Loss of Splicing Factor SRSF3 Impairs Lipophagy Through Ubiquitination and Degradation of Syntaxin17 in Hepatocytes.

Lipid accumulation in hepatocytes is the distinctive characteristic of nonalcoholic fatty liver disease. Serine/arginine-rich splicing factor 3 (SRSF3) is highly expressed in the liver and expression decreases in high-fat conditions. However, the role of SRSF3 in hepatic lipid metabolism needs to be clarified. Here, we showed that loss of SRSF3 was associated with lipid accumulation. We determined that SRSF3 regulated lipophagy, the process of selective degradation of lipid droplets by autophagy. Mechanistically, loss of SRSF3 impaired the fusion of the autophagosome and lysosome by promoting the proteasomal degradation of syntaxin 17 (STX17), a key autophagosomal SNARE protein. We found that ubiquitination of STX17 was increased and upregulation of seven in absentia homolog 1 was responsible for the increased posttranslational modification of STX17. Taken together, our data primarily demonstrate that loss of SRSF3 weakens the clearance of fatty acids by impairing lipophagy in the progression of nonalcoholic fatty liver disease, indicating a novel potential therapeutic target for fatty liver disease treatment.

HIF-2α-induced upregulation of CD36 promotes the development of ccRCC.

Clear cell renal cell carcinoma (ccRCC) is characterized by the abundance of lipid droplets and the activation of the hypoxia-inducible factor (HIF) signaling pathway. However, the lipid reprogramming induced by HIF signaling in ccRCC is not fully understood. In this study, we found that the fatty acid receptor CD36 was highly expressed in human ccRCC tissues and ccRCC cell lines. CD36 overexpression increased fatty acid uptake and lipid droplet formation, and enhanced the proliferation and migration of ccRCC cells in a DGAT1-dependent manner. In contrast, the disruption of endogenous CD36 showed the opposite effects. The upregulated expression of CD36 in ccRCC was associated with hypoxia and HIF-2α activation. Furthermore, we identified CD36 as a new target of the transcription factor HIF-2α. The knockdown of CD36 in ccRCC cells reduced lipid accumulation and also blocked the tumor-promoting effects induced by HIF-2α under hypoxia. Our findings suggest that hypoxia-dependent HIF-2α promotes the remodeling of lipid metabolism and the malignant phenotype of ccRCC via CD36, providing a certain theoretical basis for clarifying the mechanism of ccRCC.

Src-mediated Tyr353 phosphorylation of IP3R1 promotes its stability and causes apoptosis in palmitic acid-treated hepatocytes.

Palmitic acid (PA)-induced hepatocyte apoptosis is critical for the progression of nonalcoholic fatty liver disease (NAFLD). Inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is an intracellular Ca2+-release channel and is involved in PA-induced hepatocyte apoptosis. While the expression of IP3R1 is elevated in patients with NAFLD and in hepatocytes treated with PA, it remains unclear how PA promotes the expression of IP3R1. In present study, our results showed that PA induced mitochondrial dysfunction and apoptosis, which is accompanied with the increase of the IP3R1 expression in hepatic cells. The inhibition of IP3R1 expression using siRNA ameliorated the PA-induced mitochondrial dysfunction. Furthermore, PA enhanced the stability of the IP3R1 protein instead of an increase in its mRNA levels. PA also promoted the phosphorylation of IP3R1 at the Tyr353 site and increased the phosphorylation of src in hepatic cells. Moreover, an inhibitor of src kinase (SU6656) significantly reduced the Tyr353 phosphorylation of IP3R1 and decreased its stability. In addition, SU6656 improved mitochondrial function and reduced apoptosis in hepatocytes. Conclusion: PA promotes the Tyr353 phosphorylation of IP3R1 by activating the src pathway and increasing the protein stability of IP3R1, which consequently results in mitochondrial Ca2+ overload and mitochondrial dysfunction in hepatic cells. Our results also suggested that inhibition of the src/IP3R1 pathway, such as by SU6656, may be a novel potential therapeutic approach for the treatment of NAFLD.

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.

Loss of CD36 impairs hepatic insulin signaling by enhancing the interaction of PTP1B with IR.

A contradictory role of CD36 in insulin resistance was found to be related to the nutrient state. Here, we examined that the physiological functions of CD36 in insulin signal transduction in mice fed a low-fat diet. CD36 deficiency led to hepatic insulin resistance and decreased insulin-stimulated tyrosine phosphorylation of insulin receptor ß (IRß) in mice fed a low-fat diet. The ability of insulin to bind with IR did not differ between WT and CD36-deficient hepatocytes. CD36 formed a complex with IRß and dissociation of CD36/Fyn complex or inhibition of Fyn only partially reversed the effects of CD36 on hepatic insulin signaling. Furthermore, we found that CD36 deficiency led to abnormally increased hepatic protein-tyrosine phosphatase 1B (PTP1B) expression and enhanced PTP1B and IR interactions, which contributed to the decreased insulin signaling and disordered glucose metabolism. In addition, increased endoplasmic reticulum (ER) stress was found in the livers of the CD36-deficient mice, while inhibited ER stress normalized the PTP1B expression and restored insulin signaling in the CD36-deficient mice. Our findings suggest that the loss of CD36 impairs hepatic insulin signaling by enhancing the PTP1B/IR interaction that is induced by ER stress, indicating a possible critical step in the progression of hepatic insulin resistance.

