Inhibitory effect of trans-tiliroside on very low-density lipoprotein secretion in HepG2 cells and mouse liver.

An acylated flavonol glycoside, trans-tiliroside (1), is found in certain parts of different herbs, including the seeds of Rosa canina (Rosaceae). Previous studies on compound 1 have focused on triglyceride (TG) metabolism, including its anti-obesity and intracellular TG reduction effects. In the present study, the effects of compound 1 on cholesterol (CHO) metabolism were investigated using human hepatocellular carcinoma-derived HepG2 cells and mice. Compound 1 decreased CHO secretion in HepG2 cells, which was enhanced by mevalonate in a concentration-dependent manner and decreased the secretion of apoprotein B (apoB)-100, a marker of very low-density lipoprotein (VLDL). Compound 1 also inhibited the activity of microsomal triglyceride transfer proteins, which mediate VLDL formation from cholesterol and triglycerides in the liver. In vivo, compound 1 inhibited the accumulation of Triton WR-1339-induced TG in the blood of fasted mice and maintained low levels of apoB-100. These results suggest that compound 1 inhibits the secretion of CHO as VLDL from the liver and has the potential for use for the prevention of dyslipidemia.

Phospholipase D mediates very low-density lipoprotein-induced aldosterone production, in part, via lipin-1.

Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of very low-density lipoprotein (VLDL). VLDL has been shown to induce aldosterone production in multiple adrenal zona glomerulosa models, mediated in part by phospholipase D (PLD). PLD is an enzyme that hydrolyzes phosphatidylcholine to produce phosphatidic acid (PA), a lipid second messenger that can also be dephosphorylated by lipin to yield diacylglycerol (DAG), yet another lipid signal. However, it is unclear which of the two lipid second messengers, PA or DAG, underlies PLD's mediation of aldosterone production. We hypothesized that the key signal produced by PLD (indirectly) is DAG such that PLD mediates VLDL-induced aldosterone production via lipin-mediated metabolism of PA to DAG. To assess the role of lipin in VLDL-induced aldosterone production, lipin-1 was overexpressed (using an adenovirus) or inhibited (using propranolol) in HAC15 cells followed by treatment with or without VLDL. Lipin-1 overexpression enhanced the VLDL-stimulated increase in CYP11B2 expression (by 75%), and lipin-1 inhibition decreased the VLDL-stimulated increase in CYP11B2 expression (by 66%). Similarly, the VLDL-stimulated increase in aldosterone production was enhanced by lipin-1 overexpression (182%) and was decreased by lipin inhibition (80%). Our results are suggestive of DAG being the key lipid signal since manipulating lipin-1 levels/activity affects VLDL-stimulated steroidogenic gene expression and ultimately, aldosterone production. Our study warrants further investigation into VLDL-stimulated steroidogenic signaling pathways which may lead to the identification of novel therapeutic targets, such as lipin-1 and its downstream pathways, to potentially treat obesity-associated hypertension.

Nanocurcumin Improves Lipid Status, Oxidative Stress, and Function of the Liver in Aluminium Phosphide-Induced Toxicity: Cellular and Molecular Mechanisms.

