Hyperphosphatemia in chronic kidney disease exacerbates atherosclerosis via a mannosidases-mediated complex-type conversion of SCAP N-glycans.

Blood phosphate levels are linked to atherosclerotic cardiovascular disease in patients with chronic kidney disease (CKD), but the molecular mechanisms remain unclear. Emerging studies indicate an involvement of hyperphosphatemia in CKD accelerated atherogenesis through disturbed cholesterol homeostasis. Here, we investigated a potential atherogenic role of high phosphate concentrations acting through aberrant activation of sterol regulatory element-binding protein (SREBP) and cleavage-activating protein (SCAP)-SREBP2 signaling in patients with CKD, hyperphosphatemic apolipoprotein E (ApoE) knockout mice, and cultured vascular smooth muscle cells. Hyperphosphatemia correlated positively with increased atherosclerotic cardiovascular disease risk in Chinese patients with CKD and severe atheromatous lesions in the aortas of ApoE knockout mice. Mice arteries had elevated SCAP levels with aberrantly activated SCAP-SREBP2 signaling. Excess phosphate in vitro raised the activity of α-mannosidase, resulting in delayed SCAP degradation through promoting complex-type conversion of SCAP N-glycans. The retention of SCAP enhanced transactivation of SREBP2 and expression of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, boosting intracellular cholesterol synthesis. Elevated α-mannosidase II activity was also observed in the aortas of ApoE knockout mice and the radial arteries of patients with uremia and hyperphosphatemia. High phosphate concentration in vitro elevated α-mannosidase II activity in the Golgi, enhanced complex-type conversion of SCAP N-glycans, thereby upregulating intracellular cholesterol synthesis. Thus, our studies explain how hyperphosphatemia independently accelerates atherosclerosis in CKD.

Nonesterified free fatty acids enhance the inflammatory response in renal tubules by inducing extracellular ATP release.

In proteinuric renal diseases, excessive plasma nonesterified free fatty acids bound to albumin can leak across damaged glomeruli to be reabsorbed by renal proximal tubular cells and cause inflammatory tubular cells damage by as yet unknown mechanisms. The present study was designed to investigate these mechanisms induced by palmitic acid (PA; one of the nonesterified free fatty acids) overload. Our results show that excess PA stimulates ATP release through the pannexin 1 channel in human renal tubule epithelial cells (HK-2), increasing extracellular ATP concentration approximately threefold compared with control. The ATP release is dependent on caspase-3/7 activation induced by mitochondrial reactive oxygen species. Furthermore, extracellular ATP aggravates PA-induced monocyte chemoattractant protein-1 secretion and monocyte infiltration of tubular cells, enlarging the inflammatory response in both macrophages and HK-2 cells via the purinergic P2X7 receptor-mammalian target of rapamycin-forkhead box O1-thioredoxin-interacting protein/NOD-like receptor protein 3 inflammasome pathway. Hence, PA increases mitochondrial reactive oxygen species-induced ATP release and inflammatory stress, which cause a "first hit," while ATP itself is a "second hit" in amplifying the renal tubular inflammatory response. Thus, inhibition of ATP release or the purinergic P2X7 receptor may be an approach to reduce renal inflammation and improve renal function.

SCAP knockdown in vascular smooth muscle cells alleviates atherosclerosis plaque formation via up-regulating autophagy in ApoE-/- mice.

Sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) is a cholesterol sensor that plays a critical role in regulating intracellular cholesterol levels, but the association between SCAP and foam cell formation in vascular smooth muscle cells (VSMCs) is poorly understood. Using tissue-specific SCAP knockdown in apolipoprotein E (ApoE)-/- mice, we sought to search the mechanism through which SCAP signaling affects VSMC foam cell development. VSMC-specific SCAP knockdown mice were generated by Cre/LoxP-mediated gene targeting in ApoE-/- mice. Breeding SCAPflox/flox mice with SM22α-Cre mice resulted in no viable offspring with the homozygote SM22-Cre: SCAPflox/flox genotype due to embryonic lethality. We found that the heterozygote SM22α-Cre:SCAPflox/+:ApoE-/- mice fed a Western diet for 12 wk had significantly fewer atherosclerotic plaques in their aortas than the control mice due to reduced cholesterol uptake and synthesis. Furthermore, we found that autophagy in VSMCs was increased in SM22α-Cre:SCAPflox/+:ApoE-/- mice. Similarly, in vitro, SCAP knockdown in human coronary artery VSMCs by RNA interference reduced lipid accumulation and increased autophagy under LDL cholesterol loading. SCAP knockdown in VSMCs reduced oxidative stress and increased AMPK phosphorylation, which contributed to the up-regulation of autophagy in vivo and in vitro. VSMC-specific SCAP knockdown decreased the lipid accumulation and intracellular oxidative stress, increased excessive lipid clearance by enhancing lipid autophagy mediated by the reactive oxygen species/AMPK pathway in VSMCs, and consequently alleviated atherosclerosis plaque formation.-Li, D., Chen, A., Lan, T., Zou, Y., Zhao, L., Yang, P., Qu, H., Wei, L., Varghese, Z., Moorhead, J. F., Chen, Y., Ruan, X. Z. SCAP knockdown in vascular smooth muscle cells alleviates atherosclerosis plaque formation via up-regulating autophagy in ApoE-/- mice.

