Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor gamma expression

Liver X receptors (LXRs) are nuclear hormone receptors that regulate cholesterol and fatty acid metabolism in liver tissue and in macrophages. Although LXR activation enhances lipogenesis, it is not well understood whether LXRs are involved in adipocyte differentiation. Here, we show that LXR activation stimulated the execution of adipogenesis, as determined by lipid droplet accumulation and adipocyte-specific gene expression in vivo and in vitro. In adipocytes, LXR activation with T0901317 primarily enhanced the expression of lipogenic genes such as the ADD1/SREBP1c and FAS genes and substantially increased the expression of the adipocyte-specific genes encoding PPARγ (peroxisome proliferator-activated receptor γ) and aP2. Administration of the LXR agonist T0901317 to lean mice promoted the expression of most lipogenic and adipogenic genes in fat and liver tissues. It is of interest that the PPARγ gene is a novel target gene of LXR, since the PPARγ promoter contains the conserved binding site of LXR and was transactivated by the expression of LXRα. Moreover, activated LXRα exhibited an increase of DNA binding to its target gene promoters, such as ADD1/SREBP1c and PPARγ, which appeared to be closely associated with hyperacetylation of histone H3 in the promoter regions of those genes. Furthermore, the suppression of LXRα by small interfering RNA attenuated adipocyte differentiation. Taken together, these results suggest that LXR plays a role in the execution of adipocyte differentiation by regulation of lipogenesis and adipocyte-specific gene expression.     

Palmitic Acid Induces Osteoblastic Differentiation in Vascular Smooth Muscle Cells through ACSL3 and NF-κB,Novel Targets of Eicosapentaenoic Acid

Free fatty acids (FFAs), elevated in metabolic syndrome and diabetes, play a crucial role in the development of atherosclerotic cardiovascular disease, and eicosapentaenoic acid (EPA) counteracts many aspects of FFA-induced vascular pathology. Although vascular calcification is invariably associated with atherosclerosis, the mechanisms involved are not completely elucidated. In this study, we tested the hypothesis that EPA prevents the osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC) induced by palmitic acid (PA), the most abundant long-chain saturated fatty acid in plasma. PA increased and EPA abolished the expression of the genes for bone-related proteins, including bone morphogenetic protein (BMP)-2, Msx2 and osteopontin in human aortic smooth muscle cells (HASMC). Among the long-chain acyl-CoA synthetase (ACSL) subfamily, ACSL3 expression was predominant in HASMC, and PA robustly increased and EPA efficiently inhibited ACSL3 expression. Importantly, PA-induced osteoblastic differentiation was mediated, at least in part, by ACSL3 activation because acyl-CoA synthetase (ACS) inhibitor or siRNA targeted to ACSL3 completely prevented the PA induction of both BMP-2 and Msx2. Conversely, adenovirus-mediated ACSL3 overexpression enhanced PA-induced BMP-2 and Msx2 expression. In addition, EPA, ACSL3 siRNA and ACS inhibitor attenuated calcium deposition and caspase activation induced by PA. Notably, PA induced activation of NF-κB, and NF-κB inhibitor prevented PA-induction of osteoblastic gene expression and calcium deposition. Immunohistochemistry revealed the prominent expression of ACSL3 in VSMC and macrophages in human non-calcifying and calcifying atherosclerotic plaques from the carotid arteries. These results identify ACSL3 and NF-κB as mediators of PA-induced osteoblastic differentiation and calcium deposition in VSMC and suggest that EPA prevents vascular calcification by inhibiting such a new molecular pathway elicited by PA.     

