment indicated that 200 μmol/l oleic acid, 50 μmol/l linoleic acid, and 2 μmol/l linolenic acid were the optimal doses for TAG accumulation (online Supplementary Table S3). Compared with the control treatment, C18-UFAs treatment significantly increased the intracellular TAG content in BMECs (Fig. 1).
The effects of exogenous C18 unsaturated fatty acids on triglyceride contents in bovine mammary epithelial cells. The control treatment contained only fatty acid-free BSA without unsaturated fatty acids. *Indicate significant differences from values obtained in control treatment at P < 0.05.
Expression of genes related to FA synthesis
There was no significant difference in the mRNA expression of either ACACA or FASN between the treatments (P > 0.05). The abundance of SCD1 and ACSL1 was higher after C18-UFAs treatment than after control treatment, but the gene expression of SREBF1 was reduced (P < 0.05). However, treatment did not affect the expression of PPARG and LPL (Fig. 2).
The effects of exogenous C18 unsaturated fatty acids on lipogenic genes mRNA abundance in cells. FASN, fatty acid synthase; ACACA, acetyl-coenzyme A carboxylase-α; SCD, stearoyl CoA desaturase; LPL, lipoprotein lipase; ACSL1, acyl-CoA synthetase long chain 1; SREBF1, sterol regulatory element binding factor 1; PPARG, peroxisome proliferator activated receptor-γ. *Indicate significant differences from values obtained in control treatment at P < 0.05.
Intracellular FA composition
The effects of C18-UFAs treatment on FA composition in BMECs are presented in Table 1. The C16 : 0 and SFA content in BMECs was decreased with C18-UFAs treatment compared to the control treatment, whereas the cis-9 C18 : 1 and UFA content was significantly increased with C18-UFAs treatment (P < 0.05).
Effect of exogenous C18 unsaturated fatty acids on fatty acid composition in bovine mammary epithelial cells
a Control treatment contained only fatty acid-free BSA without unsaturated fatty acids (0 μM), C 18-UFAs contained 200 μmol/l of oleic acid, 50 μmol/l of linoleic acid, and 3 μmol/l of linolenic acid. SFA, saturated fatty acids; UFA, unsaturated fatty acids.
a,bValues in the same row with different superscript letters differ significantly from each other.
Discussion
Several studies have suggested that unsaturated LCFAs play an important role in the proliferation of BMECs (Rose et al., Reference Rose, Hatala, Connolly and Rayburn1993; Yonezawa et al., Reference Yonezawa, Sanosaka, Haga, Kobayashi, Katoh and Obara2008). In our study, oleic acid, linoleic acid, or linolenic acid at certain concentrations promoted the proliferation of mammary epithelial cells. TAG accumulated in the cytosol of BMECs, and the TAG content directly reflects fat synthesis in BMECs. In this study, TAG accumulation after C18-UFAs combination treatment was higher than that following control treatment. Oleate and linoleate were reported to significantly upregulate TAG synthesis in BMECs, and C18 UFAs at a concentration lower than 100 μM could increase the accumulation of TAG (Yonezawa et al., Reference Yonezawa, Yonekura, Kobayashi, Hagino, Katoh and Obara2004; Cui et al., Reference Cui, Wang, Bu, Wei, Nan, Hu and Zhou2012). A recent study in Mac-T cells showed similar results and that UFA treatment increased cytosolic TAG accumulation (Vargas-Bello-Pérez et al., Reference Vargas-Bello-Pérez, Juan and Garnsworthy2018). Moreover, Green et al. (Reference Green, Ozguden-Akkoc, Wang, Jump and Olson2010) observed that oleic acid is the preferred substrate for TAG synthesis. Therefore, the availability of preformed FAs is important for cellular TAG formation.
The lipogenic genes ACACA and FASN are involved in the metabolic pathway of de novo FA synthesis to produce SMFAs in bovine mammary tissue, whereas LPL is related to the uptake of plasma FAs (Bernard et al., Reference Bernard, Leroux and Chilliard2008; Shingfield et al., Reference Shingfield, Bernard, Leroux and Chilliard2010). Previous studies found either a significant decrease or no change in ACACA and FASN abundance in cells treated with exogenous long-chain UFAs (Jacobs et al., Reference Jacobs, Dijkstra, Liesman, Vandehaar, Lock, van Vuuren, Hendriks and van Baal2013; Li et al., Reference Li, Xing, Li, Wang, Hou and Gao2018). In the current study, C18-UFAs treatment had no influence on the expression of ACACA or FASN, and SMFA levels in cells thus remained unchanged, further suggesting that long-chain UFAs do not positively regulate de novo FA synthesis in BMECs. In accordance with the results of Sheng et al. (Reference Sheng, Yan, Qi and Zhao2015), no difference in the abundance of LPL was observed after C18-UFAs treatment, suggesting that combination UFAs treatment had little effect on the uptake of exogenous FAs. In ruminant milk fat synthesis, ACACA, FASN and LPL are thought to be putative PPARG target genes, indicating PPARG as a key factor that manipulates lipid biosynthesis in BMECs (Kadegowda et al., Reference Kadegowda, Bionaz, Piperova, Erdman and Loor2009). We found that like that of ACACA, FASN, and LPL, the mRNA level of PPARG was not altered by exogenous UFA treatment, which again suggests that these genes are at least in part controlled by PPARG.