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.

Smooth muscle cells differentiated from mesenchymal stem cells are regulated by microRNAs and suitable for vascular tissue grafts.

Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-ß1 (TGFß1), human umbilical cord-derived MSCs abundantly express the SMC markers α-smooth muscle actin (αSMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indicating the potential of utilizing human MSC-derived SMCs to generate vascular grafts. Of note, microRNA (miR) array analysis and TaqMan microRNA assays identified miR-503 and miR-222-5p as potential regulators of MSC differentiation into SMCs at early time points. Mechanistically, miR-503 promoted SMC differentiation by directly targeting SMAD7, a suppressor of SMAD-related, TGFß1-mediated signaling pathways. Moreover, miR-503 expression was SMAD4-dependent. SMAD4 was enriched at the miR-503 promoter. Furthermore, miR-222-5p inhibited SMC differentiation by targeting and down-regulating ROCK2 and αSMA. In conclusion, MSC differentiation into SMCs is regulated by miR-503 and miR-222-5p and yields functional SMCs for use in vascular grafts.

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.

Knocking down Stard3 decreases adipogenesis with decreased mitochondrial ROS in 3T3-L1 cells.

Start domain-containing protein 3 (Stard3) plays roles in intracellular cholesterol distribution, however, the role of Stard3 in the adipogenesis of 3T3-L1 preadipocytes remains unclear. We demonstrated that Stard3 expression was significantly increased during the adipogenesis of 3T3-L1 preadipocytes, accompanied by an increase of mitochondrial Reactive oxygen species (ROS). Stard3 knocking-down inhibited 3T3-L1 preadipocyte adipogenesis with decreased mitochondrial ROS levels, while ROS inducer rescued the stard3 silencing 3T3 cells with increased ROS. Moreover, Stard3 silencing reduced the expression of peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein (C/EBP)α in 3T3- L1 cells. In conclusion, Stard3 enhanced the adipogenesis of preadipocytes by enhancement of cholesterol redistribution to the mitochondrial, increasing mitochondrial ROS production. These results suggest that Stard3 is an essential factor for the 3T3-L1 cells' differentiation.

CD36 and lipid metabolism in the evolution of atherosclerosis.

Background: CD36 is a multi-functional class B scavenger receptor, which acts as an important modulator of lipid homeostasis and immune responses. Sources of data: This review uses academic articles. Areas of agreement: CD36 is closely related to the development and progression of atherosclerosis. Areas of controversy: Both persistent up-regulation of CD36 and deficiency of CD36 increase the risk for atherosclerosis. Abnormally up-regulated CD36 promotes inflammation, foam cell formation, endothelial apoptosis, macrophage trapping and thrombosis. However, CD36 deficiency also causes dyslipidemia, subclinical inflammation and metabolic disorders, which are established risk factors for atherosclerosis. Growing points: There may be an 'optimal protective window' of CD36 expression. Areas timely for developing research: In addition to traditionally modulating protein functions using gene overexpression or deficiency, the modulation of CD36 function at post-translational levels has recently been suggested to be a potential therapeutic strategy.

Fatty acid activates NLRP3 inflammasomes in mouse Kupffer cells through mitochondrial DNA release.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in many developed and developing countries worldwide. It has been well established that the chronic sterile inflammation caused by the NLRP3 inflammasome is closely related to NAFLD development. Kupffer cells (KCs) are involved in the pathogenesis of various liver diseases. We used methionine choline-deficient diets to establish a mouse nonalcoholic steatohepatitis (NASH) model. The expression and formation of the NLRP3 inflammasome in the KCs from the mouse and cell models were determined by Western blotting and co-immunoprecipitation. Evidence of mitochondrial DNA (mtDNA) release was determined by live cell labeling and imaging. KCs and the NLRP3 inflammasome exerted proinflammatory effects on the development and progression of NASH through secretion of the proinflammatory cytokine IL-1ß. NLRP3, ASC and Caspase-1 protein expression levels in KCs from NASH mouse livers were significantly higher than those in KCs from NLRP3-/- mice, and the number of NLRP3 inflammasome protein complexes was significantly higher in KCs from NASH mouse livers, whereas these protein complexes could not be formed in NLRP3-/- mice. In in vitro experiments, palmitic acid (PA) decreased the mitochondrial membrane potential and subsequently induced mtDNA release from the mitochondria to the cytoplasm. NLRP3 inflammasome expression was substantially increased, and mtDNA-NLRP3 inflammasome complexes formed upon PA stimulation. Our data suggest that mtDNA released from mitochondria during PA stimulation causes NLRP3 inflammasome activation, providing a missing link between NLRP3 inflammasome activation and NASH development, via binding of cytosolic mtDNA to the NLRP3 inflammasome.