CD36 plays a negative role in the regulation of lipophagy in hepatocytes through an AMPK-dependent pathway.

Fatty acid translocase cluster of differentiation (CD36) is a multifunctional membrane protein that facilitates the uptake of long-chain fatty acids. Lipophagy is autophagic degradation of lipid droplets. Accumulating evidence suggests that CD36 is involved in the regulation of intracellular signal transduction that modulates fatty acid storage or usage. However, little is known about the relationship between CD36 and lipophagy. In this study, we found that increased CD36 expression was coupled with decreased autophagy in the livers of mice treated with a high-fat diet. Overexpressing CD36 in HepG2 and Huh7 cells inhibited autophagy, while knocking down CD36 expression induced autophagy due to the increased autophagosome formation in autophagic flux. Meanwhile, knockout of CD36 in mice increased autophagy, while the reconstruction of CD36 expression in CD36-knockout mice reduced autophagy. CD36 knockdown in HepG2 cells increased lipophagy and ß-oxidation, which contributed to improving lipid accumulation. In addition, CD36 expression regulated autophagy through the AMPK pathway, with phosphorylation of ULK1/Beclin1 also involved in the process. These findings suggest that CD36 is a negative regulator of autophagy, and the induction of lipophagy by ameliorating CD36 expression can be a potential therapeutic strategy for the treatment of fatty liver diseases through attenuating lipid overaccumulation.

High olive oil diets enhance cervical tumour growth in mice: transcriptome analysis for potential candidate genes and pathways.

BACKGROUND: Numerous epidemiologic studies have found a close association between obesity and cancer. Dietary fat is a fundamental contributor to obesity and is a risk factor for cancer. Thus far, the impact of dietary olive oil on cancer development remains inconclusive, and little is known about its underlying mechanisms. METHODS: Nude mouse xenograft models were used to examine the effects of high olive oil diet feeding on cervical cancer (CC) development and progression. Cell proliferation, migration and invasion were observed by the methods of EdU incorporation, Wound healing and Transwell assay, separately. RNA-sequencing technology and comprehensive bioinformatics analyses were used to elucidate the molecular processes regulated by dietary fat. Differentially expressed genes (DEGs) were identified and were functionally analyzed by Gene Ontology (GO), Kyoto Enrichment of Genes and Genomes (KEGG). Then, protein-protein interaction (PPI) network and sub-PPI network analyses were conducted using the STRING database and Cytoscape software. RESULTS: A high olive oil diet aggravated tumourigenesis in an experimental xenograft model of CC. Oleic acid, the main ingredient of olive oil, promoted cell growth and migration in vitro. Transcriptome sequencing analysis of xenograft tumour tissues was then performed to elucidate the regulation of molecular events regulated by dietary fat. Dietary olive oil induced 648 DEGs, comprising 155 up-regulated DEGs and 493 down-regulated DEGs. GO and pathway enrichment analysis revealed that some of the DEGs including EGR1 and FOXN2 were involved in the transcription regulation and others, including TGFB2 and COL4A3 in cell proliferation. The 15 most strongly associated DEGs were selected from the PPI network and hub genes including JUN, TIMP3, OAS1, OASL and EGR1 were confirmed by real-time quantitative PCR analysis. CONCLUSIONS: Our study suggests that a high olive oil diet aggravates CC progression in vivo and in vitro. We provide clues to build a potential link between dietary fat and cancerogenesis and identify areas requiring further investigation.

CD36 palmitoylation disrupts free fatty acid metabolism and promotes tissue inflammation in non-alcoholic steatohepatitis.

