Intracellular regulation of lipoprotein lipase in human monocyte-derived macrophages

The intracellular pathway of lipoprotein lipase (LPL) has been examined in human monocyte-derived macrophages in culture. These cells were previously shown to synthesize and constitutively secrete LPL. The secretion is dependent on new enzyme synthesis. 6-d-old human monocytes have stores of mRNA for linear release of LPL up to 24 h. Enzyme activity in cells and in culture medium was almost completely inhibited by 24 h treatment with tunicamycin, an inhibitor of glycosylation. In monensin-treated cells a pronounced increase in enzyme activity was found, whereas the secreted activity was markedly reduced. This indicates that LPL in human monocytes is processed through a pH sensitive part of the Golgi complex and that the terminal glycosylation is not needed for the expression of its catalytic activity. Our results suggest that lysosomal function is not important in secretion of the enzyme, whereas vesicular transport seem to be involved in regulating LPL in human monocyte-derived macrophages in culture.     

Bacterial lipopolysaccharide and gamma interferon induce transcription of beta interferon mRNA and interferon secretion in murine macrophages

Bacterial lipopolysaccharide (LPS) induces interferon (IFN) secretion and an antiviral state in murine peritoneal macrophages (PM). These cells secrete predominantly IFN-beta, as shown by neutralization assays with monoclonal antibodies. Secretion of IFN-beta is also induced in PM by IFN-gamma. LPS and IFN-gamma synergistically stimulated PM to produce IFN in amounts almost comparable to those induced by infection with Newcastle disease virus. Low levels of IFN-beta mRNA can be detected in freshly harvested PM by hybridization assays. The accumulation of this mRNA is markedly increased in PM treated with LPS or IFN-gamma, and it is further enhanced in the presence of the inhibitor of protein synthesis, cycloheximide. Similar studies were carried out on the RAW 264.7 line of transformed macrophages. These cells are induced to secrete IFN-beta by LPS but not by IFN-gamma, suggesting that this cytokine may elicit such specific response only in PM. IFN-beta mRNA is undetectable in untreated RAW 264.7 cells, and accumulation of this mRNA is induced by LPS but not by IFN-gamma. The secretion of IFN induced by these agents in PM and by LPS in RAW 264.7 cells and the corresponding accumulation of IFN-beta mRNA are blocked by an inhibitor of protein kinase C, staurosporine. The activity of this kinase is apparently necessary to stimulate accumulation of IFN-beta mRNA. The induction of IFN-beta by IFN-gamma appears to be a characteristic response of PM and may be at least in part responsible for the resistance of these cells to viral infections.     

Involvement of both the tyrosine kinase and the phosphatidylinositol-3' kinase signal transduction pathways in the regulation of lipoprotein lipase expression in J774.2 macrophages by cytokines and lipopolysaccharide.

The regulation of macrophage lipoprotein lipase (LPL) by cytokines and lipopolysaccharide (LPS) is of potentially crucial importance in the pathogenesis of atherosclerosis and in the responses to endotoxin challenge. We show here that the reduction of LPL activity in J774.2 macrophages observed in the presence of interleukin (IL-1) and IL-11 was sensitive to herbimycin A, with the effect of LPS, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on LPL activity being sensitive to both herbimycin A and wortmannin. The action of the inhibitors on the IFN-gamma-dependent reduction of LPL activity was mediated at the level of LPL mRNA metabolism, with translational and/or post-translational levels of regulation being involved in the action of all the other mediators tested. These observations suggest that both the tyrosine kinase and the phosphatidylinositol-3'-kinase signalling pathways are involved in the suppression of macrophage LPL expression by LPS and cytokines.     

