Liver x receptors stimulate lipogenesis in bovine mammary epithelial cell culture but do not appear to be involved in diet-induced milk fat depression in cows

Abstract

Abstract Milk fat synthesis of ruminants can be inhibited by intermediates of ruminal fatty acid biohydrogenation including trans-10, cis-12 conjugated linoleic acid (CLA). These biohydrogenation intermediates signal a coordinated downregulation of genes involved in mammary FA synthesis, transport, and esterification. We have previously reported decreased mammary expression of sterol response element-binding protein 1 (SREBP1), SREBP1-activating proteins, and thyroid hormone-responsive spot 14 (S14) in the cow during diet-induced milk fat depression (MFD), and treatment with trans-10, cis-12 CLA. Liver x receptors (LXR) and retinoid x receptors (RXR) regulate lipogenesis and are known to bind polyunsaturated FA and LXR agonist increases lipid synthesis in mammary epithelial cell culture. The current studies investigated if biohydrogenation products of rumen origin inhibit mammary lipogenesis through LXR and/or RXR. Expression of LXRs was not different in lactating compared to nonlactating bovine mammary tissue, and expression of LXRs, RXRα, and selected LXR and RXR target genes was not changed in mammary tissue during diet-induced or CLA-induced MFD in the cow. In bovine mammary epithelial cell culture, LXR agonist stimulated lipogenesis and expression of LXRß, ATP-binding cassette 1 (ABCA1), SREBP1c, and S14, but LXR activation did not overcome CLA inhibition of lipogenesis and downregulation of LXRß, SREBP1c, and S14 expression. Lastly, expression of the LXR-regulated carbohydrate-responsive element-binding protein (ChREBP) was higher in lactating than nonlactating tissue and was decreased during CLA-induced MFD. We conclude that changes in mammary LXR expression in dairy cows are not involved in MFD and that trans-10, cis-12 CLA inhibition of lipogenesis and diet-induced MFD appears independent of direct LXR signaling.

Keywords: Carbohydrate‐responsive element‐binding protein; conjugated linoleic acid; lipogenesis; liver x receptor; milk fat depression; retinoid x receptors.

Figure 1.

Working model of the relationship between liver x receptor (LXR) and regulation of mammary lipid synthesis tested in the current experiments. Transcriptional downregulation of lipogenic enzymes, such as fatty acid synthase (FASN), results in decreased mammary lipogenic capacity and reduced milk fat synthesis during milk fat depression in the cow. In other model systems, ligand‐activated LXR/RXR heterodimers increase expression of lipogenic enzymes such as fatty acid synthase (FASN) directly (solid arrows) and indirectly (dashed arrows) through increased expression of thyroid hormone‐responsive spot 14 (S14), sterol response element‐binding protein 1 (SREBP1), and carbohydrate response element‐binding protein (ChREBP). Additionally, secondary signals include autoregulation of SREBP1 (bold arrow) and SREBP1 regulation of S14 expression. Lastly, ABCA1 is more specific marker of LXR activity as it is predominantly regulated by LXR and is not regulated by other major lipogenesis regulators (SREBP1, S14, and ChREBP).

Figure 2.

Tissue profile of liver x receptor alpha and beta (LXRα and LXRβ) and carbohydrate response element‐binding protein (ChREBP) in the cow. Panel A‐C: Tissue expression in mid‐to‐late lactation [n = 6 for subcutaneous adipose tissue (AT), liver (Liv), and lactating mammary gland (Lact), and n = 3 for uterus (Uter), lung, brain, skeletal muscle (Musc), and heart; n signifies tissue from different animals]. Panel D: Expression in lactating and nonlactating tissue (n = 7 cows sampled in both states). Values represent least‐square means ± SEM. Means are scaled relative to lactating tissue (control set to 100). Panels A, B, and D are linear plots, and Panel C is a semilog plot. Means within panels A to C that differed by P < 0.05 are denoted by different letters and difference between lactating and nonlactating tissue in panel D indicated (*P < 0.05).

Figure 3.

