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Lipoprotein lipase (LPL) hydrolyzes lipids in plasma lipoproteins so that the fatty acids can be taken up and used by cells. The activity of LPL changes rapidly in response to changes in nutrition, physical activity and other conditions. Angiopoietin-like protein 4 (ANGPTL4) is an important controller of LPL activity. Both LPL and ANGPTL4 are produced and secreted by adipocytes. When the transcription blocker Actinomycin D was added to cultures of 3T3-L1 adipocytes, LPL activity in the medium increased several-fold. LPL mRNA decreased moderately during 5h, while ANGPTL4 mRNA and protein declined rapidly, explaining that LPL activity was increased. TNF-α is known to reduce LPL activity in adipose tissue. We have shown that TNF-α increased ANGPTL4 both at the mRNA and protein level. Expression of ANGPTL4 is known to be under control of Foxo1. Use of the Foxo1-specific inhibitor AS1842856, or knockdown of ANGPTL4 by RNAi, resulted in increased LPL activity in the medium. Both with ActD and with the Foxo1 inhibitor the cells became unresponsive to TNF-α. This study shows that TNF-α, by a Foxo1 dependent pathway, increases the transcription of ANGPTL4 which is secreted by the cells and causes inactivation of LPL.

Lipoprotein lipase (LPL) and angiopoietin-like protein 4 (Angptl4) were studied in 3T3-L1 adipocytes. Transfections of the adipocytes with Angptl4 esiRNA caused reduction of the expression of Angptl4 to about one fourth of that in cells treated with vehicle only. This resulted in higher levels of LPL activity both on cell surfaces (heparin-releasable) and in the medium, while LPL activity within the cells remained unaffected. This demonstrated that even though both proteins are made in the same cell, Angptl4 does not inactivate LPL during intracellular transport. Most of the Angptl4 protein was present as covalent dimers and tetramers on cell surfaces, while within the cells there were only monomers. LPL gradually lost activity when incubated in medium, but there was no marked difference between conditioned medium from normal cells (rich in Angptl4) and medium after knockdown of Angptl4. Hence Angptl4 did not markedly accelerate inactivation of LPL in the medium. Experiments with combinations of different cells and media indicated that inactivation of LPL occurred on the surfaces of cells producing Angptl4.

We investigated a family from northern Sweden in which three of four siblings have congenital chylomicronemia. LPL activity and mass in pre- and postheparin plasma were low, and LPL release into plasma after heparin injection was delayed. LPL activity and mass in adipose tissue biopsies appeared normal. [(35)S]Methionine incorporation studies on adipose tissue showed that newly synthesized LPL was normal in size and normally glycosylated. Breast milk from the affected female subjects contained normal to elevated LPL mass and activity levels. The milk had a lower than normal milk lipid content, and the fatty acid composition was compatible with the milk lipids being derived from de novo lipogenesis, rather than from the plasma lipoproteins. Given the delayed release of LPL into the plasma after heparin, we suspected that the chylomicronemia might be caused by mutations in GPIHBP1. Indeed, all three affected siblings were compound heterozygotes for missense mutations involving highly conserved cysteines in the Ly6 domain of GPIHBP1 (C65S and C68G). The mutant GPIHBP1 proteins reached the surface of transfected Chinese hamster ovary cells but were defective in their ability to bind LPL (as judged by both cell-based and cell-free LPL binding assays). Thus, the conserved cysteines in the Ly6 domain are crucial for GPIHBP1 function.

27-Hydroxycholesterol (27OH) is a strong suppressor of cholesterol synthesis and a weak activator of LXR in vitro. The regulatory importance of 27OH in vivo is controversial. Here we utilized male mice with increased levels of 27OH either due to increased production (CYP27A1 transgenic mice) or reduced metabolism (Cyp7b1-/- mice). We also used mice lacking 27OH due to a knockout of Cyp27a1. The latter mice were treated with cholic acid to compensate for reduced bile acid synthesis. The effects of the different levels of 27OH on Srebp- and other LXR-regulated genes in the liver were investigated. In the liver of CYP27tg mice we found a modest increase of the mRNA levels corresponding to the LXR target genes Cyp7b1 and Abca1. A number of other LXR-regulated genes were not affected. The effect on Abca1 mRNA was not seen in the liver of Cyp7b1-/- mice. There were little or no effects on cholesterol synthesis. In the liver of the Cyp27-/- mice treated with 0.025% cholic acid there was no significant effect of the knockout on the LXR target genes. In a previous work triple-knockout mice deficient in the biosynthesis of 24S-hydroxycholesterol, 25-hydroxycholesterol and 27OH were shown to have impaired response to dietary cholesterol, suggesting side-chain oxidized oxysterols to be mediators in cholesterol-induced effects on LXR target genes at a transcriptional level (Chen W. et al., Cell Metab. 5 (2007) 73-79). The hydroxylated oxysterol responsible for the effect was not defined. We show here that treatment of wildtype mice with dietary cholesterol under the same conditions as in the above study induced the LXR target genes Lpl, Abcg8 and Srebp1c in wild type mice but failed to activate the same genes in mice lacking 27-hydroxycholesterol due to a knockout of Cyp27. We failed to demonstrate the above effects at the protein level (Abcg8) or at the activity level (Lpl). The results suggest that 27OH is not an important regulator of Srebp- or LXR regulated genes under basal conditions in mouse liver. On the other hand 27OH appears to mediate cholesterol-induced effects on some LXR target genes at a transcriptional level under some in vivo conditions.