BACKGROUND AND AIMS: Fatty acid translocase CD36 (CD36) is a membrane protein with multiple immuno-metabolic functions. Palmitoylation has been suggested to regulate the distribution and functions of CD36, but little is known about its significance in non-alcoholic steatohepatitis (NASH). METHODS: Human liver tissue samples were obtained from patients undergoing liver biopsy for diagnostic purposes. CD36 knockout mice were injected with lentiviral vectors expressing wild-type CD36 or CD36 with mutated palmitoylation sites. Liver histology, immunofluorescence, mRNA expression profile, subcellular distributions and functions of CD36 protein were assessed. RESULTS: The localization of CD36 on the plasma membrane of hepatocytes was markedly increased in patients with NASH compared to patients with normal liver and those with simple steatosis. Increased CD36 palmitoylation and increased localization of CD36 on the plasma membrane of hepatocytes were also observed in livers of mice with NASH. Furthermore, inhibition of CD36 palmitoylation protected mice from developing NASH. The absence of palmitoylation decreased CD36 protein hydrophobicity reducing its localization on the plasma membrane as well as in lipid raft of hepatocytes. Consequently, a lack of palmitoylation decreased fatty acid uptake and CD36/Fyn/Lyn complex in HepG2 cells. Inhibition of CD36 palmitoylation not only ameliorated intracellular lipid accumulation via activation of the AMPK pathway, but also inhibited the inflammatory response through the inhibition of the JNK signaling pathway. CONCLUSIONS: Our findings demonstrate the key role of palmitoylation in regulating CD36 distributions and its functions in NASH. Inhibition of CD36 palmitoylation may represent an effective therapeutic strategy in patients with NASH. LAY SUMMARY: Fatty acid translocase CD36 (CD36) is a multifunctional membrane protein which contributes to the development of liver steatosis. In the present study, we demonstrated that the localization of CD36 on the plasma membrane of hepatocytes is increased in patients with non-alcoholic steatohepatitis. Blocking the palmitoylation of CD36 reduces CD36 distribution in hepatocyte plasma membranes and protects mice from non-alcoholic steatohepatitis. The inhibition of CD36 palmitoylation not only improved fatty acid metabolic disorders but also reduced the inflammatory response in vitro and in vivo. The present study suggests that CD36 palmitoylation is important for non-alcoholic steatohepatitis development and inhibition of CD36 palmitoylation could be used to cure non-alcoholic steatohepatitis.

Inflammatory stress induces statin resistance by disrupting 3-hydroxy-3-methylglutaryl-CoA reductase feedback regulation.

OBJECTIVE: The risk of cardiovascular disease is increased by up to 33 to 50× in chronic inflammatory states and convention doses of statins may not provide the same cardiovascular protection as in noninflamed patients. This study investigated whether the increase in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA-R)-mediated cholesterol synthesis observed under inflammatory stress was resistant to the action of statins and if so, whether this was because of interference with the sterol regulatory element binding protein cleavage-activating protein pathway. APPROACH AND RESULTS: Inflammatory stress was induced by adding cytokines (interleukin-1ß, tumor necrosis factor-α, and interleukin-6) and lipopolysaccharides to vascular smooth muscle cells in vitro and by subcutaneous casein injection in apolipoprotein E/scavenger receptors class A/CD36 triple knockout mice in vivo. Inflammatory stress exacerbated cholesterol ester accumulation and was accompanied in vitro and in vivo by increased HMGCoA-R mRNA and protein expression mediated via activation of the sterol regulatory element binding protein cleavage-activating protein/sterol regulatory element binding protein-2 pathway. Atorvastatin reduced HMGCoA-R enzymatic activity and intracellular cholesterol synthesis in vitro. However, inflammatory stress weakened these suppressive effects. Atorvastatin at concentrations of 16 µmol/L inhibited HMGCoA-R activity by 50% in vascular smooth muscle cells, but the same concentration resulted in only 30% of HMGCoA-R activity in vascular smooth muscle cells in the presence of interleukin-1ß. Knocking down sterol regulatory element binding protein cleavage-activating protein prevented statin resistance induced by interleukin-1ß, and overexpression of sterol regulatory element binding protein cleavage-activating protein induced statin resistance even without inflammatory stress. In vivo, the amount of atorvastatin required to lower serum cholesterol and decrease aortic lipid accumulation rose from 2 to 10 mg/kg per day in the presence of inflammatory stress. CONCLUSIONS: Increased cholesterol synthesis mediated by HMGCoA-R under inflammatory stress may be one of the mechanisms for intracellular lipid accumulation and statin resistance.

Rapamycin-mediated CD36 translational suppression contributes to alleviation of hepatic steatosis.

