VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in mouse skeletal muscle cells.

AIM/HYPOTHESIS: Here, our aim was to examine whether VLDL and apolipoprotein (apo) CIII induce endoplasmic reticulum (ER) stress, inflammation and insulin resistance in skeletal muscle. METHODS: Studies were conducted in mouse C2C12 myotubes, isolated skeletal muscle and skeletal muscle from transgenic mice overexpressing apoCIII. RESULTS: C2C12 myotubes exposed to VLDL showed increased levels of ER stress and inflammatory markers whereas peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and AMP-activated protein kinase (AMPK) levels were reduced and the insulin signalling pathway was attenuated. The effects of VLDL were also observed in isolated skeletal muscle incubated with VLDL. The changes caused by VLDL were dependent on extracellular signal-regulated kinase (ERK) 1/2 since they were prevented by the ERK1/2 inhibitor U0126 or by knockdown of this kinase by siRNA transfection. ApoCIII mimicked the effects of VLDL and its effects were also blocked by ERK1/2 inhibition, suggesting that this apolipoprotein was responsible for the effects of VLDL. Skeletal muscle from transgenic mice overexpressing apoCIII showed increased levels of some ER stress and inflammatory markers and increased phosphorylated ERK1/2 levels, whereas PGC-1α levels were reduced, confirming apoCIII effects in vivo. Finally, incubation of myotubes with a neutralising antibody against Toll-like receptor 2 abolished the effects of apoCIII on ER stress, inflammation and insulin resistance, indicating that the effects of apoCIII were mediated by this receptor. CONCLUSIONS/INTERPRETATION: These results imply that elevated VLDL in diabetic states can contribute to the exacerbation of insulin resistance by activating ERK1/2 through Toll-like receptor 2.

Decreased paraoxonase 2 enzymatic activity in monocyte/macrophages cells. A comparative in vivo and in vitro study for diabetes.

AIM: To investigate peripheral blood monocytes/macrophages (Mo/Mᴓ) paraoxonase 2 (PON2) in diabetes and the factors modulating its activity. METHODS: One hundred and eighteen patients with newly diagnosed uncomplicated type 2 diabetes mellitus were compared regarding clinical, biochemical and oxidative stress parameters with 80 healthy subjects. The capacity of the peripheral blood mononuclear cells (PBMNC) to release pro-oxidants and to neutralise them was determined by measuring the respiratory burst (RB) and the intracellular antioxidant enzyme PON2. In vitro experiments were conducted on a differentiated monocytes cell line (dU937) that was exposed to serum deprivation followed by addition of isolated lipoproteins (VLDL or LDL). RESULTS: Paraoxonase 2 activity in Mo/Mᴓ was significantly lower in type 2 diabetes patients (0.042 ± 0.044 vs 0.165 ± 0.133U lactonase activity/mg protein in controls, p < .0005) and decreased in the obese in all groups. It was inversely correlated to parameters of adiposity (BMI and Waist Circumference), of glucose control (blood glucose, fructosamine and HbA1c) and insulin resistance (HOMA-IR). In multivariate regression models, 15-34% of the PON2 variance was explained by diabetes. The in vitro addition of VLDL normalised the RB of serum deprived dU937 cells, S- (to 82 ± 18% of the cells incubated with serum, S+) and PON2 activity (from 0.524 ± 0.061 in S - to 0.298 ± 0.048 U/mg protein). In contrast, when LDL was added, the RB remained lower (61 ± 12% of S+, p = .03) and PON2 higher (0.580 ± 0.030 U/mg protein, p = .003). CONCLUSIONS: The decrease in monocyte/macrophage PON2 enzymatic activity observed in type 2 diabetes cannot be totally explained by abdominal obesity and insulin resistance. The underlying molecular mechanisms need to be identified.

Inhibition of long-chain acyl coenzyme A synthetases during fatty acid loading induces lipotoxicity in macrophages.

