Xiong Z. Ruan

PPAR Agonists Protect Mesangial Cells from Interleukin 1β-Induced Intracellular Lipid Accumulation by Activating the ABCA1 Cholesterol Efflux Pathway

Previous studies have demonstrated that inflammatory cytokines such as interleukin-1beta (IL-1beta) promote lipid accumulation in human mesangial cells (HMC) by dysregulating the expression of lipoprotein receptors. Intracellular lipid accumulation is governed by both influx and efflux; therefore, the effect of IL-1beta on the efflux of lipid from HMC was investigated. IL-1beta was shown to inhibit (3)H-cholesterol efflux from HMC and increase total intracellular cholesterol concentration, probably as a result of reduced expression of the adenosine triphosphate (ATP) binding cassette A1 (ABCA1), a transporter protein involved in apolipoprotein-A1 (apo-A1)-mediated lipid efflux. To ascertain the molecular mechanisms involved, expression of peroxisome proliferator-activated receptors (PPAR) and liver X receptoralpha (LXRalpha) were examined. IL-1beta (5 ng/ml) reduced PPARalpha, PPARgamma, and LXRalpha mRNA expression. Activation of PPARgamma with the agonist prostaglandin J2 (10 micro M) and of PPARalpha with either bezafibrate (100 micro M) or Wy14643 (100 micro M) both increased LXRalpha and ABCA1 gene expression also and enhanced apoA1-mediated cholesterol efflux from lipid-loaded cells, even in the presence of IL-1beta. A natural ligand of LXRalpha, 25-hydroxycholesterol (25-OHC), had similar effects; when used together with PPAR agonists, an additive effect was observed, indicating co-operation between PPAR and LXRalpha in regulating ABCA1 gene expression. This was supported by the observation that overexpression of either PPARalpha or PPARgamma by transfection enhanced LXRalpha and ABCA1 gene induction by PPAR agonists. Taken together with previous data, it appears that, in addition to increasing lipid uptake, inflammatory cytokines promote intracellular lipid accumulation by inhibiting cholesterol efflux through the PPAR-LXRalpha-ABCA1 pathway. These results suggest potential mechanisms whereby inflammation may exacerbate lipid-mediated cellular injury in the glomerulus and in other tissues and indicate that PPAR agonists may have a protective effect.

Mechanisms of Dysregulation of Low-Density Lipoprotein Receptor Expression in Vascular Smooth Muscle Cells by Inflammatory Cytokines

Objective—Although inflammation is a recognized feature of atherosclerosis, the impact of inflammation on cellular cholesterol homeostasis is unclear. This study focuses on the molecular mechanisms by which inflammatory cytokines disrupt low-density lipoprotein (LDL) receptor regulation. Methods and Results—IL-1&bgr; enhanced transformation of vascular smooth muscle cells into foam cells by increasing uptake of unmodified LDL via LDL receptors and by enhancing cholesterol esterification as demonstrated by Oil Red O staining and direct assay of intracellular cholesterol concentrations. In the absence of IL-1&bgr;, a high concentration of LDL decreased LDL receptor promoter activity, mRNA synthesis and protein expression. However, IL-1&bgr; enhanced LDL receptor expression, overriding the suppression usually induced by a high concentration of LDL and inappropriately increasing LDL uptake. Exposure to IL-1&bgr; also caused overexpression of the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP), and enhanced its translocation from the endoplasmic reticulum to the Golgi, where it is known to cleave SREBP, thereby enhancing LDL receptor gene expression. Conclusions—These observations demonstrate that IL-1&bgr; disrupts cholesterol-mediated LDL receptor feedback regulation, permitting intracellular accumulation of unmodified LDL and causing foam cell formation. The implication of these findings is that inflammatory cytokines may contribute to intracellular LDL accumulation without previous modification of the lipoprotein.

Sirolimus modifies cholesterol Homeostasis in hepatic cells: A potential molecular mechanism for Sirolimus-Associated Dyslipidemia

Background. Sirolimus is a potent immunosuppressive agent, which is associated with dyslipidemia in clinical transplantation. The present study was undertaken to investigate the potential hepatocyte mechanisms by which sirolimus causes dyslipidemia. Methods. Using both a quantitative assay of intracellular cholesterol and an [3H]-labeled cholesterol efflux assay, we studied the effect of sirolimus on cholesterol accumulation and cholesterol efflux in HepG2 cells in the absence or presence of inflammatory stress induced by interleukin-1&bgr;. The gene and protein expression of molecules involved in cholesterol homeostasis were examined by real-time reverse-transcription polymerase chain reaction and Western blotting. Results. Sirolimus inhibited low-density lipoprotein (LDL) receptor (LDLr)-mediated cholesterol ester accumulation induced by interleukin-1&bgr; in HepG2 cells. This inhibitory effect was mediated by down-regulation of sterol regulatory element-binding proteins (SREBP) cleavage activating protein (SCAP) and SREBP-2 mRNA expression. Using confocal microscopy, we demonstrated that sirolimus reduced translocation of SCAP-SREBP2 complex from endoplasmic reticulum to Golgi for activation, thereby inhibiting LDLr gene transcription. Reduction of LDLr in the liver may result in a delay of LDL-cholesterol clearance from circulation causing an increase of plasma cholesterol concentration. Furthermore, sirolimus increased cholesterol efflux mediated by adenosine triphosphate-binding cassette transporter A1 gene expression by increasing peroxisome proliferator-activated receptor-&agr; and liver X receptor-&agr; gene and protein expression. Increased cholesterol efflux from HepG2 cells may increase high-density lipoprotein cholesterol level and also contribute to apolipoprotein B lipoprotein formation by enhancing transfer of high-density lipoprotein cholesterol to apolipoprotein B lipoproteins. Conclusions. This study demonstrates that the increase of LDL cholesterol by sirolimus is partly due to the reduction of LDLr on hepatocytes.