Rapamycin, a mammalian target of rapamycin (mTOR)-specific inhibitor, has the effect of anti-lipid deposition on non-alcoholic fatty liver disease (NAFLD), but the mechanisms with which rapamycin alleviates hepatic steatosis are not fully disclosed. CD36 is known to facilitate long-chain fatty acid uptake and contribute to NAFLD progression. Hepatic CD36 expression is closely associated with hepatic steatosis, while mTOR pathway is involved in CD36 translational control. This study was undertaken to investigate whether rapamycin alleviates hepatic steatosis via the inhibition of mTOR pathway-dependent CD36 translation. Human hepatoblastoma HepG2 cells were treated with palmitate and C57BL/6J mice were fed with high fat diet (HFD) to induce hepatic steatosis. Hepatic CD36 protein expression was significantly increased with lipid accumulation in palmitate-treated HepG2 cells or HFD-fed C57BL/6J mice. Rapamycin reduced hepatic steatosis and CD36 protein expression, but it had no influence on CD36 mRNA expression. Rapamycin had no effect on CD36 protein stability, but it significantly decreased CD36 translational efficiency. We further confirmed that rapamycin inhibited the phosphorylation of mTOR and its downstream translational regulators including p70 ribosomal protein S6 kinase (p70S6K), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor 4E (eIF4E). This study demonstrates that rapamycin inhibits hepatic CD36 translational efficiency through the mTOR pathway, resulting in reduction of CD36 protein expression and alleviation of hepatic steatosis.

Inflammatory stress reduces the effectiveness of statins in the kidney by disrupting HMGCoA reductase feedback regulation.

BACKGROUND: Patients with chronic kidney disease (CKD) are unlikely to gain the same benefit from conventional doses of statins as do patients with cardiovascular disease alone. This study investigated whether inflammation accompanying CKD causes statin resistance. METHODS: Inflammatory stress was induced by adding cytokines and lipopolysaccharide (LPS) to human mesangial cells (HMCs) in vitro, and in vivo by subcutaneous casein injection in apolipoprotein E, scavenger receptors class A and CD36 triple knockout mice. RESULTS: Inflammatory stress exacerbated cholesterol accumulation and was accompanied in vitro and in vivo by increased HMGCoA reductase (HMGCoA-R) mRNA and protein expression mediated via activation of the sterol regulatory element-binding protein cleavage-activating protein (SCAP)/sterol regulatory element-binding protein 2 pathway. Atorvastatin reduced HMGCoA-R enzymatic activity and intracellular cholesterol synthesis in vitro; however, inflammatory stress weakened these suppressive effects. Atorvastatin at concentrations of 15 µM inhibited HMGCoA-R activity by 50% (IC50) in HMCs, but the same concentration in the presence of interleukin (IL)-1ß resulted in only 30% inhibition of HMGCoA-R activity in HMCs. Knocking down SCAP prevented statin resistance induced by IL-1ß, and overexpression of SCAP-induced statin resistance even without inflammatory stress. In vivo, the amount of atorvastatin required to lower serum cholesterol and decrease kidney lipid accumulation rose from 2 to 10 mg/kg/day in the presence of inflammatory stress. CONCLUSIONS: Inflammatory stress can disrupt HMGCoA-R-mediated cholesterol synthesis resulting in intracellular lipid accumulation and statin resistance.

Cross-talk between TLR4-MyD88-NF-κB and SCAP-SREBP2 pathways mediates macrophage foam cell formation.

Myeloid differentiation factor 88 (MyD88) and NF-κB play central roles in mediating signal transduction of the Toll-like receptor (TLR) superfamily in human macrophages. The feedback regulation of LDL receptor (LDLR) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR) are mediated by the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP)-SREBP2 pathway and are key regulatory elements for cholesterol homeostasis in human cells. This study was designed to investigate cross-talk between TLR4-MyD88-NF-κB and SCAP-SREBP2 pathways in macrophage foam cell formation. phorbol 12-myristate 13-acetate-activated THP-1 macrophages were transfected with negative control or MyD88 small interfering (si)RNA. Transfected cells were incubated with LPS in the absence or presence of LDL or IκB kinase (IKK) inhibitor (BMS-345541). Intracellular cholesterol content was assessed. mRNA and protein expression of LDLR, HMG-CoAR, SCAP, and SREBP2 were examined by real-time RT-PCR and Western blot analysis. Intracellular translocation of SCAP in the organelles was detected by immunofluorecence and confocal microscopy. We demonstrated that LPS-induced cholesterol accumulation was attenuated by applying siRNA against MyD88 in the absence or presence of LDL. LPS increased both gene and protein expression of LDLR and HMG-CoAR by increasing expression and abnormal translocation of SCAP from the endoplasmic reticulum to the Golgi. These effects were blocked by knockdown of MyD88 or blockade of IKK or by knockdown of SCAP, suggesting that the cross-talk between NF-κB and SCAP plays an important role in macrophage foam cell formation and that interfering with the cross-talk might be a potential approach in preventing LPS-induced macrophage foam cell formation.

