Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes

Recently, a novel oxysterol, 5-cholesten-3beta, 25-diol 3-sulfate (25HC3S) was identified in primary rat hepatocytes following overexpression of the cholesterol transport protein, StarD1. This oxysterol was also detected in human liver nuclei. In the present study, 25HC3S was chemically synthesized. Addition of 25HC3S (6 microM) to human hepatocytes markedly inhibited cholesterol biosynthesis. Quantitative RT-PCR and Western blot analysis showed that 25HC3S markedly decreased HMG-CoA reductase mRNA and protein levels. Coincidently, 25HC3S inhibited the activation of sterol regulatory element binding proteins (SREBPs), suggesting that inhibition of cholesterol biosynthesis occurred via blocking SREBP-1 activation, and subsequently by inhibiting the expression of HMG CoA reductase. 25HC3S also decreased SREBP-1 mRNA levels and inhibited the expression of target genes encoding acetyl CoA carboxylase-1 (ACC-1) and fatty acid synthase (FAS). In contrast, 25-hydroxycholesterol increased SREBP1 and FAS mRNA levels in primary human hepatocytes. The results imply that 25HC3S is a potent regulator of SREBP mediated lipid metabolism.     

Identification of Novel Regulatory Cholesterol Metabolite, 5-Cholesten, 3β,25-Diol,Disulfate

Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3β, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.     

Cholesterol metabolite, 5-cholesten-3β-25-diol-3-sulfate, promotes hepatic proliferation in mice

Oxysterols are well known as physiological ligands of liver X receptors (LXRs). Oxysterols, 25-hydroxycholesterol (25HC) and 27-hydroxycholesterol as endogenous ligands of LXRs, suppress cell proliferation via LXRs signaling pathway. Recent reports have shown that sulfated oxysterol, 5-cholesten-3β-25-diol-3-sulfate (25HC3S) as LXRs antagonist, plays an opposite direction to oxysterols in lipid biosynthesis. The present report was to explore the effect and mechanism of 25HC3S on hepatic proliferation in vivo. Following administration, 25HC3S had a 48h half life in the circulation and widely distributed in mouse tissues. Profiler? PCR array and RTqPCR analysis showed that either exogenous or endogenous 25HC3S generated by overexpression of oxysterol sulfotransferase (SULT2B1b) plus administration of 25HC significantly up-regulated the proliferation gene expression of Wt1, Pcna, cMyc, cyclin A, FoxM1b, and CDC25b in a dose-dependent manner in liver while substantially down-regulating the expression of cell cycle arrest gene Chek2 and apoptotic gene Apaf1. Either exogenous or endogenous administration of 25HC3S significantly induced hepatic DNA replication as measured by immunostaining of the PCNA labeling index and was associated with reduction in expression of LXR response genes, such as ABCA1 and SREBP-1c. Synthetic LXR agonist T0901317 effectively blocked 25HC3S-induced hepatic proliferation. Conclusions: 25HC3S may be a potent regulator of hepatocyte proliferation and oxysterol sulfation may represent a novel regulatory pathway in liver proliferation via inactivating LXR signaling.     

Selective up-regulation of LXR-regulated genes ABCA1, ABCG1, and APOE in macrophages through increased endogenous synthesis of 24(S),25-epoxycholesterol

Liver X receptor (LXR) activation represents a mechanism to prevent macrophage foam cell formation. Previously, we demonstrated that partial inhibition of oxidosqualene:lanosterol cyclase (OSC) stimulated synthesis of the LXR agonist 24(S),25-epoxycholesterol (24(S),25-epoxy) and enhanced ABCA1-mediated cholesterol efflux. In contrast to a synthetic, nonsteroidal LXR activator, TO-901317, triglyceride accumulation was not observed. In the present study, we determined whether endogenous 24(S),25-epoxy synthesis selectively enhanced expression of macrophage LXR-regulated cholesterol efflux genes but not genes that regulate fatty acid metabolism. THP-1 human macrophages incubated with the OSC inhibitor (OSCi) RO0714565 (15 nM) significantly reduced cholesterol synthesis and maximized synthesis of 24(S),25-epoxy. Endogenous 24(S),25-epoxy increased ABCA1, ABCG1, and APOE mRNA abundance and consequently increased cholesterol efflux to apoAI. In contrast, OSCi had no effect on LXR-regulated genes LPL (lipoprotein lipase) and FAS (fatty acid synthase). TO-901317 (>or=10 nM) significantly enhanced expression of all genes examined. OSCi and TO-901317 increased the mRNA and precursor form of SREBP-1c, a major regulator of fatty acid and triglyceride synthesis. However, conversion of the precursor to the active form (nSREBP-1c) was blocked by OSCi-induced 24(S),25-epoxy but not by TO-901317 (>or=10 nm), which instead markedly increased nSREBP-1c. Disruption of nSREBP-1c formation by 24(S),25-epoxy accounted for diminished FAS and LPL expression. In summary, endogenous synthesis of 24(S),25-epoxy selectively up-regulates expression of macrophage LXR-regulated cholesterol efflux genes without stimulating genes linked to fatty acid and triglyceride synthesis.     