Synthesis and regulation of lipoprotein lipase in the hippocampus

Lipoprotein lipase (LPL) expression was determined in adult rat hippocampus and compared to enzyme expression in other brain regions. Hippocampus LPL mRNA levels were at least 2.5-fold higher than those detected in the cerebral cortex, cerebellum, and remaining brain regions. Enzyme mass and activity levels in the hippocampus were also increased to a similar degree. De novo synthesis of LPL in the hippocampus was confirmed by [35S]methionine-labeling of the tissue and identification of a 57 kDa protein obtained by immunoprecipitation. Addition of an excess amount of bovine LPL completely prevented the immunoprecipitation of this protein. The effect of nutritional modulations on brain LPL activity was determined after a 12-h fast. While no significant changes were observed in other regions of the brain, hippocampus LPL activity in fasted rats increased by 60% compared to the fed control group. Simultaneously, fasting reduced adipose LPL activity by 60%. Intraperitoneal injection of ACTH over a 5-day period had no effect on hippocampus LPL activity, while adipose LPL levels increased 2.3-fold and heart LPL levels decreased 1.4-fold. We conclude that LPL is synthesized, active and regulated in a tissue-specific manner in the adult rat hippocampus.     

Control of lipoprotein lipase secretion in mouse macrophages

The regulation of secretion of lipoprotein lipase (LPL) was studied in in vitro-derived mouse bone marrow macrophages (BMM), peritoneal exudate and resident macrophages and in the macrophage-like tumor cell line J774.1. BMM in cultures initiated with low concentrations of bone marrow cells (LC-BMC cultures) secrete more LPL per cell than BMM in cultures initiated with high concentrations of bone marrow cells (HC-BMC cultures). The suppressed state of LPL secretion in HC-BMC cultures could be alleviated by the addition of a colony-stimulating factor source (L-cell-conditioned medium; L-CM) onto the culture medium or exchanging the medium of HC-BMC cultures with medium from LC-BMC cultures for short periods (4 h). Addition of L-CM increased LPL secretion also in LC-BMC cultures. Addition of L-CM to fresh culture medium had little or no effect, suggesting that, in addition to requirement for L-CM, optimal expression depended also on factors released by the growing cells, probably providing optimal growth conditions. L-CM enhanced LPL secretion by thioglycollate-elicited peritoneal macrophages and had no effect on LPL secretion by resident peritoneal macrophages. Secretion of LPL from adherent J774.1 cells showed a biphasic effect. Secretion increased with cell density up to the point when growth inhibition was observed. In dense cultures in which cell proliferation was almost arrested, LPL secretion was remarkably suppressed (80-90%). Change of medium of dense cultures to fresh medium or medium conditioned by sparse cultures (for the last 4 h of culture) led to enhancement of LPL secretion to levels similar to those optimally expressed by sparse cultures. L-CM did not enhance LPL secretion from J774.1 cells. Dense cultures of both BMM and J774.1 cells did not contain a stable inhibitor of LPL secretion and medium from sparse cultures did not contain an inducer of LPL secretion. The data suggest that proliferating macrophages secrete large amounts of LPL, whereas in nonproliferating, quiescent cells, this activity is much reduced. L-CM enhances LPL secretion in quiescent BMM and peritoneal exudate cells to levels expressed by proliferating cells. Since this effect is already expressed after a 4 h incubation period, it is not dependent on cell cycling but could be one of the early responses to this macrophage mitogen. In J774.1 cells, a change of medium is a sufficient signal for enhancement of LPL secretion in quiescent cells.     

Post-translational regulation of lipoprotein lipase activity in adipose tissue.

Changes in adipose-tissue lipoprotein lipase activity that are independent of protein synthesis were investigated in an incubation system in vitro. Under appropriate conditions at 25 degrees C a progressive increase in the enzyme activity occurs that is energy-dependent. Part of the enzyme is rapidly inactivated when the tissue is incubated with adrenaline or adrenaline plus theophylline. The mechanism of this inactivation appears to be distinct from, and to follow, the activation of the enzyme. A hypothesis is presented to account for the results in terms of an activation of the enzyme during obligatory post-translational processing and a catecholamine-regulated inactivation of the enzyme as an alternative to secretion from the adipocyte.     