Background: The present study aimed to evaluate the effect of nanocurcumin and curcumin on liver transaminases, lipid profile, oxidant and antioxidant system, and pathophysiological changes in aluminium phosphide (ALP) induced hepatoxicity. Material and Methods. In this experimental study, thirty-six male Wistar rats were randomly divided into six groups curcumin (Cur), nanocurcumin (Nanocur), ALP, ALP+Cur, and ALP+Nanocur. All treatments were performed by oral gavage for seven days. After treatment, animals were sacrificed, and liver and blood samples were taken. Serum levels of aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (AP), total bilirubin, cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) were measured by photometric methods. Total antioxidant capacity (TAC) and malondialdehyde (MDA) as parameters of oxidative stress and mRNA expression of the nonenzyme protein including Sirtuin 1 (STR1), Forkhead box protein O1 (FOXO1) and protein O3 (FOXO3), catalase (CAT), and glutathione peroxidase (GPX) as the enzyme protein in homogenized tissues have been investigated. A histologist analyzed liver tissue sections after staining with hematoxylin-eosin. Results: In the aluminium phosphide group, there was a significant increase in MDA, ALT, AST, and AP and total bilirubin, cholesterol, triglyceride, LDL, and VLDL; AST, ALT, total bilirubin, LDL, VLDL, cholesterol, and MDA were significantly decreased; and HDL and TAC were significantly increased compared to ALP (P < 0.05). In the ALP+Nanocur group, ALT, AST, ALP, total bilirubin, cholesterol, LDL, VLDL, triglyceride, and MDA were significantly decreased and HDL and TAC were increased significantly (P < 0.05). The effect of nanocurcumin on controlling serum levels of LDL, VLDL, triglyceride, and MDA in ALP-poisoned rats was significantly more than curcumin (P < 0.05). The ALP group had significant changes in genes SIRT1, FOXO1a, FOXO3a, CAT, and GPX compared to healthy controls (P < 0.05). Nanocurcumin mice expressed more SIRT1, FOXO1a, CAT, and GPX genes than controls, and curcumin-treated mice expressed more SIRT1 and FOXO1a genes (P < 0.05). Histopathological findings also indicated a more significant protective effect of nanocurcumin relative to curcumin against ALP-induced hepatotoxicity. Conclusion: Nanocurcumin significantly protects the liver against aluminum phosphide toxicity. It is suggested that nanocurcumin-based drugs be developed to reduce the toxic effects of ALP in poisoned patients.

Effect of Native and Modified Apolipoprotein A-I on DNA Synthesis in Cultures of Different Cells.

Culturing of bone marrow cells in serum-free RPMI-1640 medium for 24 h was accompanied by a decrease in the rate of [3H]-thymidine incorporation into DNA. Addition of native apolipoprotein A-I (apoA-I) or plasma LDL and HDL to the culture medium increased this parameter. In contrast to native apoA-I, its modified form decelerated DNA synthesis in bone marrow cells. A similar inhibitory effect of modified protein was observed in cultures of human embryonic kidney cells (HEK293) and in rapidly proliferating mouse macrophage cell line ANA-1. The only exclusion was human myeloid cell line U937: neither native nor modified apoA-I affected DNA synthesis in these cells. Thus, the regulatory effects of apoA-I are tissue-specific; this protein can produce either stimulatory or inhibitory effect on DNA biosynthesis in cells depending on its conformation.

Very-Low-Density Lipoprotein of Metabolic Syndrome Modulates Gap Junctions and Slows Cardiac Conduction.

Very-low-density lipoproteins (VLDL) is a hallmark of metabolic syndrome (MetS) and each manifestation of MetS is related to atrial fibrillation (AF) risks. Slowed atrial conduction is a mechanism of AF in MetS. We hypothesized that VLDL can modulate and reduce atrial gap junctions. VLDLs were separated from normal (Normal-VLDL) and MetS (MetS-VLDL) individuals. VLDLs (15 µg/g) and equivalent volumes of saline (CTL) were injected respectively to C57BL/6 mice for 6 weeks. Electrocardiograms demonstrated that MetS-VLDL induced prolongation of P wave (P = 0.041), PR intervals (P = 0.014), QRS duration and QTc interval (both P = 0.003), but Normal-VLDL did not. Optical mapping of perfused hearts confirmed slowed conduction on atria and ventricles of MetS-VLDL mice. Slowed cardiac conduction was associated with significant atrial and ventricular remodeling, along with systolic dysfunction and comparable intra-cardiac fibrosis. MetS-VLDL induced downregulation of Cx40 and Cx43 at transcriptional, translational and tissue levels, and it also enhanced O-GlcNAcylation of Cx40 and Cx43. Protein structure analyses predicted O-GlcNAcylation at serine 18 of Cx40 and Cx43 which may impair stability of gap junctions. In conclusion, MetS-VLDL modulates gap junctions and delays both atrial and ventricular conduction. VLDL may contribute to the pathophysiology of atrial fibrillation and ventricular arrhythmias in MetS.

Impaired stimulation of glucose transport in cardiac myocytes exposed to very low-density lipoproteins.