Fatty acid translocase promoted hepatitis B virus replication by upregulating the levels of hepatic cytosolic calcium.

Hepatitis B virus (HBV) is designated a "metabolovirus" due to the intimate connection between the virus and host metabolism. The nutrition state of the host plays a relevant role in the severity of HBV infection. Metabolic syndrome (MS) is prone to increasing HBV DNA loads and accelerating the progression of liver disease in patients with chronic hepatitis B (CHB). Cluster of differentiation 36 (CD36), also named fatty acid translocase, is known to facilitate long-chain fatty acid uptake and contribute to the development of MS. We recently found that CD36 overexpression enhanced HBV replication. In this study, we further explored the mechanism by which CD36 overexpression promotes HBV replication. Our data showed that CD36 overexpression increased HBV replication, and CD36 knockdown inhibited HBV replication. RNA sequencing found some of the differentially expressed genes were involved in calcium ion homeostasis. CD36 overexpression elevated the cytosolic calcium level, and CD36 knockdown decreased the cytosolic calcium level. Calcium chelator BAPTA-AM could override the HBV replication increased by CD36 overexpression, and the calcium activator thapsigargin could improve the HBV replication reduced by CD36 knockdown. We further found that CD36 overexpression activated Src kinase, which plays an important role in the regulation of the store-operated Ca2+ channel. An inhibitor of Src kinase (SU6656) significantly reduced the CD36-induced HBV replication. We identified a novel link between CD36 and HBV replication, which is associated with cytosolic calcium and the Src kinase pathway. CD36 may represent a potential therapeutic target for the treatment of CHB patients with MS.

Suppression of CD36 attenuates adipogenesis with a reduction of P2X7 expression in 3T3-L1 cells.

Adipogenesis is a process of differentiation from preadipocyte into adipocyte, and is regulated by several transcription factors, including the peroxisome proliferator-activated receptor gamma (PPARγ) and the CCAAT-enhancer-binding protein alpha (C/EBPα). CD36 is a membrane protein which contributes to the metabolic disorders such as obesity. Although the previous study demonstrated CD36 participated in the progression of adipogenesis, the mechanism is still unclear. We report here that knockdown of CD36 expression by CD36 small interfering RNA (siRNA) resulted in a reduction of adipocyte differentiation and adipogenic protein expression. In addition, purinergic receptor P2X, ligand-gated ion channel 7 (P2X7) was downregulated in CD36-knockdown 3T3-L1 cells, suggesting that the suppression of CD36 attenuates adipogenesis via the P2X7 pathway in 3T3-L1 cells.

Lipid disorder and intrahepatic renin-angiotensin system activation synergistically contribute to non-alcoholic fatty liver disease.

BACKGROUND: This study aimed to investigate the possible synergistic effects of lipid disorder with renin-angiotensin system (RAS) activation in non-alcoholic fatty liver disease (NAFLD). METHODS: Apolipoprotein E gene-knockout mice, angiotensin II (Ang II) type 1 receptor (AT1) gene-knockout mice and human hepatoblastoma cell line (HepG2) were used for experiments. Lipid accumulation was examined by Filipin staining and intracellular cholesterol quantitative assay. The gene and protein expression of molecules involved in RAS and low-density lipoprotein receptor (LDLr) pathway was examined by real-time PCR, immunofluorescent staining and Western blot. RESULTS: There was significantly increased expression of RAS components and extracellular matrix (ECM) in livers of high-fat-diet-fed apolipoprotein E gene-knockout mice compared with controls. Upregulation of RAS components was positively associated with increased plasma levels of lipid profile. The in vitro study further confirmed that cholesterol loading increased supernatant renin activity and Ang II level of HepG2 cells, accompanied by increased ECM production that was positively associated with increased expression of intracellular RAS components. Interestingly, Ang II treatment increased lipid accumulation in livers of C57BL/6 mice and HepG2 cells. Furthermore, Ang II treatment increased gene and protein expression of sterol regulatory element-binding protein (SREBP) cleavage activating protein (SCAP), SREBP-2 and LDLr, which were mediated by enhanced SCAP/SREBP-2 complex translocation from endoplasmic reticulum to Golgi. However, LDLr pathway was accordingly downregulated in livers of AT1 gene-knockout C57BL/6 mice or in HepG2 cells treated by telmisartan. CONCLUSION: These findings demonstrate that lipid disorder and intrahepatic RAS activation synergistically accelerate NAFLD progression.

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.