OBJECTIVE: Obesity is often associated with hypertriglyceridemia and elevated free fatty acids (FFAs), which are independent risk factors for cardiovascular disease and diabetes. Although impairment of cholesterol homeostasis is known to induce toxicity in macrophages, the consequence of altered fatty acid homeostasis is not clear. METHODS AND RESULTS: Long-chain acyl CoA synthetases (ACSLs) play a critical role in fatty acid homeostasis by channeling fatty acids to diverse metabolic pools. We treated mouse peritoneal macrophages (MPMs) with VLDL or FFAs in the presence of triacsin C, an inhibitor of the 3 ACSL isoforms present in macrophages. Treatment of macrophages with VLDL and triacsin C resulted in reduced TG accumulation but increased intracellular FFA levels, which induced lipotoxicity characterized by apoptosis. Treatment of MPMs with the saturated fatty acid stearic acid in the presence of triacsin C increased intracellular stearic acid and induced apoptosis. Stromal vascular cells collected from high-fat diet-fed mice displayed foam cell morphology and exhibited increased mRNA levels of macrophage markers and ACSL1. Importantly, all of these changes were associated with increased FFA level in AT. CONCLUSIONS: Inhibition of ACSLs during fatty acid loading results in apoptosis via accumulation of FFAs. Our data have implications in understanding the consequences of dysregulated fatty acid metabolism in macrophages.

Up-regulation of VLDL receptor expression and its signaling pathway induced by VLDL and beta-VLDL.

Very low density lipoprotein receptor (VLDLR) is thought to participate in the pathogenesis of atherosclerosis induced by VLDL and beta-VLDL. The present study was undertaken to elucidate the effects of VLDL and beta-VLDL on VLDLR expression and its signaling pathway. RAW264.7 cells were incubated with VLDL and beta-VLDL. The expression of VLDLR mRNA was detected by RT-PCR. The transcriptional activity of VLDLR gene was detected in recombinant plasmid pGL4.2VR-luciferase transfected RAW264.7. Western blot assay was used to detect the changes of phosphorylated ERK1/2 protein. Inhibitors or activators were used to observe the signal pathway involving VLDLR expression regulation. The results showed that VLDL and beta-VLDL stimulated ERK1/2 activity in a PKC-dependent manner. VLDL or beta-VLDL-induced VLDLR expression on macrophages was extremely abolished by inhibitors ERK1/2 or PKC. Our findings revealed that VLDL or beta-VLDL-induced VLDLR expression via PKC/ERK cascades and the effect was linked to the transcriptional activation of VLDLR gene promoter.

Apolipoprotein CIII induces expression of vascular cell adhesion molecule-1 in vascular endothelial cells and increases adhesion of monocytic cells.

BACKGROUND: Activation of vascular endothelial cells (ECs) plays an important role in atherogenesis and plaque instability. Lipoproteins containing apolipoprotein CIII (apoCIII) predict coronary heart disease (CHD). We recently reported that apoCIII has a proinflammatory effect on human monocytes. In this study, we looked for a direct effect of apoCIII on EC expression of adhesion molecules, leading to monocytic cell adhesion. METHODS AND RESULTS: Treatment of ECs with apoCIII or apoCIII-rich VLDL caused human monocytic THP-1 cells to adhere to them under static condition or under laminar sheer stress (1.0 dyne/cm2). ApoCIII increased EC expression of vascular cell adhesion molecule-1 (VCAM-1) protein and intercellular cell adhesion molecule-1 (ICAM-1) protein (4.9 +/- 1.5-fold and 1.4 +/- 0.5-fold versus control, respectively). Furthermore, apoCIII remarkably increased membrane-bound protein kinase C (PKC) beta in ECs, indicating activation. A selective inhibitor of PKCbeta prevented the rise in VCAM-1 and THP-1 cell adhesion to ECs. Moreover, exposure of ECs to apoCIII induced nuclear factor-kappaB (NF-kappaB) activation. PKCbeta inhibition abolished apoCIII-induced NF-kappaB activation, and NF-kappaB inhibition reduced expression of VCAM-1, each resulting in reduced THP-1 cell adhesion. ApoCIII-rich VLDL also activated PKCbeta and NF-kappaB in ECs and increased expression of VCAM-1. Pretreatment of ApoCIII-rich VLDL with anti-apoCIII neutralizing antibody abolished its effect on PKCbeta activation. CONCLUSIONS: Our findings provide the first evidence that apoCIII increases VCAM-1 and ICAM-1 expression in ECs by activating PKCbeta and NF-kappaB, suggesting a novel mechanism for EC activation induced by dyslipidemia. Therefore, apoCIII-rich VLDL may contribute directly to atherogenesis by activating ECs and recruiting monocytes to them.

Very low density lipoprotein-mediated signal transduction and plasminogen activator inhibitor type 1 in cultured HepG2 cells.

