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

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.

A novel peroxisome proliferator response element modulates hepatic low-density lipoprotein receptor gene transcription in response to PPARδ activation.

The hepatic expression of low-density lipoprotein (LDL) receptor (LDLR) gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative peroxisome proliferator-activated receptor (PPAR)-response element (PPRE) sequence motif located at -768 to -752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin (RSV)-mediated transactivation. EMSA and ChIP assay further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression.

Identification of PPREs and PPRE associated genes in the human genome: insights into related kinases and disease implications.

Introduction: "Peroxisome Proliferator-Activated Receptors" (PPARs) belong to the class of transcription factors (TF) identified as Nuclear Receptors (NR). Upon activation by peroxisome proliferators (PPs), PPARs modulate a diverse range of genes, consequently regulating intra-cellular lipid metabolism, glucose uptake, apoptosis, and cell proliferation. Subsequent to the heterodimerization of Retinoid X Receptors (RXR) with PPARs induced by the binding of activators to PPARs, facilitates the binding of the resulting complex to Peroxisome Proliferator-Activated Receptors Response Elements (PPRE), with a consensus sequence 5'AGGTCANAGGTCA-3', and regulate the transcription of the targeted genes. Methods: A comprehensive screening of PPRE within the whole human genome was performed using the Genome Workbench and UCSC Genome Browser to find the associated genes. Subsequently, the kinase subset was isolated from the extracted list of PPRE-related genes. Functional enrichment of the kinases was performed using FunRich, ToppGene, and ShinyGO. Network analysis and enrichment studies were then further performed using NDEx to elucidate these identified kinases' connections and significance. Additionally, the disease association of the PPRE kinases was analyzed using DisGeNET data in R studio and the COSMIC dataset. Results: A comprehensive analysis of 1002 PPRE sequences within the human genome (T2T), yielded the identification of 660 associated genes, including 29 kinases. The engagement of these kinases in various biological pathways, such as apoptosis, platelet activation, and cytokine pathways, revealed from the functional enrichment analysis, illuminates the multifaceted role of PPAR in the regulation of cellular homeostasis and biological processes. Network analysis reveals the kinases interact with approximately 5.56% of the Human Integrated Protein-Protein Interaction rEference (HIPPIE) network. Disease association analysis using DisGeNET and COSMIC datasets revealed the significant roles of these kinases in cellular processes and disease modulation. Discussion: This study elucidates the regulatory role of PPAR-associated genes and their association with numerous biological pathways. The involvement of the kinases with disease-related pathways highlights new potential for the development of therapeutic strategies designed for disease management and intervention.

Fatty acids regulate perilipin5 in muscle by activating PPARδ.

The surface of lipid droplets (LDs) in various cell types is coated with perilipin proteins encoded by the Plin genes. Perilipins regulate LD metabolism by selectively recruiting lipases and other proteins to LDs. We have studied the expression of perilipins in mouse muscle. The glycolytic fiber-enriched gastrocnemius muscle expresses predominantly Plin2-4. The oxidative fiber-enriched soleus muscle expresses Plin2-5. Expression of Plin2 and Plin4-5 is elevated in gastrocnemius and soleus muscles from mice fed a high-fat diet. This effect is preserved in peroxisome proliferator-activated receptor (PPAR)α-deficient mice. Mouse muscle derived C2C12 cells differentiated into glycolytic fibers increase transcription of these Plins when exposed to various long chain fatty acids (FAs). To understand how FAs regulate Plin genes, we used specific activators and antagonists against PPARs, Plin promoter reporter assays, chromatin immunoprecipitation, siRNA, and animal models. Our analyses demonstrate that FAs require PPARδ to induce transcription of Plin4 and Plin5. We further identify a functional PPAR binding site in the Plin5 gene and establish Plin5 as a novel direct PPARδ target in muscle. Our study reveals that muscle cells respond to elevated FAs by increasing transcription of several perilipin LD-coating proteins. This induction renders the muscle better equipped to sequester incoming FAs into cytosolic LDs.