Regulation of astrocyte lipid metabolism and ApoE secretion by the microglial oxysterol, 25-hydroxycholesterol

Neuroinflammation, a major hallmark of Alzheimer’s disease and several other neurological and psychiatric disorders, is often associated with dysregulated cholesterol metabolism. Relative to homeostatic microglia, activated microglia express higher levels of Ch25h, an enzyme that hydroxylates cholesterol to produce 25-hydroxycholesterol (25HC). 25HC is an oxysterol with interesting immune roles stemming from its ability to regulate cholesterol metabolism. Since astrocytes synthesize cholesterol in the brain and transport it to other cells via ApoE-containing lipoproteins, we hypothesized that secreted 25HC from microglia may influence lipid metabolism as well as extracellular ApoE derived from astrocytes. Here, we show that astrocytes take up externally added 25HC and respond with altered lipid metabolism. Extracellular levels of ApoE lipoprotein particles increased after treatment of astrocytes with 25HC without an increase in Apoe mRNA expression. In mouse astrocytes-expressing human ApoE3 or ApoE4, 25HC promoted extracellular ApoE3 better than ApoE4. Increased extracellular ApoE was due to elevated efflux from increased Abca1 expression via LXRs as well as decreased lipoprotein reuptake from suppressed Ldlr expression via inhibition of SREBP. 25HC also suppressed expression of Srebf2, but not Srebf1, leading to reduced cholesterol synthesis in astrocytes without affecting fatty acid levels. We further show that 25HC promoted the activity of sterol-o-acyl transferase that led to a doubling of the amount of cholesteryl esters and their concomitant storage in lipid droplets. Our results demonstrate an important role for 25HC in regulating astrocyte lipid metabolism.     

Tryptase promotes human monocyte-derived macrophage foam cell formation by suppressing LXRα activation

Accumulated mast cells in atherosclerotic plaques secrete a high level of tryptase that may participate in the pathogenesis of atherosclerotic disease by diverse pathways. However, the role of tryptase in the lipid metabolism of macrophages remains to be defined. In the present study, we found that the addition of tryptase into THP-1-derived macrophages increased both intracellular lipid accumulation and total cholesterol level. Tryptase promoting foam cell formation was also observed by transmission electron microscope. These effects were resisted by APC366, a selective inhibitor of mast cell tryptase. Tryptase dramatically resisted 22RHC induced activation of LXRα protein expression, which can be reversed by SAM-11 (a PAR-2-specific neutralizing antibody) and reduced LXRα, ABCG1, ABCA1 and SREBP-1c mRNA levels and ABCG1 protein level, which were all blocked by APC366. PAR-2 agonist also redeemed 22RHC stimulation to activate LXRα, ABCG1 protein expression, and mRNA levels of LXRα and its target genes in both THP-1-derived macrophages and primary human monocyte-derived macrophages. In primary macrophages that were first transfected with PAR-2 siRNA and then treated with tryptase, both the ABCG1 protein level and mRNA levels of LXRα and ABCG1 were higher than those in the control siRNA-treated cells. Taken together, our data clarified the PAR-2 expression of human macrophages and suggested that tryptase might promote lipid accumulation in macrophages and foam cell formation by suppressing LXRα activation via PAR-2/LXRα/LXRα target genes signaling pathway. This investigation sheds a new light on the role of tryptase in foam cell formation and pathogenesis of atherosclerosis.     