Production of gamma interferon by human T and null cells and its regulation by macrophages

Human mononuclear subpopulations were tested for the capacity to produce interferon after mitogenic stimulation with protein A from Staphylococcus aureus. Mononuclear cells were separated into highly enriched macrophage, T-, B-, and null-cell subpopulations by Sephadex G-10 adherence, anti-human IgG F(ab′) two-column chromatography, and rosetting with sheep erythrocytes. Interferon (IFN) production was observed in both T- and null-cell preparations, but not in macrophage or B-cell preparations. Physicochemical and antigenic characterization of IFN from T- and null-cell preparations showed that both mononuclear subpopulations produced gamma IFN (IFNγ). Regulatory studies showed that IFNγ production was differentially regulated by macrophages. Macrophage addition to T lymphocytes augmented both cellular proliferation and IFNγ production, whereas macrophage addition to null cells suppressed IFNγ production and had no effect on the minimal proliferative response observed for these cells.     

Atorvastatin decreases lipoprotein lipase and endothelial lipase expression in human THP-1 macrophages

Macrophage-derived lipases are associated with atherosclerosis in human and animal studies. Despite numerous non-lipid-lowering effects of statins, their effect on macrophage LPL and endothelial lipase (EL) expression has not been investigated. In the present study, atorvastatin and simvastatin dose-dependently decreased LPL and EL expression as well as Rho, liver X receptor alpha (LXRalpha), and nuclear factor kappaB (NF-kappaB) activation in THP-1 macrophages. Atorvastatin-reduced LPL and EL expression was only partially recovered by mevalonate cotreatment, indicating that mechanisms independent of reductase inhibition may be present. By contrast, Rho activation by lysophosphatidyl acid further decreased LPL and EL expression in the presence or absence of atorvastatin. Another Rho activator, farnysyl pyrophosphate, decreased EL expression only in the absence of atorvastatin. LXRalpha activation by T0901317 and 22(R)-hydroxycholesterol not only rescued but also significantly increased LPL expression in the presence and absence of atorvastatin, respectively, whereas LXRalpha inhibition by 22(S)-hydroxycholesterol decreased LPL expression. By contrast, EL expression was suppressed by LXRalpha activation in the presence or absence of atorvastatin. NF-kappaB inhibition by SN50 was associated with an approximately 30% reduction of EL expression. Furthermore, atorvastatin treatment significantly attenuated the lipid accumulation in macrophages treated with oxidized LDL. We conclude that atorvastatin reduces LPL and EL expression by reducing the activation of LXRalpha and NF-kappaB, respectively.     

脂蛋白脂酶调控因子研究进展

脂蛋白脂酶(Lipoprotein lipase, LPL)是脂质代谢的关键酶, 其正常调控对于机体向组织提供脂质营养至关重要。作为LPL重要的调控因子, 糖基化磷脂酰肌醇锚定高密度脂蛋白结合蛋白1(Glycosylphosphatidylinositol- anchored high density lipoprotein-binding protein 1, GPIHBP1)能与LPL结合起脂解平台的作用, 并作为载体参与LPL向毛细血管内皮细胞的转运。另外, 近年来也鉴定出其他几个LPL活性调控因子, 包括microRNAs、A型重复排序蛋白相关受体(Sortilin-related receptor with A-type repeats, SorLA)和载脂蛋白(Apolipoproteins, apo)。这些LPL调控因子的成功鉴定, 有助于人们深入认识机体脂解代谢和乳糜微粒血症发生的内在机制。文章重点综述了LPL的调控因子GPIHBP1的研究进展, 同时也对其他几个调控因子的研究进展进行了讨论。     

Nutrient regulation of post-heparin lipoprotein lipase activity in obese subjects.