We recently observed that free fatty acids impair the stimulation of glucose transport into cardiomyocytes in response to either insulin or metabolic stress. In vivo, fatty acids for the myocardium are mostly obtained from triglyceride-rich lipoproteins (chylomicrons and Very Low-Density Lipoproteins). We therefore determined whether exposure of cardiac myocytes to VLDL resulted in impaired basal and stimulated glucose transport. Primary adult rat cardiac myocytes were chronically exposed to VLDL before glucose uptake was measured in response to insulin or metabolic stress, provoked by the mitochondrial ATP synthase inhibitor oligomycin. Exposure of cardiac myocytes to VLDL reduced both insulin-and oligomycin-stimulated glucose uptake. The reduction of glucose uptake was associated with a moderately reduced tyrosine phosphorylation of the insulin receptor. No reduction of the phosphorylation of the downstream effectors of insulin signaling Akt and AS160 was however observed. Similarly only a modest reduction of the activating phosphorylation of the AMP-activated kinase (AMPK) was observed in response to oligomycin. Similar to our previous observations with free fatty acids, inhibition of fatty acid oxidation restored oligomycin-stimulated glucose uptake. In conclusions, VLDL-derived fatty acids impair stimulated glucose transport in cardiac myocytes by a mechanism that seems to be mediated by a fatty acid oxidation intermediate. Thus, in the clinical context of the metabolic syndrome high VLDL may contribute to enhancement of ischemic injury by reduction of metabolic stress-stimulated glucose uptake.

Very-low and low-density lipoproteins induce neutral lipid accumulation and impair migration in monocyte subsets.

Blood monocytes are heterogeneous effector cells of the innate immune system. In circulation these cells are constantly in contact with lipid-rich lipoproteins, yet this interaction is poorly characterised. Our aim was to examine the functional effect of hyperlipidaemia on blood monocytes. In the Ldlr(-/-) mouse monocytes rapidly accumulate cytoplasmic neutral lipid vesicles during hyperlipidaemia. Functional analysis in vivo revealed impaired monocyte chemotaxis towards peritonitis following high fat diet due to retention of monocytes in the greater omentum. In vitro assays using human monocytes confirmed neutral lipid vesicle accumulation after exposure to LDL or VLDL. Neutral lipid accumulation did not inhibit phagocytosis, endothelial adhesion, intravascular crawling and transmigration. However, lipid loading led to a migratory defect towards C5a and disruption of cytoskeletal rearrangement, including an inhibition of RHOA signaling. These data demonstrate distinct effects of hyperlipidaemia on the chemotaxis and cytoskeletal regulation of monocyte subpopulations. These data emphasise the functional consequences of blood monocyte lipid accumulation and reveal important implications for treating inflammation, infection and atherosclerosis in the context of dyslipidaemia.

High-density and very-low-density lipoprotein have opposing roles in regulating tumor-initiating cells and sensitivity to radiation in inflammatory breast cancer.

PURPOSE: We previously demonstrated that cholesterol-lowering agents regulate radiation sensitivity of inflammatory breast cancer (IBC) cell lines in vitro and are associated with less radiation resistance among IBC patients who undergo postmastectomy radiation. We hypothesized that decreasing IBC cellular cholesterol induced by treatment with lipoproteins would increase radiation sensitivity. Here, we examined the impact of specific transporters of cholesterol (ie lipoproteins) on the responses of IBC cells to self-renewal and to radiation in vitro and on clinical outcomes in IBC patients. METHODS AND MATERIALS: Two patient-derived IBC cell lines, SUM 149 and KPL4, were incubated with low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL), or high-density lipoproteins (HDL) for 24 hours prior to irradiation (0-6 Gy) and mammosphere formation assay. Cholesterol panels were examined in a cohort of patients with primary IBC diagnosed between 1995 and 2011 at MD Anderson Cancer Center. Lipoprotein levels were then correlated to patient outcome, using the log rank statistical model, and examined in multivariate analysis using Cox regression. RESULTS: VLDL increased and HDL decreased mammosphere formation compared to untreated SUM 149 and KPL4 cells. Survival curves showed enhancement of survival in both of the IBC cell lines when pretreated with VLDL and, conversely, radiation sensitization in all cell lines when pretreated with HDL. In IBC patients, higher VLDL values (>30 mg/dL) predicted a lower 5-year overall survival rate than normal values (hazard ratio [HR] = 1.9 [95% confidence interval [CI]: 1.05-3.45], P=.035). Lower-than-normal patient HDL values (<60 mg/dL) predicted a lower 5-year overall survival rate than values higher than 60 mg/dL (HR = 3.21 [95% CI: 1.25-8.27], P=.015). CONCLUSIONS: This study discovered a relationship among the plasma levels of lipoproteins, overall patient response, and radiation resistance in IBC patients and IBC patient-derived cell lines. A more expansive study is needed to verify these observations.