The bacterial pathogen Yersinia pestis expresses a potential adhesin, the pH6 antigen (pH6-Ag), which appears as fimbria-like structures after exposure of the bacteria to low pH. pH6-Ag was previously shown to agglutinate erythrocytes and to bind to certain galactocerebrosides. We demonstrate that purified pH6-Ag selectively binds to apolipoprotein B (apoB)-containing lipoproteins in human plasma, mainly LDL. Binding was not prevented by antibodies to apoB. pH6-Ag interacted also with liposomes and with a lipid emulsion, indicating that the lipid moiety of the lipoprotein was responsible for the interaction. Both apoB-containing lipoproteins and liposomes prevented binding of pH6-Ag to THP-I monocyte-derived macrophages as well as pH6-Ag-mediated agglutination of erythrocytes. Binding of pH6-Ag to macrophages was not dependent on the presence of LDL receptors. Treatment of the cells with Triton X-100 or with methyl-beta-cyclodextrin indicated that the binding of pH6-Ag was partly dependent on lipid rafts. We suggest that interaction of pH6-Ag with apoB-containing lipoproteins could be of importance for the establishment of Y. pestis infections. Binding of lipoproteins to the bacterial surface could prevent recognition of the pathogen by the host defence systems. This might be important for the ability of the pathogen to replicate in the susceptible host.

Lipoprotein lipase (LPL) efficiently mediates the binding of lipoprotein particles to lipoprotein receptors and to proteoglycans at cell surfaces and in the extracellular matrix. It has been proposed that LPL increases the retention of atherogenic lipoproteins in the vessel wall and mediates the uptake of lipoproteins in cells, thereby promoting lipid accumulation and plaque formation. We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B (apoB). Chemical modification of lysines and arginines in apoB or mutation of its main proteoglycan binding site did not abolish the interaction of LDL with LPL as shown by surface plasmon resonance (SPR) and by experiments with THP-I macrophages. Recombinant LDL with either apoB100 or apoB48 bound with similar affinity. In contrast, partial delipidation of LDL markedly decreased binding to LPL. In cell culture experiments, phosphatidylcholine-containing liposomes competed efficiently with LDL for binding to LPL. Each LDL particle bound several (up to 15) LPL dimers as determined by SPR and by experiments with THP-I macrophages. A recombinant NH(2)-terminal fragment of apoB (apoB17) bound with low affinity to LPL as shown by SPR, but this interaction was completely abolished by partial delipidation of apoB17. We conclude that the LPL-apoB interaction is not significant in bridging LDL to cell surfaces and matrix components; the main interaction is between LPL and the LDL lipids.

Lipoprotein lipase (LPL) is present in cells involved in development of atherosclerosis (endothelial cells, smooth muscle cells, and macrophages). A direct involvement of LPL in atherogenesis has been suggested. Previously we used the surface plasmon resonance technique to study the interaction of lipoproteins with surfaces covered by heparan sulfate proteoglycans (HSPG) and LPL [A. Lookeneet al.(1997)Biochemistry36, 5267–5275]. The binding was much increased by the presence of LPL. Here we demonstrate that mild oxidation of low-density-lipoprotein (LDL) and very-low-density lipoprotein (VLDL)in vitroincreases their binding to surfaces covered by HSPG and LPL, while extensive oxidation decreases it. Similar results were obtained with a lipid emulsion (Intralipid), indicating that oxidation-induced changes of the lipid part could explain the effects. LPL increased binding and uptake of the mildly oxidized (compared to nonoxidized) LDL by THP-I monocyte-derived macrophages. Our studies indicate that LPL has the highest affinity for mildly oxidized LDL and support its involvement in development of atherosclerosis.