This study examines the immediate effect of ingestion of oral carbohydrate and fat on lipoprotein lipase (LPL) activity post-heparin in six lean and six obese age-matched women. Subjects were given, on two separate occasions, 340 kcal carbohydrate or an equicaloric amount of fat, both in 300 ml of water. Post-heparin LPL activity (10,000 U) was measured on each occasion 120 minutes after ingestion of the meal. Following oral carbohydrate postprandial plasma insulin levels were significantly higher in obese subjects than in lean (p < 0.01). Impaired glucose tolerance was seen in the obese group. GIP secretion was similar in lean and obese subjects both during oral fat and carbohydrate ingestion. GLP-1 secretion post-carbohydrate was lower in obese subjects. Total LPL activity unadjusted for body weight was similar in the two groups after carbohydrate administration but was significantly lower when adjusted per kg body weight. Total LPL activity was lower in the lean group at 130 minutes after fat administration (p < 0.02). Fasting serum triglycerides were higher in the obese group and were inversely related to the post-carbohydrate LPL activity (r = - 0.65, p < 0.02). Intraluminal lipoprotein lipase activity is not increased in established obesity. Fat and carbohydrate nutrients may affect LPL activity differently in lean and obese subjects.     

Regulation of the secretion of lipoprotein lipase by mouse macrophages

The regulation of the secretion of lipoprotein lipase was studied in primary cultures of mouse peritoneal macrophages and in the murine macrophage cell line J774. As previously reported, both cell types secrete a lipase with the characteristics of lipoprotein lipase. Incubation of macrophages with insulin, insulin-like growth factor, and L-thyroxine had no effect on lipoprotein lipase secretion. Incubation with dexamethasone and with several agents which increase intracellular cyclic AMP led to a decrease in lipoprotein lipase secretion by mouse peritoneal macrophages. These results suggest that the hormonal regulation of lipoprotein lipase in macrophages is different from that in adipose tissue and heart muscle. Incubation of the macrophages with heparin caused a marked increase in the secretion of lipoprotein lipase. Short incubations with heparin (5 min) caused a release of the enzyme into the media, while longer incubations caused a 2-8-fold increase in net lipoprotein lipase secretion which was maximal after 2-16 h depending on cell type, and persisted for 24 h. The effect of heparin was dose-dependent and specific (it was not duplicated by other glycosaminoglycans). The mechanism of heparin-induced increase in lipoprotein lipase secretion was explored. The increase was not caused by the release of a presynthesized intracellular pool of lipoprotein lipase or by the stabilization of lipoprotein lipase by heparin after secretion. The heparin-induced increase in lipoprotein lipase secretion was dependent on protein synthesis. The secretion of lipoprotein lipase by macrophages in response to low levels of heparin may be a significant factor in the formation of atherosclerotic lesions.     

Differential regulation of indoleamine 2,3-dioxygenase by lipopolysaccharide and interferon gamma in murine bone marrow derived dendritic cells

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme in the L-tryptophan-kynurenine pathway, which converts an essential amino acid, L-tryptophan, to N-formylkynurenine. The expression of IDO increases when inflammation is induced by wounding, infection or tumor growth. Although recent studies have suggested that IDO expression is up-regulated by IFN-gamma in various cell types and that the induction of IDO can also be mediated through an IFN-gamma-independent mechanism, these mechanisms still remain unknown. In this study, we investigated whether lipopolysaccharide (LPS) induces the expression of IDO through an IFN-gamma-mediated signaling pathway or not. IFN-gamma-induced expression of IDO expression was inhibited only by JAK inhibitor I. However, LPS-induced expression of IDO was inhibited by LY294002 and SP600125 but not by JAK inhibitor I, SB203580, or U0126. These findings clearly indicate that LPS can induce the IDO expression via an IFN-gamma-independent mechanism and PI3 kinase and JNK in the LPS-induced pathway leading to IDO expression.     