Stimulation of phagocyte adhesion to endothelial cells by modified VLDL and HDL requires scavenger receptor BI.

Hyperglycemia- and oxidative stress-induced modification of circulating lipoproteins is being increasingly recognized as an important pathogenetic factor for diabetic cardiovascular damages. This study was designed to investigate the impact of modified very low-density lipoprotein and high-density lipoprotein on phagocyte adhesion to endothelial cells and the involvement of scavenger receptor class B type 1 (SR-BI) in this process. Native lipoproteins were isolated by density gradient ultracentrifugation and in vitro glycoxidative or oxidative modification was performed in the presence of glucose or sodium hypochlorite, respectively. One hour co-incubation experiments with lipoproteins, freshly prepared polymorphonuclear leukocytes (PMN), and venous endothelial cells (HUVEC) were performed in the presence or absence of different scavenger receptors and signal transduction inhibitors. PMN adhesion to HUVEC was quantified fluorimetrically. We demonstrated that oxidized and glycoxidized lipoproteins promote adhesion of PMN to HUVEC from 1.5- to 2.5-fold with oxidized lipoproteins having the greatest effect. Treatment with the highly specific SR-BI inhibitor, BLT-1 produced substantial reduction of lipoprotein-induced adhesion to endothelial cells. Native and modified lipoproteins recruited extracellular signal-regulated kinase (ERK 1/2), p38 mitogen-activated protein kinase, and Janus kinase 2 as downstream signaling pathways for adhesion. From this study, it could be concluded that modification of lipoproteins plays a crucial role in atherosclerotic progression and SR-BI may be considered as a potential therapeutic target for the prevention of diabetic cardiovascular complications.

Very low density lipoproteins derived from periodontitis patients facilitate macrophage activation via lipopolysaccharide function.

OBJECTIVE: Periodontitis, a chronic oral infection caused mainly by gram-negative bacteria, induces endotoxemia and associates with the risk for atherosclerosis. We investigated the effect of periodontal treatment on proatherogenic properties of very low density lipoproteins (VLDL). METHODS: VLDL were isolated from 30 systemically healthy periodontitis patients before (pre-treatment) and 3 months after treatment (post-treatment). The mass compositions were analyzed, and VLDL-induced changes in cellular cholesterol content and expression of selected genes of human THP-1 macrophages were measured. RESULTS: Periodontal treatment decreased the local inflammation in the periodontium, but did not have a significant effect on C-reactive protein (CRP) levels, VLDL composition, or VLDL potential to induce cholesterol uptake or gene expression by the macrophages. Incubation of macrophages in the presence of VLDL resulted in more than twofold increase in their cellular cholesterol content. Uptake of VLDL with ensuing macrophage cholesterol accumulation correlated positively with VLDL-associated lipopolysaccharide (LPS) activity (r=0.436, P=.016) and apolipoprotein E content (r=0.374, P=.046). Pre-treatment VLDL derived from the patients with high CRP levels displayed higher LPS activity than that of VLDL derived from patients with low CRP (above vs. below median, P=.007). In addition, pre-treatment VLDL isolated from patients with high systemic inflammation induced higher relative mRNA expression of CD14, TNF-α, MCP-1, and IL-6 in the macrophages. CONCLUSION: Inflammation and endotoxemia induced by severe periodontitis may increase VLDL-dependent macrophage activation and cellular cholesterol accumulation, and thereby atherogenesis.

Activation of peroxisome proliferator-activated receptor δ inhibits human macrophage foam cell formation and the inflammatory response induced by very low-density lipoprotein.