Effects of milk fat depression induced by trans‐10, cis‐12 conjugated linoleic acid (CLA) or a low‐forage, high‐oil diet (LF/HO) on mammary mRNA abundance in dairy cows. Panel A: Liver x receptor alpha and beta (LXRα and LXRβ) and the LXR‐responsive gene ATP‐binding cassette 1 (ABCA1). Panel B: retinoid X receptor alpha (RXRα), the RXR‐responsive gene protein kinase C substrate 80K‐H (PRKCSH), and carbohydrate response element‐binding protein (ChREBP). Infusion of trans‐10, cis‐12 CLA decreased milk fat concentration and yield by 23 and 24%, respectively, and the LF/HO diet decreased milk fat concentration and yield 31 and 38%, respectively (Harvatine and Bauman 2006). Values represent least‐square means ± SEM. Means are scaled relative to control (control set to 100; n =8–9 samples per treatment). Significant difference between lactating and nonlactating tissue indicated (*P <0.05).

Figure 4.

Effect of trans‐10, cis‐12 conjugated linoleic acid (CLA) and LXR agonist (TO9) on lipogenesis in a bovine mammary epithelial cell line (MAC‐T). Lipogenesis was measured by incorporation of ¹⁴C acetate into lipids during the last 4 h of treatment and data are expressed as a percent of control. Panel A: Cells were treated for 24 h in basal media with 75 µmol/L CLA, 5 µmol/L LXR agonist (TO‐901317), 10 µmol/L of 9‐cis retinoic acid (9c RA), or combinations of these treatments. Panel B: Cells were treated for 24h in basal media supplemented with 5% fetal bovine serum and 2.5 µg/mL of bovine insulin with 75 µmol/L CLA, 5 µmol/L LXR agonist (TO‐901317), or combinations of these treatments. Values represent least‐square means ± SEM (includes intra‐ and interexperimental run error). Means are scaled relative to control (control set to 100; n =6 wells per treatment across two independent experiments Panel A and n =3 wells per treatment for panel B). Means within panels that differed by P <0.05 are denoted by different letters.

Figure 5.

Effect of trans‐10, cis‐12 conjugated linoleic acid (CLA) and LXR agonist (TO9) on gene expression in a bovine mammary epithelial cell line (MAC‐T). Cells were treated for 24 h in basal media with 75 µmol/L CLA, 5 µmol/L LXR agonist (TO‐901317), 10 µmol/L of 9‐cis retinoic acid (9c RA) or combinations of these treatments. Panel A: Expression of liver x receptor beta (LXRß); Panel B: Expression of fatty acid synthase (FASN) known to be responsive to LXR and SREBP1c in rodent liver; Panel C: Expression of SREBP1c known to be regulated by LXR and SREBP1c; Panel D: Expression of thyroid hormone‐responsive spot 14 (S14) known to be regulated by LXR and SREBP1c; and Panel E: Expression of ABCA1 that is predominantly regulated by LXR. Values represent least‐square means ± SEM (includes intra and inter experimental run error). Means are scaled relative to control (control set to 100; n =5‐8 per treatment). Panel A to D are linear plots and Panel E is a semilog plot. Means within panels that differed by P <0.05 are denoted by different letters.

Figure 6.

Regulation of the SREBP1c promoter by trans‐10, cis‐12 conjugated linoleic acid (CLA). Luciferase plasmids containing approximately a 1.3 kb fragment of the murine SREBP1C promoter with site‐specific mutations in the sterol response element (Δ SRE), LXR response element (Δ LXRE), or both (Δ SRE&LXRE) from Chen et al. (; plasmids D, m24, m31, and m34). Activity of the wild‐type (WT) and mutated promoters was tested in basal media with vehicle control (BSA) or with the 75 µmol/L CLA for 24 h. Values represent least‐sqaure means ± SEM. Means are scaled relative to control (control set to 100; n =6 per treatment). Means that differed during control treatment by P <0.05 are denoted by different letters and inhibition by CLA within each promoter construct is shown along with the percent inhibition relative to control media (*P <0.05 and **P <0.01).