INTRODUCTION
Milk protein and fat content as well as milk fatty acids (FA) composition are the important signals for milk quality measurement. Approximately fifty percent of milk FA, including short- and medium-chain FA (SMCFA) (C4:0 to C14:0) as well as ca. one-half of the C16:0 are synthesized
de novo from acetate and β-hydroxybutyrate in the mammary gland of dairy cows. The remaining C16:0 and almost all of the longer chain FA are considered to be derived from the diet, depending on the diet composition (
Palmquist, 2006).
Bionaz and Loor (2008) reported that peroxisome proliferator-activated receptor
gamma (PPARG) which is one of members of the nuclear receptor transcription factors was up-regulated and the expression of genes involved in
de novo fatty acid synthesis (acetyl-coenzyme A carboxylase alpha (ACACA) and fatty acid synthase (FASN), fatty acid uptake and transport (Cluster of differentiation 36 (CD36) and Fatty acid-binding protein 3 (FABP3)) and desaturation (Stearoyl-CoA desaturase [SCD]) was stimulated during lactation (
Bionaz and Loor, 2008b). The results suggested that the expression of genes related to milk fat synthesis could be regulated though PPARG.
Kadegowda et al. (2009) demonstrated that the role of PPARG and long-chain fatty acids (LCFA) in regulating milk fat synthesis. Studies indicated that LCFA significantly suppressed
de novo synthesis of SMCFA (
Banks et al., 1976;
Jenkins, 1999;
Warntjes et al., 2008) and inhibited ACACA and FASN mRNA expression (
Kadegowda et al., 2009). Therefore, the expression of genes involved in
de novo synthesis of FA could probably be regulated by PPARG, and further affected the synthesis of milk fat.
Studies indicated that the change of milk fat was usually accompanied by a decrease in milk protein content when fat was added in the cow diets (
Cant et al., 1991;
Jenkins and Mcguiret, 2006;
Weisbjerg et al., 2008).
Weisbjerg et al. (2008) reported that when medium and high yielding cows were fed the diets with 29, 40, and 52 g palm fatty acid distillate fat by substituting barley, general linear responses per 10 g increase in FA ration were 0.039 (p = 0.07) and −0.071 (p<0.0001) for fat and protein concentration, respectively. These data suggested that milk fat synthesis was improved by addition of exogenous LCFA whereas milk protein synthesis was inhibited. However,
Yonezawa et al. (2004) indicated that exogenous LCFA such as palmitate, stearate, oleate, or linoleate stimulated the accumulation of triacylglycerol (TAG) as well as αs1-casein (CSN1S1) mRNA expression in bovine mammary epithelial cells (BMECs). Little data regarding the mechanism of the effect of LCFA on milk fat and protein synthesis is available. Some studies suggested that the mammalian target of rapamycin (mTOR) played a role in FA and TAG synthesis (
Soliman, 2011), together with mammary protein synthesis (
Burgos et al., 2010). Rapamycin inhibited the expression and the transactivation activity of PPARG by blocking mTOR (
Kim and Chen, 2004). Milk fat and protein synthesis might be co-regulated by signal transducer and activator of transcription 5 (STAT5) (
Bernard et al., 2008).
The present study examined the effects of exogenous saturated LCFA on cell proliferation and the accumulation of TAG, together with mRNA expression of CSN1S1 and genes involved in lipid and protein synthesis in BMECs, to provide a theoretical basis for further elucidating the mechanism by which LCFA regulates milk fat and protein synthesis.
DISCUSSION
Compared with the control, all LCFA had no significant effects on cell proliferation at 0.78 to 100 μM, and a significant suppression was observed at 200 to 400 μM when BMECs were cultured with 0.78 to 400 μM stearate, oleate, linoleate, or linolenate (Cui et al., 2012).
Yonezawa et al. (2008b) indicated that there was no significant differences in cell proliferation when BMECs were incubated with or without 100 μM palmitate or stearate, whereas a significant increase occurred compared with the control when incubated with 100 μM oleate or linoleate. However, the present study indicated that BMECs proliferation tended to increase quadratically with increasing addition of palmitate or stearate in a dose-dependent manner, and that the addition of 200 to 500 μM palmitate or 200 to 400 μM stearate promoted more effectively than the other groups, but the positive effects tended to be suppressed when the addition of palmitate or stearate was increased to 500 and 600 μM, respectively. It would therefore appear that a low dose of palmitate or stearate had a stimulatory effect on BMECs proliferation, and that overdose of palmitate or stearate had adverse effects, and inhibited the proliferation of BMECs
in vitro. The reason LCFA could mediate cell proliferation and survival was unknown, and it is probably related to bGPR40, GPR40 is one of the G protein-coupled receptors, and it could be activated by medium and long-chain fatty acids (
Briscoe et al., 2003), and then regulated cell proliferation and survival in mammary epithelial cells (
Yonezawa et al., 2008b). The reason the present result was inconsistent with previous research was probably that the action of LCFA on cell proliferation was dependent on the kinds and amount of exogenous LCFA. There is very little data that examines the relationship between LCFA and cell proliferation in BMECs and further studies are needed to investigate the mechanism.
