Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyl-transferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARbeta/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.
The mineralocorticoid receptor (MR) plays a central role in electrolyte homeostasis and in cardiovascular disease. We have previously reported a ligand-dependent N/C-interaction in the MR. In the present study we sought to fully characterize the MR N/C-interaction. By using a range of natural and synthetic MR ligands in a mammalian two-hybrid assay we demonstrate that in contrast to aldosterone, which strongly induces the interaction, the physiological ligands deoxycorticosterone and cortisol weakly promote the interaction but predominantly inhibit the aldosterone-mediated N/C-interaction. Similarly, progesterone and dexamethasone antagonize the interaction. In contrast, the synthetic agonist 9alpha-fludrocortisol robustly induces the interaction. The ability of the N/C interaction to discriminate between MR agonists suggests a subtle conformational difference in the ligand-binding domain induced by these agonists. We also demonstrate that the N/C interaction is not cell specific, consistent with the evidence from a glutathione-S-transferase pull-down assay, of a direct protein-protein interaction between the N- and C-terminal domains of the MR. Examination of a panel of deletions in the N terminus suggests that several regions may be critical to the N/C-interaction. These studies have identified functional differences between physiological MR ligands, which suggest that the ligand-specific dependence of the N/C-interaction may contribute to the differential activation of the MR that has been reported in vivo.
High plasma high-density lipoproteins (HDL) protect against neurodegenerative disease. However, the underlying mechanisms are largely unexplored. Phospholipid transfer protein (PLTP) is a key protein involved in plasma HDL remodeling and is thought to influence pathogenesis of Alzheimer's disease. We reported earlier that liver-X receptor (LXR) activation promotes cellular cholesterol efflux and formation of HDL-like particles in an in vitro model of the blood-brain barrier (BBB) consisting of porcine brain capillary endothelial cells. We here hypothesized that BCEC may express PLTP which may be involved in HDL metabolism in the brain. Immunohisto/cytochemical staining of porcine brain and cultured primary pBCEC.mRNA expression levels were determined by real-time PCR.PL transfer activity in pBCEC supernatants, cell lysates and in transwell supernatants was determined by radiometric assay. PLTP is expressed in cerebrovascular endothelial cells PLTP expression and activity are up-regulated by LXR activation and in a polarized manner PLTP-mediated modification increases the cholesterol removal capacity of HDL 3 LXR activation up-regulates PLTP activity in vivo Endogenous PLTP remodels HDL and pre β-HDL formation is enhanced by LXR agonist PLTP silencing disrupts the biogenesis of HDL particles Phospholipid transfer protein is expressed in cerebrovascular endothelial cells and involved in HDL biogenesis and remodeling at the blood brain barrier.
Please cite this paper as: Barrett, Parham, Pippal, Cockshell, Moretti, Brice, Pitson, and Bonder (2011). Over‐Expression of Sphingosine Kinase‐1 Enhances a Progenitor Phenotype in Human Endothelial Cells. Microcirculation 18 ( 7 ), 583–597. Abstract Objectives: The use of endothelial progenitor cells in vascular therapies has been limited due to their low numbers present in the bone marrow and peripheral blood. The aim of this study was to investigate the effect of sphingosine kinase on the de‐differentiation of mature human endothelial cells toward a progenitor phenotype. Methods: The lipid enzyme sphingosine kinase‐1 was lentivirally over‐expressed in human umbilical vein endothelial cells and cells were analyzed for progenitor phenotype and function. Results: Sphingosine kinase‐1 mRNA expression was induced approximately 150‐fold with a resultant 20‐fold increase in sphingosine kinase‐1 enzymatic activity. The mRNA expression of the progenitor cell markers CD34, CD133, and CD117 and transcription factor NANOG increased, while the endothelial cell markers analyzed were largely unchanged. The protein level of mature endothelial cell surface markers CD31, CD144, and von Willebrand factor significantly decreased compared to controls. In addition, functional assays provided further evidence for a de‐differentiated phenotype with increased viability, reduced uptake of acetylated low‐density lipoprotein and decreased tube formation in Matrigel in the cells over‐expressing sphingosine kinase‐1. Conclusions: These findings suggest that over‐expression of sphingosine kinase‐1 in human endothelial cells promotes, in part, their de‐differentiation to a progenitor cell phenotype, and is thus a potential tool for the generation of a large population of vascular progenitor cells for therapeutic use.