Inflammatory stress exacerbates hepatic cholesterol accumulation via disrupting cellular cholesterol export.

BACKGROUND AND AIM: Both inflammation and cholesterol accumulation play important roles in the development of non-alcoholic fatty liver disease. This study was undertaken to investigate whether inflammation aggravated cholesterol accumulation via disrupting hepatic cholesterol export and we explored the underlying mechanisms. METHODS: We used casein injection in C57BL/6J mice, and tumor necrosis factor alpha (TNF-α) stimulation in human hepatoblastoma cell line (HepG2) cells to induce inflammation. Intracellular cholesterol level was examined by Oil Red O staining and quantitative analysis. Bile acid level was quantified by colorimetric analysis. (3)[H] cholesterol assay by scintillation counting was performed to evaluate the cholesterol efflux. The mRNA and protein expression was examined by real-time polymerase chain reaction and Western blot. RESULTS: Inflammation increased cholesterol accumulation in livers of C57BL/6J mice and in HepG2 cells. High-fat diet in mice and low-density lipoprotein (LDL) loading in HepG2 cells increased bile acid synthesis and cholesterol efflux, enhanced the mRNA and protein expression of liver X receptor α (LXRα), peroxisome proliferator-activated receptors (PPARα, γ), cholesterol 7α-hydroxylase (CYP7A1) and ATP-binding cassette transporter A1 (ABCA1). However, inflammation reduced bile acid synthesis and cholesterol efflux even in high-fat-diet-fed mice and HepG2 cells in the presence of LDL loading. The enhanced effects of these genes and proteins expression due to high-fat diet and LDL loading were inhibited by inflammation both in vivo and in vitro. CONCLUSIONS: Inflammation disrupted PPAR-LXR-CYP7A1/ABCA1-mediated bile acid synthesis and cholesterol efflux resulting in exacerbated cholesterol accumulation in livers of C57BL/6J mice and HepG2 cells.

CD36-mediated metabolic crosstalk between tumor cells and macrophages affects liver metastasis.

Liver metastasis is highly aggressive and treatment-refractory, partly due to macrophage-mediated immune suppression. Understanding the mechanisms leading to functional reprogramming of macrophages in the tumor microenvironment (TME) will benefit cancer immunotherapy. Herein, we find that the scavenger receptor CD36 is upregulated in metastasis-associated macrophages (MAMs) and deletion of CD36 in MAMs attenuates liver metastasis in mice. MAMs contain more lipid droplets and have the unique capability in engulfing tumor cell-derived long-chain fatty acids, which are carried by extracellular vesicles. The lipid-enriched vesicles are preferentially partitioned into macrophages via CD36, that fuel macrophages and trigger their tumor-promoting activities. In patients with liver metastases, high expression of CD36 correlates with protumoral M2-type MAMs infiltration, creating a highly immunosuppressive TME. Collectively, our findings uncover a mechanism by which tumor cells metabolically interact with macrophages in TME, and suggest a therapeutic potential of targeting CD36 as immunotherapy for liver metastasis.

Cholesterol sensor SCAP contributes to sorafenib resistance by regulating autophagy in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most malignant tumors and the fourth leading cause of cancer-related death worldwide. Sorafenib is currently acknowledged as a standard therapy for advanced HCC. However, acquired resistance substantially limits the clinical efficacy of sorafenib. Therefore, further investigations of the associated risk factors are highly warranted. METHODS: We analysed a group of 78 HCC patients who received sorafenib treatment after liver resection surgery. The expression of SCAP and its correlation with sorafenib resistance in HCC clinical samples were determined by immunohistochemical analyses. Overexpression and knockdown approaches in vitro were used to characterize the functional roles of SCAP in regulating sorafenib resistance. The effects of SCAP inhibition in HCC cell lines were analysed in proliferation, apoptosis, and colony formation assays. Autophagic regulation by SCAP was assessed by immunoblotting, immunofluorescence and immunoprecipitation assays. The combinatorial effect of a SCAP inhibitor and sorafenib was tested using nude mice. RESULTS: Hypercholesterolemia was associated with sorafenib resistance in HCC treatment. The degree of sorafenib resistance was correlated with the expression of the cholesterol sensor SCAP and consequent deposition of cholesterol. SCAP is overexpressed in HCC tissues and hepatocellular carcinoma cell lines with sorafenib resistance, while SCAP inhibition could improve sorafenib sensitivity in sorafenib-resistant HCC cells. Furthermore, we found that SCAP-mediated sorafenib resistance was related to decreased autophagy, which was connected to decreased AMPK activity. A clinically significant finding was that lycorine, a specific SCAP inhibitor, could reverse acquired resistance to sorafenib in vitro and in vivo. CONCLUSIONS: SCAP contributes to sorafenib resistance through AMPK-mediated autophagic regulation. The combination of sorafenib and SCAP targeted therapy provides a novel personalized treatment to enhance sensitivity in sorafenib-resistant HCC.