Cardiac lipoprotein lipase: effects of lipopolysaccharide and tumor necrosis factor

Summary Lipopolysaccharide (LPS), the active principle of certain endotoxins, protein-free perfused in rat hearts leads in 3 h to a considerable loss of lipoprotein lipase (LPL) activity. In the presence of albumin LPS has virtually no effect. Tumor necrosis factor (TNF) added instead of LPS had no effects on LPL activity during 3 hin vitro perfusion. LPS injected into rats intravenously leads within 3 h to severe toxic phenomena amongst which increased capillary permeability. This was visualized as increased rate of interstitial fluid formation in Langendorff hearts mounted 3 h after rats had been treated with LPS. LPL activity did not decline in 3 h lasting endotoxemia. Six hours after LPS injection, however, cardiac LPL activity was considerably lowered, although immunoblotting and immunohistochemistry still showed LPL protein to be present. These date indicate the presence of a considerable pool of inactive LPL protein in addition to active LPL, that can be released in the presence of heparin. The LPL activity is lowered by LPS injection after a lag phase of at least 3 h, while capillary endothelial cells are influenced more rapidly. The relatively late expression of TNF toxicity in cardiomyocytes of the intact heart is discussed.     

Linkage of low-density lipoprotein size to the lipoprotein lipase gene in heterozygous lipoprotein lipase deficiency.

Small low-density lipoprotein (LDL) particles are a genetically influenced coronary disease risk factor. Lipoprotein lipase (LpL) is a rate-limiting enzyme in the formation of LDL particles. The current study examined genetic linkage of LDL particle size to the LpL gene in five families with structural mutations in the LpL gene. LDL particle size was smaller among the heterozygous subjects, compared with controls. Among heterozygous subjects, 44% were classified as affected by LDL subclass phenotype B, compared with 8% of normal family members. Plasma triglyceride levels were significantly higher, and high-density lipoprotein cholesterol (HDL-C) levels were lower, in heterozygous subjects, compared with normal subjects, after age and sex adjustment. A highly significant LOD score of 6.24 at straight theta=0 was obtained for linkage of LDL particle size to the LpL gene, after adjustment of LDL particle size for within-genotype variance resulting from triglyceride and HDL-C. Failure to adjust for this variance led to only a modest positive LOD score of 1.54 at straight theta=0. Classifying small LDL particles as a qualitative trait (LDL subclass phenotype B) provided only suggestive evidence for linkage to the LpL gene (LOD=1. 65 at straight theta=0). Thus, use of the quantitative trait adjusted for within-genotype variance, resulting from physiologic covariates, was crucial for detection of significant evidence of linkage in this study. These results indicate that heterozygous LpL deficiency may be one cause of small LDL particles and may provide a potential mechanism for the increase in coronary disease seen in heterozygous LpL deficiency. This study also demonstrates a successful strategy of genotypic specific adjustment of complex traits in mapping a quantitative trait locus.     

Nutritional regulation of lipoprotein lipase in guinea pig tissues

Glucose transport in guinea pig adipocytes has been shown to be markedly resistant to stimulation by insulin. Lipoprotein lipase is another transport catalyst in adipose tissue which is believed to be regulated by insulin. We have therefore studied how feeding-fasting affects lipoprotein lipase activity in guinea pig tissues. There was an even more marked decrease in adipose tissue lipoprotein lipase activity on fasting in guinea pigs (10-20 fold) than in rats or mice (4-5 fold). In adipocytes, the activity decreased only 2.5-4.5 fold; most of the change was in extracellular lipoprotein lipase. On glucose refeeding, the activity was rapidly restored. In the first 4 hours after glucose administration extracellular lipoprotein lipase activity increased to more than 10 times the amount present in adipocytes. After cycloheximide, lipoprotein lipase activity decreased with a half-life of 22 min. It is concluded that lipoprotein lipase is rapidly produced and turned over in guinea pig adipose tissue, and that the system is quite sensitive to feeding-fasting. In contrast to adipose tissue, there was no significant change in lipoprotein lipase activity in any other tissue on fasting. There was a strong correlation between the activities in heart and diaphragm muscle, but this correlation was independent of feeding-fasting.