The overproduction and accumulation of amyloid-beta (Aβ) plays a crucial role in the pathogenesis of Alzheimer's Disease (AD). Aβ accumulation in cerebral capillaries can lead to cerebral amyloid angiopathy, indicating a thus far underestimated role of the blood-brain barrier (BBB) in the pathogenesis of the disease. In addition, it has become increasingly evident that apoJ, also known as clusterin, is involved in cholesterol/lipid trafficking in the brain. Previous studies revealed an increase of apoJ expression in AD. ApoJ can bind to Aβ peptides, prevents their fibrillization, and thereby enhances endocytosis by glial cells. Although evidence exists for a close connection between peripheral and cerebral apolipoprotein-, cholesterol- and amyloid precursor protein (APP)/Aβ metabolism, information about the interconnecting mechanisms occurring in or mediated by brain capillary endothelial cells (BCEC) is lacking. Our central aim is to define the involvement of apoJ in cholesterol, and APP/ Aβ metabolism at the BBB. Primary porcine (p)BCEC were incubated with modulators of cellular cholesterol metabolism, i.e. the endogenous LXR agonists 24(S)OH-cholesterol and 27OH-cholesterol, the synthetic LXR ligand TO901317, cholesterol, and the HMGCo-A reductase inhibitor simvastatin. Protein and mRNA expression levels of apoJ, APP/Aβ and enzymes of the amyloid cascade were part of the examinations. Furthermore, to obtain polarized secretion of apoJ and APP products to the basolateral (mimicking the brain-parenchymal side of the BBB) and apical compartments (mimicking the blood-side of the BBB), pBCEC are cultured on multiwell transwell filters in the absence or presence of modulators of cholesterol metabolism. Real-time PCR and immunoblotting analyses suggest a higher expression of both apoJ and APP mRNA and protein, respectively, in response to simvastatin treatment. Results obtained during transwell studies suggest a release of apoJ primarily to the basolateral as compared to the apical compartment, which is altered by modulating cellular cholesterol metabolism. ApoJ synthesis and secretion towards the basolateral compartment of the BBB may suggest a beneficial role of apoJ in APP/Aβ metabolism. Since APP processing and production of Aβ is linked to cholesterol homeostasis, future experiments are aiming to elucidate the role of apoJ in APP/Aβ metabolism at the BBB in vitro (and in vivo).
Nuclear hormone receptors (NRs) are agonist-dependent transcription factors that translate nutritional and physiological signals into gene regulation. The significance of NRs in human health is highlighted by the variety of medicinal drugs associated with dysfunctional hormone signalling, in the context of inflammation, endocrine and metabolic diseases (including dyslipidemia and diabetes). A subgroup of the NR family controls metabolism in a tissue/cell specific manner. For example, the Peroxisome Proliferator Activated Receptor (PPAR) subgroup comprising PPARa, â/a and a isoforms, regulate lipid storage, adipogenesis and lipid catabolism in an organ specific manner. For example, agonists such as the hypolipidemicfibrates and insulin sensitizer-thiazolidinedione (TZD), that modulate PPARa and PPARa respectively, have utility in the treatment of dyslipidemia and diabetes, respectively. Many NRs are expressed in skeletal muscle, a major mass tissue that accounts for ~40% of the total body mass and energy expenditure. This lean tissue is a major site for lipid mobilization and catabolism, cholesterol efflux, and insulin-stimulated glucose disposal. Moreover, this tissue expresses cytokines that regulate adiposity, and energy expenditure, in a process that involves reciprocal signalling between adipose tissue and muscle. Therefore, this peripheral organ plays a critical role in insulin sensitivity, the blood lipid profile, and energy balance. Accordingly, skeletal muscle has an important role in the progression of dyslipidemia, diabetes and obesity. Further, several studies demonstrate that NRs in muscle regulate carbohydrate, lipid and energy homeostasis. Therefore, NR and skeletal muscle are therapeutic targets in the battle against metabolic disorders. PPARa is abundantly expressed in skeletal muscle, surprisingly, the precise role and function of PPARa in skeletal muscle cell metabolism has not been examined. Nevertheless, given the utility of hypolipidemic fibrate drugs, and the contribution of muscle to lipid homeostasis, the role of PPARa in skeletal muscle needs to be further investigated. Correspondingly, the