Advanced glycation end products (AGEs) increase human mesangial foam cell formation by increasing Golgi SCAP glycosylation in vitro.

Advanced glycation end products (AGEs) is one of the causative factors of diabetic nephropathy, which is associated with lipid accumulation in glomeruli. This study was designed to investigate whether N(ε)-(carboxymethyl) lysine (CML; a member of the AGEs family) increases lipid accumulation by impairing the function of sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) in human mesangial cells (HMCs). Intracellular cholesterol content was assessed by Oil Red O staining and quantitative assay. The expression of molecules controlling cholesterol homeostasis was examined using real-time quantitative RT-PCR and Western blotting. The activity of Golgi-processing enzymes was determined using enzyme-based methods, and the translocation of SCAP from the endoplasmic reticulum (ER) to the Golgi was detected by confocal microscopy. CML increased cholesterol accumulation in HMCs. Exposure to CML increased expression and abnormal translocation of SCAP from the ER to the Golgi even in the presence of a high concentration of LDL. The increased SCAP translocation carried more SREBP-2 to the Golgi for activation by proteolytic cleavages, enhancing transcription of 3-hydroxy-3-methylclutaryl-CoA reductase and the LDL receptor. CML increased Golgi mannosidase activity, which may enhance glycosylation of SCAP. This prolonged the half-life and enhanced recycling of SCAP between the ER and the Golgi. The effects of CML were blocked by inhibitors of Golgi mannosidases. AGEs (CML) increased lipid synthesis and uptake, thereby causing foam cell formation via increasing transcription and protein glycosylation of SCAP in HMCs. These data imply that inhibitors of Golgi-processing enzymes might have a potential renoprotective role in prevention of mesangial foam cell formation.

Inflammatory stress exacerbates lipid-mediated renal injury in ApoE/CD36/SRA triple knockout mice.

Both lipids and inflammation play important roles in the progression of kidney disease. This study was designed to investigate whether inflammation exacerbates lipid accumulation via LDL receptors (LDLr), thereby causing renal injury in C57BL/6J mice, apolipoprotein E (ApoE) knockout (KO) mice, and ApoE/CD36/scavenger receptor A triple KO mice. The mice were given a subcutaneous casein injection to induce inflammatory stress. After 14 wk, terminal blood samples were taken for renal function, lipid profiles, amyloid A (SAA), and IL-6 assays. Lipid accumulation in kidneys was visualized by oil red O staining. Fibrogenic molecule expression in kidneys was examined. There was a significant increase in serum SAA and IL-6 in the all casein-injected mice compared with respective controls. Casein injection reduced serum total cholesterol, LDL cholesterol, and HDL cholesterol and caused lipid accumulation in kidneys from three types of mice. The expression of LDLr and its regulatory proteins sterol-responsive element-binding protein (SREBP) 2 and SREBP cleavage-activating protein (SCAP) were upregulated in inflamed mice compared with controls. Casein injection induced renal fibrosis accompanied by increased expression of fibrogenic molecules in the triple KO mice. These data imply that inflammation exacerbates lipid accumulation in the kidney by diverting lipid from the plasma to the kidney via the SCAP-SREBP2-LDLr pathway and causing renal injury. Low blood cholesterol levels, resulting from inflammation, may be associated with high risk for chronic renal fibrosis.

Inflammatory stress exacerbates hepatic cholesterol accumulation via increasing cholesterol uptake and de novo synthesis.