Milk fat is composed mainly of TAG secreted mammary epithelial cells in the form of droplets of variable size. Cells cultured with LCFA (palmitate, stearate or oleate) at 200 to 400 μM or linoleate at 50 to 400 μM markly increased TAG accumulation (
Yonezawa et al., 2004).
Yonezawa et al. (2008a) showed that 100 μM oleate and linoleate caused a significant increase in the accumulation of TAG, whereas palmitate and stearate failed to do so. In contrast, intracellular TAG contents were 140, 80, and 250% greater than the control, respectively, when 100 μM palmitate, stearate or t10c12 CLA was supplemented in culture medium (
Kadegowda et al., 2009). These results suggested that addition of exogenous LCFA could accelerate the accumulation of intracellular TAG in BMECs. The present experiment observed similar results with palmitate or stearate, and indicated that TAG contents were increased at 0 to 600 μM in a concentration-dependent manner, and the addition of 600 μM was less effective in improving TAG accumulation.
ACACA and
FASN are two important genes implicated in
de novo synthesis of milk fatty acid in bovine mammary tissues.
De novo synthesis of fatty acid, via acetyl-CoA and butyryl-CoA, is executed by
ACACA and
FASN (
Bionaz and Loor, 2008). The present results suggested that expressions of
ACACA and
FASN were suppressed markedly when palmitate was added in the culture medium. The results explained the previous observations by
Noble et al. (1969), who found that the supplementation of 10% palmitic acid in the cow diet decreased the concentrations and yields of 6:0, 10:0, 12:0 and 14:0 and the concentrations of 8:0 and 14:1, but increased the concentrations of 16:0 and 16:1, as well as the yields of 4:0, 16:0 and 16:1 in the milk fat. Similarly, the concentrations of short and medium chain FA (C6:0-C15:0) were decreased or tended to be decreased when C16:0 was supplemented in the diet of lactating dairy cows
in vivo (
Warntjes et al., 2008). So, the present study proposed that palmitate could inhibit
de novo synthesis of milk fatty acid through suppressing genes (
ACACA and
FASN) associated with
de novo fatty acid synthesis. A previous study reported that palmitic acid suppressed
de novo synthesis of fatty acids from acetate in bovine mammary homogenate and the possible reason was an allosteric effect on ACACA or FASN (
Wright et al., 2002). However, opposite results were observed by
Hansen and Knudsen (1987), who reported that addition of palmitic acid to the incubation medium stimulated synthesis and incorporation of fatty acids synthesized
de novo into triacylglycerols. They presumed that palmitic acid acted as a “primer” for triacylglycerol synthesis by acytation of the sn-1 position; thereby it allows medium-chain and short-chain fatty acids to be incorporated into the sn-2 and sn-3 positions (
Hansen and Knudsen., 1987). Therefore, further studies are necessary to solve this inconsistency.
Noble et al. (1969) reported that the addition of 5% or 10% stearic acid instead of starch in the diets of cows decreased the concentrations and yields of 10:0, 12:0, 14:0, 14:1, 16:0, and 16:1 in the milk fat but increased the concentrations and yields of 18:0 and 18:1. In the present study, the suppressing effects of stearate on
ACACA and
FASN mRNA abundance were shown, which was also in agreement with previous observations by
Kadegowda et al. (2009), who reported that treatment with stearate decreased the expression of
ACACA and
FASN in MacT relative to the control. Three possible reasons for inhibitory effect of
de novo fatty acid synthesis by stearate are as follows: i) Stearate inhibited
de novo synthesis of fatty acid by down-regulating the mRNA expression of
ACACA and
FASN; ii) Long-chain acyl-CoA competed with newly synthesized medium-chain acyl-CoA for the sn-2 and sn-3 positions of the triacylglycerol backbone; iii) long-chain acyl-CoA inhibited
de novo synthesis of fatty acid by suppression of acetyl-CoA carboxylase (
Hansen and Knudsen., 1987).
FABP participates in the uptake and intracellular transport of fatty acid in many tissues (
Lehner and Kuksis, 1996). The coexpression of
FABP and
CD36 in the bovine mammary gland implied that these two proteins were indeed involved in a close functional relationship (
Spitsberg et al., 1995). FABP3 involved in intracellular LCFA transport and
FABP3 mRNA abundance was up-regulated during lactation in the bovine mammary gland (
Bionaz and Loor, 2008a).