objective of this study was to examine the differential effects of clinically utilized fibrates on metabolic gene expression in skeletal muscle cells. Furthermore, we were interested in identifying the molecular mechanisms that mediated the agonist specific effects on gene expression. Consequently, we utilized a number of clinically used hypolipidemic fibrates including fenofibrate, clofibrate, ciprofibrate and gemfibrozil to demonstrate that PPARa, can functionally regulate genes involved in lipid and carbohydrate homeostasis in skeletal muscle cells. We demonstrate that fenofibrate induces genes involved in fructose uptake and glycogen metabolism in skeletal muscle cells. Interestingly, fenofibrate represses the mRNA expression of SREBP1c and ABCA1, classical LXR target genes. Moreover, we demonstrate that the different PPARa agonists have similar, but distinct effects on gene expression. Each drug appears to have a unique regulatory footprint. These studies also suggest that the differential effects of fibrate esters and acids in skeletal muscle cells involves cross talk with the oxy-sterol dependent LXR signaling pathway. In addition, we show overlapping, and distinct effects of selective ligands for PPAR-a, -â/a and a isoform in skeletal muscle cells, where PPARa regulates expression of genes implicated in, TG hydrolysis, fructose uptake and glycogen synthesis, PPARâ/a isoform regulates expression of genes implicated in preferential lipid utilization, FA catabolism and energy uncoupling and finally, PPARa regulates genes involved in glucose uptake, FA synthesis and lipid storage. We utilized cDNA microarray expression profiling technology to rigorously characterize the genes that were upregulated or downregulated in response to the PPARa agonists. Unfortunately, due to several technical shortcomings we were unable to extensively elucidate the transcriptional program induced in response to the PPARa agonists. This is elaborately discussed in Chapter-4. However, we identified two genes implicated in lipid metabolism, Melanocortin 2 receptor (MC2R) and Glutathione-S-Transferase (GST), which were differentially expressed in response to fenofibrate. In addition we observed Sortilin and Insig1 were differentially expressed in response to wyeth14643. MC2R belongs to the melanocortin pathway and is implicated in the process of lipolysis, while GST is an oxidative stress response gene that prevents the free radical formation, that are linked to the development of insulin resistance. Additionally, Sortilin and Insig1 are implicated in the insulin mediated glucose uptake and antilipogenic activity respectively. The differential expression of these genes provided us with a new direction by which PPARa may be regulating the pathogenesis of metabolic diseases as a manifestation of lipid imbalance. However, due to limited time for this study, validation of these results could not be performed. We had observed that fenofibrate repressed the expression of the LXR target genes, SREBP1c and ABCA1 in the presence and absence of the LXR agonist, T0901317. Therefore, we endeavored to investigate the molecular mechanism underlying the regulatory cross talk between PPARa and LXR signaling pathways. Our studies utilizing the GAL4 hybrid assay suggest that fenofibrate antagonizes LXR agonist mediated activation. We hypothesized that fenofibrate antagonizes LXR activity by attenuating LXR mediated corepressor displacement and/or cofactor recruitment. However, in a mammalian two-hybrid assay, we observed that fenofibrate (in the absence of LXR agonist) efficiently induced corepressor displacement from LXR. In contrast, fenofibrate had an additive effect on LXR dependent cofactor recruitment. These studies presents us with a paradox, that although fenofibrate represses LXR activity, it does not prevent corepressor displacement or inhibit coactivator recruitment by LXR. These data suggest fenofibrate-mediated attenuation of LXR activity and LXR dependent gene expression does not involve cofactor displacement/recruitment. Further studies are therefore required to elucidate the precise mechanism of fenofibrate-mediated modulation of LXR activity and LXR dependent gene expression. In conclusion, the activation of PPARa in skeletal muscle and the transcriptional regulation of the genes involved in the lipid and carbohydrate homeostasis, suggests PPARa activation in skeletal muscle is an important site for the therapeutic effect of fibrates.
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