BACKGROUND AND AIM: Cholesterol accumulation plays an important role in the progression of non-alcoholic fatty liver disease. We have demonstrated that inflammation aggravated cholesterol accumulation, causing tissue injury in the vessel and kidney. This study was undertaken to investigate whether inflammatory stress exacerbates hepatic cholesterol accumulation and we explored the underlying mechanisms. METHODS: We used casein injection in C57BL/6J mice, interleukin-1ß and interleukin-6 stimulation in human hepatoblastoma cell line (HepG2) cells to induce inflammatory stress. Oil Red O staining and intracellular cholesterol assay were used to quantify cellular cholesterol levels. Real-time reverse transcription polymerase chain reaction and Western blot were used to measure messenger RNA (mRNA) and protein expression of target genes. HMGCoA reductase (HMGCoA-r) enzymatic activity and cellular cholesterol synthesis were measured by radioactive methods. RESULTS: We demonstrated that inflammatory stress increased hepatic cholesterol accumulation and enhanced sterol regulatory element binding protein 2 (SREBP2), low-density lipoprotein receptor (LDLr) and HMGCoA-r mRNA and protein expression in livers of C57BL/6J mice and in HepG2 cells. A high-fat diet in mice or LDL loading in HepG2 cells inhibited mRNA and protein expression of these genes. However, the suppressive effect was overridden by inflammatory stress both in vivo and in vitro. Inflammatory stress increased HMGCoA-r enzymatic activity and cellular cholesterol synthesis in HepG2 cells in the absence or presence of LDL loading. CONCLUSION: Inflammatory stress disrupted hepatic SREBP2-mediated low-density lipoprotein receptor and HMGCoA-r feedback regulation resulting in exacerbated cholesterol accumulation in livers of C57BL/6J mice and HepG2 cells.

Inflammatory stress exacerbates ectopic lipid deposition in C57BL/6J mice.

BACKGROUND: Chronic systemic inflammation and abnormal free fatty acid metabolism are closely related to ectopic lipid deposition. In this study, we investigate if inflammation tissue-specifically disrupts lipogenesis and lipolysis in nonadipose tissues and adipose tissue, resulting in ectopic lipid deposition in C57BL/6J mice. METHODS: We used casein injection in C57BL/6J mice to induce a chronic systemic inflammatory stress in vivo. Serum was analyzed for free fatty acid and cytokines. Insulin sensitivities were evaluated by glucose and insulin tolerance tests. Liver, muscle, adipose tissues were taken for lipid analysis. Real-time polymerase chain reaction and western blotting were used to examine the gene and protein expression of molecules involved in adipogenesis and lipolysis in tissues. RESULTS: Casein injection elevated serum levels of IL-6 and SAA in mice, which are associated with increased lipid accumulation in liver and muscle, suggesting that chronic systemic inflammation induces ectopic lipid deposition in nonadipose tissues. The inflammatory stress upregulated mRNA and protein expression of sterol regulatory element binding protein 1, fatty acid synthase, and acetyl CoA carboxylase alpha, while inhibited these molecules expression in adipose. Interestingly, in the same experimental setting, inflammation increased triglyceride lipase and hormone-sensitive lipase expression in white adipose tissue. Inflammation also induced insulin resistance and increased serum free fatty acid levels in C57BL/6J mice. CONCLUSIONS: Chronic systemic inflammation increased lipogenesis in nonadipose tissues and lipolysis in white adipose tissue, resulting in ectopic lipid deposition in nonadipose tissues. This disturbed free fatty acid homeostasis and caused insulin resistance in C57BL/6J mice.

Sirolimus inhibits endogenous cholesterol synthesis induced by inflammatory stress in human vascular smooth muscle cells.

Inflammatory stress accelerates the progression of atherosclerosis. Sirolimus, a new immunosuppressive agent, has been shown to have pleiotropic antiatherosclerotic effects. In this study we hypothesized that sirolimus inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)-mediated cholesterol synthesis in human vascular smooth muscle cells (VSMCs) under inflammatory stress. Using radioactive assay, we demonstrated that sirolimus inhibited the increase of interleukin-1beta (IL-1beta)-induced cholesterol synthesis in VSMCs. Further studies showed that sirolimus inhibited both the HMGR gene and protein expression in VSMCs treated with or without IL-1beta. These effects were mediated by inhibiting the gene expression of sterol regulatory element-binding protein-2 (SREBP-2) and SREBP-2 cleavage-activating protein (SCAP) as checked by real-time PCR, Western blot analysis, and confocal microscopy for the observation of decreased protein translocation of the SCAP/SREBP-2 complex from the endoplasmic reticulum (ER) to the Golgi. Insulin-induced gene-1 (Insig-1) is a key ER protein controlling the feedback regulation of HMGR at transcriptional and posttranscriptional levels. We demonstrated that sirolimus increased Insig-1 expression which may bind to the SCAP, preventing the exit of SCAP-SREBP complexes from the ER. The increased Insig-1 also accelerated HMGR protein degradation in VSMCs as shown by pulse-chase analysis. In conclusion, sirolimus inhibits cholesterol synthesis induced by inflammatory stress through the downregulation of HMGR expression and the acceleration of HMGR protein degradation. These findings may improve our understanding of the molecular mechanisms of the antiatherosclerosis properties of sirolimus.