25-Hydroxycholesterol-3-sulfate attenuates inflammatory response via PPARγ signaling in human THP-1 macrophages

The nuclear receptor peroxisome proliferator-activated receptors (PPARs) are important in regulating lipid metabolism and inflammatory responses in macrophages. Activation of PPARγ represses key inflammatory response gene expressions. Recently, we identified a new cholesterol metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), as a potent regulatory molecule of lipid metabolism. In this paper, we report the effect of 25HC3S and its precursor 25-hydroxycholesterol (25HC) on PPARγ activity and on inflammatory responses. Addition of 25HC3S to human macrophages markedly increased nuclear PPARγ and cytosol IκB and decreased nuclear NF-κB protein levels. PPARγ response element reporter gene assays showed that 25HC3S significantly increased luciferase activities. PPARγ competitor assay showed that the K(i) for 25HC3S was ～1 μM, similar to those of other known natural ligands. NF-κB-dependent promoter reporter gene assays showed that 25HC3S suppressed TNFα-induced luciferase activities only when cotransfected with pcDNAI-PPARγ plasmid. In addition, 25HC3S decreased LPS-induced expression and release of IL-1β. In the PPARγ-specific siRNA transfected macrophages or in the presence of PPARγ-specific antagonist, 25HC3S failed to increase IκB and to suppress TNFα and IL-1β expression. In contrast to 25HC3S, its precursor 25HC, a known liver X receptor ligand, decreased nuclear PPARγ and cytosol IκB and increased nuclear NF-κB protein levels. We conclude that 25HC3S acts in macrophages as a PPARγ ligand and suppresses inflammatory responses via the PPARγ/IκB/NF-κB signaling pathway.     

RORα and 25-Hydroxycholesterol Crosstalk Regulates Lipid Droplet Homeostasis in Macrophages

Nuclear hormone receptors have important roles in the regulation of metabolic and inflammatory pathways. The retinoid-related orphan receptor alpha (Rorα)-deficient staggerer (sg/sg) mice display several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia, and increased susceptibility to atherosclerosis. In this study we demonstrate that macrophages from sg/sg mice have increased ability to accumulate lipids and accordingly exhibit larger lipid droplets (LD). We have previously shown that BMMs from sg/sg mice have significantly decreased expression of cholesterol 25-hydroxylase (Ch25h) mRNA, the enzyme that produces the oxysterol, 25-hydroxycholesterol (25HC), and now confirm this at the protein level. 25HC functions as an inverse agonist for RORα. siRNA knockdown of Ch25h in macrophages up-regulates Vldlr mRNA expression and causes increased accumulation of LDs. Treatment with physiological concentrations of 25HC in sg/sg macrophages restored lipid accumulation back to normal levels. Thus, 25HC and RORα signify a new pathway involved in the regulation of lipid homeostasis in macrophages, potentially via increased uptake of lipid which is suggested by mRNA expression changes in Vldlr and other related genes.     

Induction of ABCA1 and ABCG1 expression by the liver X receptor modulator cineole in macrophages

We investigated the effect of cineole on the expression of genes related to reverse cholesterol transport and hepatic fatty acid metabolism. Cineole, a small aroma compound in teas and herbs, significantly stimulated the transactivation of liver X receptor modulator (LXR)-α and LXR-β. The mRNA and protein expression of LXRs and their target genes, including ABCA1 and ABCG1, was significantly increased in macrophages stimulated with cineole. This led to the subsequent removal of cholesterol from the cells. Interestingly, cineole showed tissue-selective LXR induction: hepatocytes stimulated with cineole showed significantly reduced expression of LXR-α and LXR-α-responsive genes, including FAS and SCD-1 (P <0.05). Accordingly, hepatocytes treated with cineole displayed reduced cellular lipid accumulation compared with control cells, as assessed by Oil Red O lipid staining and cholesterol quantification. These results suggest that cineole is a selective LXR modulator that regulates the expression of key genes in reverse cholesterol transport in macrophages without inducing lipogenesis in hepatocytes. This selective LXR modulator may have practical implications for the development of hypocholesterolemic or anti-atherosclerotic agents and also suggests.     