OBJECTIVE: Hypertriglyceridemia is an important risk factor for cardiovascular disease. Elevated plasma very low-density lipoprotein (VLDL) puts insulin-resistant patients at risk for atherosclerosis. VLDL readily induces macrophage lipid accumulation and inflammatory responses, for which targeted therapeutic strategies remain elusive. We examined the ability of VLDL to induce macrophage foam cells and the inflammatory response and sought to define the cell signaling cascades involved. We further examined the potential of peroxisome proliferator-activated receptor (PPAR) δ activation to attenuate both VLDL-stimulated lipid accumulation and cytokine expression. METHODS AND RESULTS: THP-1 macrophages exposed to VLDL displayed significant triglyceride accumulation, which was attenuated by PPARδ activation. PPARδ agonists stimulated a transcriptional program resulting in inhibition of lipoprotein lipase activity, activation of fatty acid uptake, and enhanced ß-oxidation. VLDL-treated macrophages significantly increased the expression of activator protein 1 associated cytokines interleukin-1ß, macrophage inflammatory protein 1α, and intercellular adhesion molecule-1. VLDL treatment significantly increased the phosphorylation of both extracellular signal-related kinase 1 and 2 and p38. VLDL reduced AKT phosphorylation as well as its downstream effector forkhead box protein O1, concomitant with increased nuclear forkhead box protein O1. Cells treated with PPARδ agonists were completely resistant to VLDL-induced expression of inflammatory cytokines, mediated by normalization of mitogen-activated protein kinase (MAPK)(erk) and AKT/forkhead box protein O1 signaling. CONCLUSIONS: The combined PPARδ-mediated reductions of lipid accumulation and inflammatory cytokine expression suggest a novel macrophage-targeted therapeutic option in treating atherosclerosis.

NADPH oxidase 2 plays a critical role in dysfunction and apoptosis of pancreatic ß-cells induced by very low-density lipoprotein.

In type 2 diabetes, pancreatic ß-cells cannot secret enough insulin compensate for insulin resistance, which are often accompanied by abnormality in lipid metabolism such as hypertriglyceridemia. It is reported that oxidative stress is involved in pancreatic ß-cell dysfunction. However, molecular mechanisms linking between excessive generations of reactive oxygen species (ROS) and ß-cell dysfunction and apoptosis induced by high levels of very low-density lipoprotein (VLDL) are poorly understood. In this study, we test the hypothesis that NADPH oxidase 2 (NOX2)-derived ROS may play a key role in dysfunction and apoptosis of pancreatic ß-cell induced by VLDL. Our results show that the ApoCIII transgenic mice displayed increased serum TG levels, enhanced generation of ROS and impaired insulin content in pancreatic ß-cells. In vitro, the treatment of pancreatic NIT-1 cells with 1 mg/ml VLDL for 12 h stimulated NOX2-derived ROS generation, decreased expression and secretion of insulin. Furthermore, we found that VLDL induced dysfunction and apoptosis of pancreatic ß-cells through JNK and p53 pathways, which were rescued by siRNA-mediated NOX2 reduction. In conclusion, our data demonstrate a critical role of NOX2-derived ROS in dysfunction and apoptosis through JNK and p53 pathways in pancreatic ß-cells induced by VLDL.

Very-low-density lipoprotein mediates transcriptional regulation of aldosterone synthase in human adrenocortical cells through multiple signaling pathways.

Diabetic dyslipidemia is characterized by increased circulatory very-low-density lipoprotein (VLDL) levels. Aldosterone, apart from its role in fluid and electrolyte homeostasis, has also been implicated in insulin resistance and myocardial fibrosis. The impact of VLDL as a potential risk factor for aldosterone-mediated cardiovascular injury in diabetes mellitus, however, remains to be investigated. We have therefore studied native and modified VLDL-mediated steroidogenesis and its underlying molecular mechanisms in human adrenocortical carcinoma cells, NCI H295R. Native VLDL (natVLDL), isolated from healthy volunteers, was subjected to in vitro modification with glucose (200 mmol/l) or sodium hypochlorite (1.5 mmol/l) for preparation of glycoxidized and oxidized VLDL, respectively. VLDL treatment induced steroidogenesis in both a concentration- and time-dependent manner. Native and glycoxidized VLDL (50 µg/ml) were almost two-fold more potent in adrenocortical aldosterone release than angiotensin II (100 nmol/l). These forms of VLDL significantly augmented transcriptional regulation of aldosterone synthase (Cyp11B2), partially through scavenger receptor class B type I, as evident from the effect of BLT-1. In contrast to glycoxidized VLDL, oxidized VLDL significantly attenuated the stimulatory effect of natVLDL on adrenocortical hormone synthesis. Moreover, treatment with specific pharmacological inhibitors (H89, U0126, AG490) provided supporting evidence that VLDL, irrespective of modification, presumably recruited PKA, ERK1/2 and Jak-2 for steroid hormone release through modulation of Cyp11B2 mRNA level. In conclusion, this study demonstrates a novel insight into intracellular mechanism of VLDL-mediated aldosterone synthesis through transcriptional regulation of steroidogenic acute regulatory protein (StAR) and Cyp11B2 expression in human adrenocortical carcinoma cell line.