Inflammatory stress exacerbates lipid accumulation in hepatic cells and fatty livers of apolipoprotein E knockout mice.

UNLABELLED: The prevailing theory in non-alcoholic fatty liver disease (NAFLD) is the "two-hit" hypothesis. The first hit mainly consists of lipid accumulation, and the second is subsequent systemic inflammation. The current study was undertaken to investigate whether inflammatory stress exacerbates lipid accumulation in liver and its underlying mechanisms. We used interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) stimulation in human hepatoblastoma cell line (HepG2) cells and primary hepatocytes in vitro, and casein injection in apolipoprotein E knockout mice in vivo to induce inflammatory stress. The effects of inflammatory stress on cholesterol accumulation were examined by histochemical staining and a quantitative intracellular cholesterol assay. The gene and protein expressions of molecules involved in cholesterol trafficking were examined by real-time polymerase chain reaction (PCR) and western blot. Cytokine production in the plasma of apolipoprotein E knockout mice was measured by enzyme-linked immunosorbent assay. Our results showed that inflammatory stress increased cholesterol accumulation in hepatic cells and in the livers of apolipoprotein E knockout mice. Further analysis showed that inflammatory stress increased the expression of low-density lipoprotein (LDL) receptor (LDLr), sterol regulatory element-binding protein (SREBP) cleavage activating protein (SCAP), and SREBP-2. Confocal microscopy showed that IL-1beta increased the translocation of SCAP/SREBP-2 complex from endoplasmic reticulum (ER) to Golgi in HepG2 cells, thereby activating LDLr gene transcription. IL-1beta, TNF-alpha, and systemic inflammation induced by casein injection also inhibited expression of adenosine triphosphate-binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor-alpha (PPAR-alpha), and liver X receptor-alpha (LXRalpha). This inhibitory effect may cause cholesterol efflux reduction. CONCLUSION: Inflammatory stress up-regulates LDLr-mediated cholesterol influx and down-regulates ABCA1-mediated cholesterol efflux in vivo and in vitro. This may exacerbate the progression of NAFLD by disrupting cholesterol trafficking control, especially during the second hit phase of liver damage.

Inflammatory stress increases unmodified LDL uptake via LDL receptor: an alternative pathway for macrophage foam-cell formation.

OBJECTIVE: To investigate if inflammatory stress increases intracellular accumulation of unmodified low-density lipoprotein (LDL) in human monocyte cell line (THP-1) macrophages by disrupting the sterol regulatory element binding proteins (SREBPs) cleavage-activating protein (SCAP)-SREBP2-mediated feedback regulation of LDL receptor. MATERIALS AND METHODS: THP-1 macrophages were incubated in serum-free medium in the absence or presence of LDL alone, LDL plus lipopolysaccharide (LPS) and LPS alone, then intracellular cholesterol content, tumor necrosis factor alpha level in the supernatants, mRNA and protein expression of LDL receptor, and SREBP2 and SCAP in the treated cells were assessed by Oil Red O staining, cholesterol enzymatic assay, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and Western blotting analysis, respectively. RESULTS: We demonstrated that LPS enhanced transformation of THP-1 macrophages into foam cells by increased uptake of unmodified LDL as evidenced by Oil Red O staining and direct assay of intracellular cholesterol. In the absence of LPS, 25 microg/ml LDL decreased LDL receptor mRNA and protein expression (p < 0.05). However, LPS enhanced LDL receptor expression, overcoming the suppression of LDL receptor induced by 25 microg/ml LDL and inappropriately increasing LDL uptake (p < 0.05). Exposure to LPS also caused overexpression of mRNA and protein of SCAP and SREBP2 (p < 0.05). These observations indicate that LPS disrupts cholesterol-mediated LDL receptor feedback regulation, permitting intracellular accumulation of unmodified LDL and causing foam-cell formation. CONCLUSION: The implication of these findings is that inflammatory stress may contribute to intracellular LDL accumulation in THP-1 macrophages without previous modification of LDL.