The role of LXRα in goose primary hepatocyte lipogenesis

In this study, we investigate the role of liver X receptor alpha (LXR alpha) in lipogenesis in geese in order to understand the differences in hepatic steatosis mechanisms between mammals and waterfowl. Primary goose hepatocytes were isolated and treated with the LXR alpha agonist T0901317. Triglyceride (TG) accumulation, acetyl-CoA carboxylase alpha (ACC alpha) and fatty acid synthase (FAS) activities, and gene expression levels of LXR alpha, sterol regulatory element-binding proteins-1 (SREBP-1), FAS, ACC alpha and lipoprotein lipase (LPL) were measured in primary hepatocytes. We found a dose-dependent up-regulation of TG accumulation, ACC, and FAS activities and the mRNA levels of LXR alpha, SREBP-1, FAS, ACC alpha, and LPL genes in the presence of To-901317. We also found that binding of nuclear SREBP-1 to ACC alpha SRE sequence was induced by To-901317 (P < 0.05). In conclusion, LXR alpha is involved in the induction of the lipogenic pathway through activation of SREBP-1 and its target genes in goose primary hepatocytes.     

Transcriptional control mechanisms of genes of lipid and fatty acid metabolism in the Atlantic salmon (Salmo salar L.) established cell line, SHK-1

The regulatory control mechanisms of lipid and fatty acid metabolism were investigated in Atlantic salmon. We identified sterol regulatory element binding protein (SREBP) genes in salmon and characterised their response, and the response of potential target and other regulatory genes including liver X receptor (LXR), to cholesterol and long-chain polyunsaturated fatty acids (LC-PUFA) in the salmon established cell line, SHK-1. Two cDNAs for SREBPs homologous to mammalian SREBP-1 and SREBP-2 were characterised. We identified three groups of genes whose expression responded differently to the treatments. One group of genes, including cholesterol biosynthetic genes, showed increased expression in response to lipid depletion but supplementary cholesterol or LC-PUFA had no further effect. The expression of a second group of genes belonging to fatty acid biosynthetic pathways, included fatty acid synthase, Δ6 and Δ5 fatty acyl desaturases, also increased after lipid depletion but this was negated by cholesterol or by LC-PUFA supplementation. The expression of a third group of genes including acyl-CoA oxidase, HMG-CoA reductase and Elovl5 elongase was increased by cholesterol treatment but was not affected by lipid depletion or by LC-PUFA. This same pattern of expression was also shown by liver X receptor (LXR), indicating that acyl-CoA oxidase, HMG-CoA reductase and Elovl5 are possible direct targets of LXR. This suggests that salmon Elovl5 may be regulated differently from mammalian Elovl5, which is an indirect target of LXR, responding to LXR-dependent increases in SREBP-1.     

Endoplasmic reticulum stress leads to lipid accumulation through upregulation of SREBP-1c in normal hepatic and hepatoma cells

Endoplasmic reticulum stress (ERS) has been found in non-alcoholic fatty liver disease. The study was to further explore the mechanistic relationship between ERS and lipid accumulation. To induce ERS, the hepatoblastoma cell line HepG2 and the normal human L02 cell line were exposed to Tg for 48 h. RT-PCR and Western blot were performed to evaluate glucose-regulated protein (GRP-78) expression as a marker of ERS. ER ultrastructure was assessed by electron microscopy. Triglyceride content was examined by Oil Red O staining and quantitative intracellular triglyceride assay. The hepatic nuclear sterol regulatory element-binding protein (SREBP-1c), liver X receptor (LXRs), fatty acid synthase (FAS), and acetyl-coA carboxylase (ACC1) expressions were examined by real-time PCR and Western blot. 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) was used to inhibit S1P serine protease inhibitor, and SREBP-1c cleavage was evaluated under ERS. SREBP-1c was knockdown and its effect on lipid metabolism was observed. Tg treatment upregulated GRP-78 expression and severely damaged the ER structure in L02 and HepG2 cells. ERS increased triglyceride deposition and enhanced the expression of SREBP-1c, FAS, and ACC1, but have no influence on LXR. AEBSF pretreatment abolished Tg-induced SREBP-1c cleavage. Moreover, SREBP-1c silencing reduced triglycerides and downregulated FAS expression. Pharmacological ERS induced by Tg leads to lipid accumulation through upregulation of SREBP-1c in L02 and HepG2 cells.