Kadegowda et al. (2009) reported that palmitate and stearate up-regulated the
FABP3 mRNA expression, whereas t10c12 CLA and cis-9 18:1 had the opposite effect. However, in the present results showed that the expression of FABP3 was inhibited by addition of palmitate or stearate in the culture medium. Less data are available regarding effect of saturated LCFA on
FABP3 mRNA expression and the reason for the discrepancy is still unknown. CD36 played an important role in FA importation in bovine mammary cells (
Bionaz and Loor, 2008b). Previous studies indicated that mRNA abundance of
CD36 was markedly increased when cells were cultured with palmitate, stearate, oleate, linoleate, 20:5 or t10c12 CLA (
Yonezawa et al., 2004;
Kadegowda et al., 2009). This trend was confirmed by the present study.
PPARG is one of members of the nuclear receptor transcription factors.
Kadegowda et al. (2008) reported that Lipogenic genes were up-regulated when MAC-T cells were incubated with the PPARG agonist rosiglitazone compared with the control and the results suggested that PPARG played a role in regulating bovine milk fat synthesis. The present findings suggested that both palmitate and stearate had no significant effect on
PPARG mRNA expression in BMECs, but
PPARG mRNA expression had a tendency to be increased in value with the increasing addition of palmitate and stearate compared with the control.
Kadegowda et al. (2009) indicated that fatty acids including 16:0, 18:0, cis-9 18:1, trans-10 18:1, trans- 10, cis-12 18:2 and 20:5, up-regulated or down-regulated the expression of the lipogenic genes without affecting mRNA abundance of
PPARG in BMEC
S. These results are supported by
in vivo study that the lipid supplement diets had no effect on the expression
PPARG in bovine mammary tissue (
Invernizzi et al., 2010). Although there is no effect of fatty acids on expression
PPARG, we cannot neglect the role for this nuclear receptor transcription factor in milk fat synthesis.
Little information is available regarding the regulation of milk protein synthesis by fatty acids. In this study, the findings indicated that palmitate or stearate elevated linearly or quadratically
CSN1S1 mRNA expression.
Yonezawa et al. (2004) observed that expression of
CSN1S1 was stimulated when saturated or unsaturated fatty acids were supplemented in culture medium. However, previous observation reported that β-casein levels were decreased in HC11 mouse mammary epithelial cells treated with unsaturated fatty acids, but not saturated fatty acid (
Pauloin et al., 2010). There are two possible reasons for this difference: i) the cells are from different species of animals, and HC11 is a cell line ii) HC11 cells are unable to produce αS1-casein (
Pauloin et al., 2010). In addition, the present study examined only the regulation of
CSN1S1 at a transcriptional level by saturated LCFA.
The mTOR protein, a conserved Ser/Thr protein kinase, is comprised of two distinct multi-protein complexes termed mTORC1 and mTORC2 (
Laplante and Sabatini, 2009). The mTOR signaling cascade integrated amino acid availability, cellular energy status, and endocrine signals to regulate protein synthesis by phosphorylating eukaryotic initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1), a translational repressor, and p70 ribosomal protein S6 kinase-1 (S6K1) (
Burgos et al., 2010). The expression of
SREBP1 target genes
ACACA,
FASN, and
SCD was suppressed by rapamycin, suggesting a role for mTORC1 in
de novo synthesis of fatty acids (
Soliman, 2011). mTOR inhibition with rapamycin reduced protein levels and the activity of PPARG
in vitro (
Kim and Chen, 2004). The present study did not investigate the effect of palmitate or stearate on
SREBP1 expression, however, there were no significant differences in mRNA abundance of
mTOR and
STAT5 between saturated fatty acid treatments and the control, although they had a tendency to be increased or be decreased in value with increasing addition of palmitate or stearate. Many protein-protein interactions are involved in regulating STAT5 activity at the level of gene expression (
Furth et al., 2011). Consequently, the fact that the expressions of
mTOR and
STAT5 were not affected by saturated LCFA in our study is not against a role for two signaling pathways in the synthesis of milk fat and protein.
Taken together, the present study revealed that BMECs viability and the accumulation of TAG was stimulated in a dose-dependent matter with increamental addition of saturated LCFA in culture medium. Palmitate and stearate suppressed linearly or quadratically the expression of ACACA, FASN, and FABP3, but had the opposite effect on CD36 and CSN1S1 mRNA abundance. Our results suggested that saturated LCFA could inhibit de novo synthesis of milk fatty acids and accelerate milk protein synthesis by regulating related genes expression. The present results also implied that LCFA regulated milk fat synthesis as well as milk protein synthesis. Significant differences in mRNA expression of PPARG, mTOR, and STAT5 were not observed in this study, so future research is needed to elucidate the role of LCFA in regulation of protein translation. Further investigation is required to examine the exact mechanism in which LCFA regulates milk fatty acids and milk protein synthesis.