High-density lipoprotein phospholipids interfere with dendritic cell Th1 functional maturation.

Lipoproteins are both lipid carriers in the blood and regulators of essential biological processes. Several studies demonstrated that lipoproteins modified during pathological conditions could alter dendritic cell (DC) maturation. Here the immune function of non-pathological lipoproteins is addressed by analysing their impact on human DC maturation triggered by TLR ligands. Upon TLR4 stimulation, low- and high-density lipoproteins (LDL and HDL) strongly inhibited the ability of DC to induce a Th1 response of T cells, characterized by high levels of IFNγ secretion, whereas the effect of very low-density lipoprotein was subject to variations. HDL also inhibited the Th1 function of DC stimulated by TLR1/2 and TLR2/6 ligands. The phospholipid fraction from HDL retained the inhibitory activity of the lipoprotein. We identified the 1-palmitoyl-2-linoleyl-phosphatidylcholine (PLPC) as one active phospholipid that inhibited the Th1 function of mature DCs whereas the dipalmitoyl-phosphatidylcholine had no significant effect. The treatment of DC by PLPC, 24h before TLR4 stimulation, resulted in reduced activation of NF-κB. This study shows that some HDL phospholipids have a direct immunoregulatory function, by modulating DC ability to activate a Th1 response of T cells.

Adrenal cell aldosterone production is stimulated by very-low-density lipoprotein (VLDL).

Very low-density lipoproteins (VLDL) are a class of large lipoprotein synthesized in the liver. The key function of VLDL, in vivo, is to carry triglyceride from the liver to adipose tissue. As a steroidogenic organ, the adrenal gland mainly uses lipoproteins as sources of cholesterol. Although VLDL receptors have been detected in the human adrenal, the function of VLDL in the adrenal gland remains unknown. Herein, we used primary cultures of human and bovine adrenal cells and the adrenocortical cell line H295R as models to determine the effects of VLDL on adrenal steroidogenesis. Our studies revealed that VLDL significantly increased aldosterone synthesis in all of the models tested. This increase was largely due to VLDL's stimulation of the expression of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2). VLDL increased CYP11B2 mRNA expression in a concentration-dependent manner. Effects of VLDL on CYP11B2 transcript levels were not additive with angiotensin II or potassium but were additive with the cAMP pathway agonists ACTH and forskolin. Nifedipine completely inhibited the effects of VLDL on CYP11B2 mRNA, suggesting that calcium is the main signal transduction pathway used by VLDL in adrenal cells. Indeed, VLDL increased cytosolic free calcium levels. An in vivo study conducted in sucrose-fed rats showed a positive correlation between elevated triglyceride (VLDL) levels in plasma and CYP11B2 expression in the adrenal. In conclusion, we have shown that VLDL can stimulate aldosterone synthesis in adrenocortical cells by increasing StAR and CYP11B2 expression, an event likely mediated by a calcium-initiated signaling cascade.

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.

Expression of the human apoE2 isoform in adipocytes: altered cellular processing and impaired adipocyte lipogenesis.