In normal subjects and in patients with cardiovascular disease, plasma triglycerides are positively correlated with plasminogen activator inhibitor type 1 (PAI-1) levels. Moreover, in vitro studies indicate that VLDLs induce PAI-1 synthesis in cultured cells, ie, endothelial and HepG2 cells. However, the signaling pathways involved in the effect of VLDL on PAI-1 synthesis have not yet been investigated. We report that VLDLs induce a signaling cascade that leads to an enhanced secretion of PAI-1 by HepG2 cells. In myo-[(3)H]inositol-labeled HepG2 cells, VLDL (100 microg/mL) caused a time-dependent increase in [(3)H]inositol phosphates, the temporal sequence being tris>bis>monophosphate. VLDL brought about a time-dependent stimulation of membrane-associated protein kinase C (PKC) activity and arachidonate release. Finally, VLDL stimulated mitogen-activated protein (MAP) kinase, and this effect was reduced by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which suggests that PKC plays a pivotal role in MAP kinase phosphorylation. VLDL-induced PAI-1 secretion was completely prevented by U73122, a specific inhibitor of phosphatidylinositol-specific phospholipase C, by H7 or by PKC downregulation, and by mepacrine (all P<0.01 versus VLDL-treated cells). 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)-octyl ester, which prevents Ca2+ release from intracellular stores, inhibited VLDL-induced PAI-1 secretion by 60% (P<0.05), and the MAP kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 completely suppressed both basal and VLDL-induced PAI-1 secretion. These data demonstrate that VLDL-induced PAI-1 biosynthesis results from a principal signaling pathway involving PKC-mediated MAP kinase activation.

Apolipoprotein E4 induces neuronal cell death under conditions of suppressed de novo cholesterol synthesis.

The presence of the apolipoprotein E (apoE) allele epsilon4 is a major risk factor for the development of Alzheimer's disease (AD); however, the molecular mechanism underlying the acceleration of AD development in individuals with epsilon4 remains to be determined. To investigate the isoform-specific effects of apoE on neurons, primary neuron cultures were prepared from fetal rat cerebral cortices. Inhibition by compactin, a 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitor of de novo cholesterol synthesis, induced premature neuronal cell death in a dose-dependent manner. In the presence of compactin at a sublethal dose to the cells, rabbit beta-migrating very low density lipoprotein (beta-VLDL) with human apoE4 (the product of epsilon4) induced premature neuronal cell death, while that with apoE3 (the product of epsilon3) did not. Neurons cultured in the presence of apoE4, beta-VLDL, and compactin were shrunken and spherical, containing condensed chromatin and fragmented DNA, features characteristic of apoptosis. The addition of intermediate metabolites of the cholesterol biosynthetic pathway, including mevalonate and squalene, rescued neuronal cells incubated with apoE4 and beta-VLDL, in the presence of compactin. These results strongly suggest that a reduction in the level of endogenously synthesized cholesterol is a prerequisite for apoE4-induced neuronal cell death.

Impact of monocyte colony-stimulating factor upon beta-very low density lipoprotein (beta-VLDL) cholesterol metabolism in tetradecanoyl phorbol acetate-derived THP-1 cells.

The effect of monocyte colony stimulating factor (M-CSF) on the beta-very low density lipoprotein (beta-VLDL) metabolism in THP-1 cells (human leukemia cell line) was studied. THP-1 cells treated with M-CSF decreased Latex Bead phagocytosis, but the cells incubated with 12-tetradecanoyl-phorbol-13-acetate (TPA) enhanced phagocytosis 2.5-fold. Binding activity of 125I-M-CSF to THP-1 cells was higher than that in THP-1 cells elicited with TPA. THP-1 cells incubated with M-CSF before TPA treatment were designated MT macrophages, and those incubated with M-CSF after TPA treatment were called TM macrophages. When these cells were incubated with beta-VLDL, the cholesterol ester content in MT macrophages was less than in TM macrophages. The uptake of [3H]cholesterol oleate-beta-VLDL in MT macrophages was the same as in TM macrophages. The released radioactivity from [3H]cholesterol oleate-beta-VLDL loaded MT macrophages was higher than that from TM macrophages. Acid cholesterol esterase activity and ACAT activity were the same in both types of macrophages. Neutral cholesterol esterase activity was higher in MT than in TM macrophages. These results suggested that beta-VLDL-induced cholesterol ester deposition in THP-1 cells-derived macrophages was suppressed by M-CSF, when M-CSF acted at the stage of monocytes (THP-1 cells), and that the reduction of cholesterol ester might be due to enhanced release of cholesterol from the cells with high neutral cholesterol esterase activity.