Fatty acid transport by vectorial acylation in mammals: roles played by different isoforms of rat long-chain acyl-CoA synthetases

Mammals express multiple isoforms of acyl-CoA synthetase (ACSL1 and ACSL3-6) in various tissues. These enzymes are essential for fatty acid metabolism providing activated intermediates for complex lipid synthesis, protein modification, and beta-oxidation. Yeast in contrast express four major ACSLs, which have well-defined functions. Two, Faa1p and Faa4p, are specifically required for fatty acid transport by vectorial acylation. Four ACSLs from the rat were expressed in a yeast faa1delta faa4delta strain and their roles in fatty acid transport and trafficking characterized. All four restored ACS activity yet varied in substrate preference. ACSL1, 4, and 6 were able to rescue fatty acid transport activity and triglyceride synthesis. ACSL5, however, was unable to facilitate fatty acid transport despite conferring robust oleoyl-CoA synthetase activity. This is the first study evaluating the role of the mammalian ACSLs in fatty acid transport and supports a role for ACSL1, 4, and 6 in transport by vectorial acylation.     

Acyl-CoA synthetase long-chain 3-mediated fatty acid oxidation is required for TGFβ1-induced epithelial-mesenchymal transition and metastasis of colorectal carcinoma

Cancer cells frequently undergo metabolic reprogramming to support tumorigenicity and malignancy, which is recognized as a hallmark of cancer. In addition to glycolysis and glutaminolysis, alterations in fatty acid (FA) metabolism have received increasing concerns in the past few years. Recently, accumulating evidence has shown that fatty acid β-oxidation (FAO) is abnormally activated in various tumors, which is associated with the machinery of proliferation, stemness, metastasis, and radiochemotherapeutic resistance of cancer cells. Acyl-CoA synthetases 3 (ACSL3) belongs to a family of enzymes responsible for converting free long-chain FAs into fatty acyl-CoA esters, which act as substrates both for lipid synthesis and FAO.Here, we demonstrate that transforming growth factor beta 1 (TGFβ1) induces the up-regulation of ACSL3 through sterol regulatory element-binding protein 1 (SREBP1) signaling to promote energy metabolic reprogramming in colorectal carcinoma (CRC) cells. ACSL3 mediates the epithelial mesenchymal transition (EMT) and metastasis of CRC cells by activation of FAO pathway to produce ATP and reduced nicotinamide adenine dinucleotide phosphate (NADPH), which sustain redox homeostasis and fuel cancer cells for invasion and distal metastasis. Thus, targeting ACSL3 and FAO metabolic pathways might be exploited for therapeutic gain for CRC and other FAs- addicted cancers.     

Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation

ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate. Although an association between elevated levels of ACSL4 and hepatosteatosis has been reported, the function of ACSL4 in hepatic FA metabolism and the regulation of its functional expression in the liver remain poorly defined. Here we provide evidence that AA selectively downregulates ACSL4 protein expression in hepatic cells. AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis. The inhibitory action of AA on ACSL4 protein stability could not be prevented by rosiglitazone or inhibitors that interfere with the cellular pathways involved in AA metabolism to biologically active compounds. In contrast, treatment of cells with inhibitors specific for the proteasomal degradation pathway largely prevented the AA-induced ACSL4 degradation. We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination. Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.     

Overexpressed FATP1, ACSVL4/FATP4 and ACSL1 Increase the Cellular Fatty Acid Uptake of 3T3-L1 Adipocytes but Are Localized on Intracellular Membranes

Long chain acyl-CoA synthetases are essential enzymes of lipid metabolism, and have also been implicated in the cellular uptake of fatty acids. It is controversial if some or all of these enzymes have an additional function as fatty acid transporters at the plasma membrane. The most abundant acyl-CoA synthetases in adipocytes are FATP1, ACSVL4/FATP4 and ACSL1. Previous studies have suggested that they increase fatty acid uptake by direct transport across the plasma membrane. Here, we used a gain-of-function approach and established FATP1, ACSVL4/FATP4 and ACSL1 stably expressing 3T3-L1 adipocytes by retroviral transduction. All overexpressing cell lines showed increased acyl-CoA synthetase activity and fatty acid uptake. FATP1 and ACSVL4/FATP4 localized to the endoplasmic reticulum by confocal microscopy and subcellular fractionation whereas ACSL1 was found on mitochondria. Insulin increased fatty acid uptake but without changing the localization of FATP1 or ACSVL4/FATP4. We conclude that overexpressed acyl-CoA synthetases are able to facilitate fatty acid uptake in 3T3-L1 adipocytes. The intracellular localization of FATP1, ACSVL4/FATP4 and ACSL1 indicates that this is an indirect effect. We suggest that metabolic trapping is the mechanism behind the influence of acyl-CoA synthetases on cellular fatty acid uptake.     

