Peroxisome proliferator-activated receptor delta regulates lipid droplet formation and transport in goat mammary epithelial cells

Abstract

Even though recent evidence in goat mammary epithelial cells (GMEC) suggest a role of peroxisome proliferator-activated receptor delta (PPARD) in regulating lipid homeostasis, its role is not fully understood. Our hypothesis was that PPARD regulates lipid transport processes in GMEC and, thus, plays a crucial role in regulating fat formation. The PPARD was overexpressed using an adenovirus system (Ad-PPARD) with recombinant green fluorescent protein (Ad-GFP) as the control. Results revealed that overexpression of PPARD markedly upregulated the mRNA abundance of PPARD. Compared with the control (Ad-GFP+dimethyl sulfoxide), overexpression of PPARD alone had no effect on mRNA expression of CD36, SCD1, FABP4, ACSL1, and ADRP. The cultures overexpressing PPARD with the PPARD ligand GW0742 (GW) upregulated the expression of CD36, FABP3, FABP4, ACSL1, and ADRP. Overexpression of PPARD in GMEC plus GW increased the concentration of 16:1 and 18:1-trans and was associated with upregulation of SCD1. Compared with the control (Ad-GFP+dimethyl sulfoxide), the decrease of triacylglycerol concentration coupled with upregulation of genes related to lipid droplet secretion (e.g., ADRP and ACSL1) induced by PPARD overexpression suggests a role in lipid droplet (LD) secretion. Luciferase assay revealed that GW increased the ADRP promoter activity in a dose-dependent manner. Knockdown of PPARD impaired the increase of ADRP promoter activity induced by GW, whereas GW enhanced the activity of ADRP promoter in GMEC overexpressing PPARD. Data with the ADRP 5'-flanking truncated luciferase reporter suggest a core region (-1,444 to -990 bp) response element for the induction of GW. This core region contains a known PPARG response element (PPRE) at -1,003 to -990 bp. When the PPRE was mutated, the overexpression of PPARD had no effect on ADRP promoter activity. Collectively, these results reveal a novel role for PPARD in lipid homeostasis via promoting fatty acid transport and LD formation through a mechanism of direct binding to the promoter of key genes. Hence, PPARD activity may contribute to fatty acid transport and LD formation during lactation.

ACSL1 is a key regulator of inflammatory and macrophage foaming induced by short-term palmitate exposure or acute high-fat feeding

“… 28 , 56 The emerging evidence, including our data, seems to be supporting the notion that other PPARs, such as PPARδ, may also regulate CD36-mediated lipid homeostasis by promoting fatty acid uptake, transport, and lipid droplet formation. 57 Similarly, another study by Nahle et al. showed that during the fasting state, activation of PPARδ subsequently upregulated the expression of CD36 to increase fatty acid utilization in the skeletal muscle.…”

Regulation of Key Genes for Milk Fat Synthesis in Ruminants

“…The mRNA abundance of CD36 was down-regulated by 56% after interfering PPARA in GMECs, which was the same as the positive regulatory effect of PPARA in the de novo synthesis of milk fat, however, interfering PPARA up-regulated the mRNA abundance of ACSL1 and also significantly increased TAG content, suggesting a stronger negative regulation of ACSL1 by PPARA and further interpreting the dominance of LCFA uptake in the blood ( 37 ). Several studies have confirmed that t10c12-CLA, PPARG and PPARD exhibit positive regulation of both CD36 ( 35 , 87 , 106 ) and ACSL1 ( 34 , 36 , 63 ), however, Shi et al ( 61 ) challenge the above view that t10c12-CLA A positively regulates CD36 and ACSL1, which may be related to factors such as the species, mode and amount of additives, the exact mechanism of action needs to be further verified by more in-depth experimental studies as well as large-scale populations. As previously described, 14-3-3γ, LXR, SREBP1 and CIDEC have positive regulatory roles in the de novo synthesis of milk fat, which perform similar functions during LCFA uptake ( 21 , 29 , 36 , 69 ).…”

Enhancement of PPARα-Inhibited Leucine Metabolism-Stimulated β-Casein Synthesis and Fatty Acid Synthesis in Primary Bovine Mammary Epithelial Cells

“…Twelve hours before treatment with the leucine-containing medium, BMECs were treated with WY14643 in an amino acid-free medium and the cells were cultured with dimethyl sulfoxide (DMSO) serving as control. PPARα was overexpressed using an adenoviral system as described previously . BMECs were incubated with the adenovirus medium Ad-PPARα and Ad-GFP.…”

“…PPARα was overexpressed using an adenoviral system as described previously. 20 BMECs were incubated with the adenovirus medium Ad-PPARα and Ad-GFP. After treatment, BMECs were harvested for RNA analysis, cholesterol assay, and other experimental examinations.…”