Angiopoietin-like protein 4 (ANGPTL4) is suggested to be a master regulator of plasma triglyceride metabolism. Our aim was to study whether the previously reported high levels of ANGPTL4 detected in serum from patients with rheumatoid arthritis (RA) by ELISA was due to any specific molecular form of this protein (oligomers, monomers or fragments). ANGPTL4 levels were first determined in serum from 68 RA patients and 43 age and sex matched control subjects and the mean values differed by a factor of 5.0. Then, ANGPTL4 was analyzed after size exclusion chromatography (SEC) of serum samples. With serum from one of the RA patients with high levels of ANGPTL4, the dominant reactivity was found in fractions corresponding to high-molecular weight proteins. In addition, a minor peak of reactivity eluting late from the column was found both in the patient and in controls. By the use of Hetero-Block r, and by careful selection of antibodies, we documented non-specific reactions for ANGPTL4 in 39% of samples from the RA patients, most likely due to cross-reactivity of the antibodies with rheumatoid factor (RF). The corresponding figure for control subjects was 6.3%. After corrections for non-specific reactions, the mean level of ANGPTL4 in serum from RA patients was still significantly higher than in control individuals (mean levels were 101 +/- 62 and 67 +/- 39 ng/ml respectively, P = 0.02). We re-analyzed samples from our previously published studies on ANGPL4 levels in patients on hemodialysis and patients with diabetes type 2. These samples did not show false positive reactions. The levels of ANGPTL4 were comparable to those detected previously.

Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

Lipoprotein lipase (LPL) is important for clearance of triacylglycerols (TG) from plasma both as an enzyme and as a bridging factor between lipoproteins and receptors for endocytosis. The amount of LPL at the luminal side of the capillary endothelium determines to what extent lipids are taken up. Mechanisms to control both the activity of LPL and its transport to the endothelial sites are regulated, but poorly understood. Angiopoietin-like proteins (ANGPTLs) 3 and 4 are potential control proteins for LPL, but plasma concentrations of ANGPTLs do not correlate with plasma TG levels. We investigated the effects of recombinant human N-terminal (NT) ANGPTLs3 and 4 on LPL-mediated bridging of TG-rich lipoproteins to primary mouse hepatocytes and found that the NT-ANGPTLs, in concentrations sufficient to cause inactivation of LPL in vitro, were unable to prevent LPL-mediated lipoprotein uptake. We therefore investigated the effects of lipoproteins (chylomicrons, VLDL and LDL) on the inactivation of LPL in vitro by NT-ANGPTLs3 and 4 and found that LPL activity was protected by TG-rich lipoproteins. In vivo, postprandial TG protected LPL from inactivation by recombinant NT-ANGPTL4 injected to mice. We conclude that lipoprotein-bound LPL is stabilized against inactivation by ANGPTLs. The levels of ANGPTLs found in blood may not be sufficient to overcome this stabilization. Therefore it is likely that the prime site of action of ANGPTLs on LPL is in subendothelial compartments where TG-rich lipoprotein concentration is lower than in blood. This could explain why the plasma levels of TG and ANGPTLs do not correlate.

Lipoprotein lipase (LPL) is produced by cells in the artery wall and can mediate binding of lipoproteins to cell surface heparan sulfate proteoglycans (HSPG), resulting in endocytosis (the bridging function). Active, dimeric LPL may dissociate to inactive monomers, the main form found in plasma. We have studied binding/internalization of human low density lipoprotein (LDL), mediated by bovine LPL, using THP-1 monocytes and macrophages. Uptake of (125)I-LDL was similar in monocytes and macrophages and was not affected by the LDL-receptor family antagonist receptor-associated protein (RAP) or by the phagocytosis inhibitor cytochalasin D. In contrast, uptake depended on HSPG and on membrane cholesterol. Incubation in the presence of dexamethasone increased the endogenous production of LPL by the cells and also increased LPL-mediated binding of LDL to the cell surfaces. Monomeric LPL was bound to the cells mostly in a heparin-resistant fashion. We conclude that the uptake of LDL mediated by LPL dimers is receptor-independent and involves cholesterol-enriched membrane areas (lipid rafts). Dimeric and monomeric LPL differ in their ability to mediate binding/uptake of LDL, probably due to different mechanisms for binding/internalization.