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-1&bgr;, tumor necrosis factor-&agr;, 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 &mgr;mol/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-1&bgr;. Knocking down sterol regulatory element binding protein cleavage–activating protein prevented statin resistance induced by interleukin-1&bgr;, 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.

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.

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.

Chronic inflammation aggravates metabolic disorders of hepatic fatty acids in high-fat diet-induced obese mice

The prevalence of nonalcoholic fatty liver disease (NAFLD) increases with increasing body mass index (BMI). However, approximately 40–50% of obese adults do not develop hepatic steatosis. The level of inflammatory biomarkers is higher in obese subjects with NAFLD compared to BMI-matched subjects without hepatic steatosis. We used a casein injection in high-fat diet (HFD)-fed C57BL/6J mice to induce inflammatory stress. Although mice on a HFD exhibited apparent phenotypes of obesity and hyperlipidemia regardless of exposure to casein injection, only the HFD+Casein mice showed increased hepatic vacuolar degeneration accompanied with elevated inflammatory cytokines in the liver and serum, compared to mice on a normal chow diet. The expression of genes related to hepatic fatty acid synthesis and oxidation were upregulated in the HFD-only mice. The casein injection further increased baseline levels of lipogenic genes and decreased the levels of oxidative genes in HFD-only mice. Inflammatory stress induced both oxidative stress and endoplasmic reticulum stress in HFD-fed mice livers. We conclude that chronic inflammation precedes hepatic steatosis by disrupting the balance between fatty acid synthesis and oxidation in the livers of HFD-fed obese mice. This mechanism may operate in obese individuals with chronic inflammation, thus making them more prone to NAFLD.

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.

Association between reductions in low-density lipoprotein cholesterol with statin therapy and the risk of new-onset diabetes: a meta-analysis.

A recent meta-analysis demonstrated that statin therapy was associated with a risk of diabetes. The present study investigated whether the relative reduction in low-density lipoprotein cholesterol (LDL-c) was a good indicator of the risk of new-onset diabetes. We searched the PubMed, Embase, Cochrane Central Register, Lilacs, Food and Drug Administration, and European Medicines Agency databases for randomized controlled trials of statins. Fourteen trials were included in the study. Eight trials with target LDL-c levels ≤100u2009mg/dL (2.6u2009mmol/L) or LDL-c reductions of at least 30% were extracted separately. The results showed that the overall risk of incident diabetes increased by 11% (ORu2009=u20091.11; 95% CI 1.03–1.20). The group with intensive LDL-c-lowering statin had an 18% increase in the likelihood of developing diabetes (ORu2009=u20091.18; 95% CI, 1.10–1.28). Furthermore, the risks of incident diabetes were 13% (ORu2009=u20091.13; 95% CI 1.01–1.26) and 29% (ORu2009=u20091.29; 95% CI 1.13–1.47) in the subgroups with 30–40% and 40–50% reductions in LDL-c, respectively, suggesting that LDL-c reduction may provide a dynamic risk assessment parameter for new-onset diabetes. In conclusion, LDL-c reduction is positively related to the risk of new-onset diabetes. When LDL-c is reduced by more than 30% during lipid-lowering therapy, blood glucose monitoring is suggested to detect incident diabetes in high-risk populations.

Inflammatory Stress Sensitizes the Liver to Atorvastatin-Induced Injury in ApoE-/- Mice.

Statins, which are revolutionized cholesterol-lowing agents, have been reported to have unfavorable effects on the liver. Inflammatory stress is a susceptibility factor for drug-induced liver injury. This study investigated whether inflammatory stress sensitized the liver to statin-induced toxicity in mice and explored the underlying mechanisms. We used casein injection in ApoE-/- mice to induce inflammatory stress. Half of the mice were orally administered atorvastatin (10mg/kg/d) for 8 weeks. The results showed that casein injection increased the levels of serum pro-inflammatory cytokines (IL-6 and TNFα). Atorvastatin treatment increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in casein injection mice. Moreover, atorvastatin treatment exacerbated hepatic steatosis, inflammation and fibrosis, as well as increased hepatic reactive oxygen species (ROS) and malondialdehyde in casein injection mice. However, above changes were not observed in atorvastatin treated alone mice. The protein expression of liver nuclear factor erythroid 2-related factor 2 (Nrf2) and the mRNA expressions of Nrf2 target genes were increased, together with the enhancement of activities of hepatic catalase and superoxide dismutase in atorvastatin treated alone mice, but these antioxidant responses were lost in mice treated with atorvastatin under inflammatory stress. This study demonstrates that atorvastatin exacerbates the liver injury under inflammatory stress, which may be associated with the loss of adaptive antioxidant response mediated by Nrf2.