Paton and Ntambi (Reference Paton and Ntambi2009) suggested the multiple roles of SCD in the regulation of lipid and carbohydrate metabolism. In bovine mammary lipid metabolism, SCD is responsible for introducing a cis double bond at the Δ9 position in a wide range of FAs. Decreased SCD abundance was observed in some studies in which BMECs were treated with preformed UFAs (Jacobs et al., Reference Jacobs, Dijkstra, Liesman, Vandehaar, Lock, van Vuuren, Hendriks and van Baal2013; Li et al., Reference Li, Xing, Li, Wang, Hou and Gao2018). The caprine SCD genotype showed suggestive associations on milk FA composition with higher percentages of PUFA (Zidi et al., Reference Zidi, Fernández-Cabanás, Urrutia, Carrizosa, Polvillo, González-Redondo, Jordana, Gallardo, Amills and Serradilla2010). In our study, the increase in the expression of SCD suggested an increase in the desaturation ability of mammary cells following UFA treatment relative to control treatment. Together with the increased intracellular total UFA and C18 : 1 content and decreased total SFA and C16 : 0 content, we propose a positive feedback mechanism that increased the abundance of SCD when the cells were supplemented with C18-UFAs and an enhanced degree of unsaturation within cells. However, the reason for the difference in SCD expression response to FA between this study and earlier researches is not clear, and further research is needed.
ACSL1 is an isozyme of the LCFA coenzyme A ligase family that plays a key role in lipid biosynthesis and FA degradation. Prior to use in TAG synthesis, FAs must be activated by ACSL1 (Rudolph et al., Reference Rudolph, McManaman, Phang, Russell, Kominsky, Serkova, Stein, Anderson and Neville2007). In the present study, the significantly higher expression of ACSL1 in C18-UFAs-treated cells suggested the stimulation of LCFA activation by preformed UFAs, which may further explain the enhanced TAG accumulation following UFAs treatment relative to control treatment.
Investigations have focused on the dominant role of SREBF1 and verified its function in the regulation of lipid metabolism (Desvergne et al., Reference Desvergne, Michalik and Wahli2006). In the ruminant mammary epithelium, SREBF1 has been revealed to play a central role in the integrated regulation of lipid biosynthesis through the regulation of key enzymes (Ma and Corl, Reference Ma and Corl2012; Xu et al., Reference Xu, Luo, Tian, Li, Zhang, Chen, Li and Loor2018). In this study, a mixture of oleic acid, linoleic acid, and linoleic acid was used to stimulate BMECs and found to markedly reduce SREBF1 expression, which suggested that SREBF1 was negatively regulated by preformed UFAs. This is in line with studies in which BMECs were treated with a single C18 UFA, resulting in a significant reduction in SREBF1 expression (Kadegowda et al., Reference Kadegowda, Bionaz, Piperova, Erdman and Loor2009; Jacobs et al., Reference Jacobs, Dijkstra, Liesman, Vandehaar, Lock, van Vuuren, Hendriks and van Baal2013; Harvatine et al., Reference Harvatine, Boisclair and Bauman2018). These results elucidated that SREBF1 is a master regulator of bovine mammary lipid synthesis that could be repressed by PUFAs.
In accordance with our study showing that the cellular oleic acid and total UFA concentrations were higher following incubation with a mixture of oleic, linoleic and linolenic acids, Vargas-Bello-Pérez et al. (Reference Vargas-Bello-Pérez, Juan and Garnsworthy2019) showed that the single addition of oleate increased cytosolic MUFAs and UFAs in Mac-T cells. Together with the present results, these findings suggest that oleic acid serves as a potent regulator of FA unsaturation in BMECs. Although exogenously supplied FAs could be affected by transcriptional and translational processes in BMECs before synthesized FAs in milk, the types and content of the FAs provided may significantly influence milk FA composition. The current experiment revealed that milk FA composition and exogenous supplemented FA composition tended to be consistent, indicating that variation in milk LCFAs could reflect changes in exogenous LCFAs to a certain extent.
In conclusion, a mixture of oleic acid, linoleic acid, and linolenic acid regulated milk lipid synthesis in BMECs by controlling genes encoding enzymes involved in FA desaturation and LCFA activation, the transcription factor SREBF1, and TAG accumulation. Exogenous C18 UFAs supplementation may have a significant benefit for human health by improving milk fat composition.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029920000722.
Acknowledgment
The authors express their gratitude to the postgraduate students of the Animal Science College of Inner Mongolia Agricultural University for their help with collection of the experimental samples. This work was supported by the National Basic Research Program of China (2011CB100803) and Inner Mongolia Natural Science Foundation (2018LH03011).