Rat long chain acyl-CoA synthetase 5 increases fatty acid uptake and partitioning to cellular triacylglycerol in McArdle-RH7777 cells

Long chain acyl-CoA synthetase (ACSL) catalyzes the initial step in long chain fatty acid metabolism. Of the five mammalian ACSL isoforms cloned and characterized, ACSL5 is the only isoform found to be located, in part, on mitochondria and thus was hypothesized to be involved in fatty acid oxidation. To elucidate the specific roles of ACSL5 in fatty acid metabolism, we used adenoviral-mediated overexpression of ACSL5 (Ad-ACSL5) in rat hepatoma McArdle-RH7777 cells. Confocal microscopy revealed that Ad-ACSL5 colocalized to both mitochondria and endoplasmic reticulum. When compared with cells infected with Ad-GFP, Ad-ACSL5-infected cells at 24 h after infection had 2-fold higher acyl-CoA synthetase activities and 30% higher rates of fatty acid uptake when incubated with 500 microM [1-(14)C]oleic acid. Metabolism of [1-(14)C]oleic acid to cellular triacylglycerol (TAG) increased 42% in Ad-ACSL5-infected cells, but when compared with control cells, metabolism to acid-soluble metabolites, phospholipids, and medium TAG did not differ substantially. The incorporation of [1-(14)C]oleate and [1,2,3-(3)H]glycerol into TAG was similar in Ad-ACSL5-infected cells, thus indicating that Ad-ACSL5 increased TAG synthesis through both de novo and reacylation pathways. However, [1-(14)C]acetic acid incorporation into cellular lipids showed that, when compared with control cells, Ad-ACSL5-infected cells did not increase the metabolism of fatty acids that were derived from de novo synthesis. These results suggest that uptake of fatty acids into cells is regulated by metabolism and that overexpressed ACSL5 partitions exogenously derived fatty acids toward TAG synthesis and storage.     

Susceptibility of Pancreatic Beta Cells to Fatty Acids Is Regulated by LXR/PPARα-Dependent Stearoyl-Coenzyme A Desaturase

Chronically elevated levels of fatty acids-FA can cause beta cell death in vitro. Beta cells vary in their individual susceptibility to FA-toxicity. Rat beta cells were previously shown to better resist FA-toxicity in conditions that increased triglyceride formation or mitochondrial and peroxisomal FA-oxidation, possibly reducing cytoplasmic levels of toxic FA-moieties. We now show that stearoyl-CoA desaturase-SCD is involved in this cytoprotective mechanism through its ability to transfer saturated FA into monounsaturated FA that are incorporated in lipids. In purified beta cells, SCD expression was induced by LXR- and PPARα-agonists, which were found to protect rat, mouse and human beta cells against palmitate toxicity. When their SCD was inhibited or silenced, the agonist-induced protection was also suppressed. A correlation between beta cell-SCD expression and susceptibility to palmitate was also found in beta cell preparations isolated from different rodent models. In mice with LXR-deletion (LXRβ^-/- and LXRαβ^-/-), beta cells presented a reduced SCD-expression as well as an increased susceptibility to palmitate-toxicity, which could not be counteracted by LXR or PPARα agonists. In Zucker fatty rats and in rats treated with the LXR-agonist TO1317, beta cells show an increased SCD-expression and lower palmitate-toxicity. In the normal rat beta cell population, the subpopulation with lower metabolic responsiveness to glucose exhibits a lower SCD1 expression and a higher susceptibility to palmitate toxicity. These data demonstrate that the beta cell susceptibility to saturated fatty acids can be reduced by stearoyl-coA desaturase, which upon stimulation by LXR and PPARα agonists favors their desaturation and subsequent incorporation in neutral lipids.