Expression of apoE in adipocytes has been shown to have an important role in modulating adipocyte triglyceride (TG) metabolism and gene expression that is independent of circulating and extracellular apoE. The impact of adipocyte expression of common human apoE isoforms was evaluated using adipocytes harvested from human apoE2, -3, and -4 knock-in mice. Expression of the apoE2 isoform was associated with an increase in adipocyte apoE gene expression and apoE synthesis. Newly synthesized apoE2 was unstable in adipocytes and demonstrated increased degradation and decreased secretion. ApoE2-expressing mice were hyperlipidemic, and had increased size of gonadal fat pads and of adipocytes, compared with apoE3 mice. In isolated cells, however, expression of the apoE2 isoform produced defective lipogenesis and increased TG hydrolysis. Incubation of adipose tissue with apoE3-containing TG-rich lipoproteins resulted in a significant increase in TG in adipose tissue from apoE3 and -E4 mice, but not apoE2 mice. Reduced capacity to internalize FFA as lipogenic substrate contributed to defective lipogenesis. Newly synthesized apoE2 is unstable in adipocytes and results in decreased adipocyte TG synthesis and defective FA uptake. These changes recapitulate those observed in apoE knockout adipocytes and have implications for understanding metabolic disturbances in humans expressing the E2 isoform.

Tissue factor pathway inhibitor 2 is induced by thrombin in human macrophages.

Tissue factor pathway inhibitor 2 (TFPI2) is a serine protease inhibitor critical for the regulation of extracellular matrix remodeling and atherosclerotic plaque stability. Previously, we demonstrated that TFPI2 expression is increased in monocytes from patients with familial combined hyperlipidemia (FCH). To gain insight into the molecular mechanisms responsible for this upregulation, we examined TFPI2 expression in THP-1 macrophages exposed to lipoproteins and thrombin. Our results showed that TFPI2 expression was not affected by treatment with very low density lipoproteins (VLDL), but was induced by thrombin (10 U/ml) in THP-1 (1.9-fold increase, p<0.001) and human monocyte-derived macrophages (2.3-fold increase, p<0.005). The specificity of the inductive effect was demonstrated by preincubation with the thrombin inhibitors hirudin and PPACK, which ablated thrombin effects. TFPI2 induction was prevented by pre-incubation with MEK1/2 and JNK inhibitors, but not by the EGF receptor antagonist AG1478. In the presence of parthenolide, an inhibitor of NFκB, but not of SR-11302, a selective AP-1 inhibitor, thrombin-mediated TFPI2 induction was blunted. Our results also show that thrombin treatment increased ERK1/2, JNK and IκBα phosphorylation. Finally, we ruled out the possibility that TFPI2 induction by thrombin was mediated by COX-2, as preincubation with a selective COX-2 inhibitor did not prevent the inductive effect. In conclusion, thrombin induces TFPI2 expression by a mechanism involving ERK1/2 and JNK phosphorylation, leading finally to NFkB activation. In the context of atherosclerosis, thrombin-induced macrophage TFPI2 expression could represent a means of avoiding excessive activation of matrix metalloproteases at sites of inflammation.

Androgens and very low density lipoprotein are essential for the growth of previtellogenic oocytes from Japanese eel, Anguilla japonica, in vitro.

To investigate the regulation of lipid uptake into the eel oocyte in more detail, effects of 11-ketotestosterone (11-KT) and lipid transporters (lipoproteins) were determined in vitro. Ovarian explants from previtellogenic Japanese eels (Anguilla japonica) were incubated for 28 days with 11-KT and/or with very low density lipoproteins (Vldl), low density lipoproteins (Ldl), or high density lipoproteins (Hdl) purified from eel plasma. The androgen 11-KT induced notable increases in oocyte diameter, which were accompanied by the appearance of vacuoles rather than lipid. Ldl and Hdl increased oocyte diameters, whereas Vldl did not. However, coincubation of 11-KT and Vldl, but not of Ldl or Hdl, resulted in dramatic increases in oocyte size and lipid droplet surface area. Effects of both 11-KT (oocyte size) and Vldl (lipid droplet surface area) were dose-dependent between 1 and 100 ng/ml and between 0.5 and 5 mg/ml, respectively. Interestingly, abnormal oocyte cytology under conditions of coculture with 11-KT and Vldl could essentially be prevented if Vldl concentrations were high enough (≥ 5 mg Vldl/ml medium). Unlike 11-KT, estradiol-17beta had no effect on oocyte diameter or lipid droplet surface area. We conclude that Vldl is a key transporter of neutral lipids that accumulate into the eel oocyte during oogenesis and that Vldl-dependent lipid uptake is stimulated by the androgen 11-KT.

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells.

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.