Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype.

BACKGROUND: Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1-6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed "LPA" throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways. METHODS: Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits. RESULTS: LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors' phosphorylation (NF-κB, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production. CONCLUSIONS: In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of pro-inflammatory mediators.

Purification and Application of Genetically Encoded Potassium Ion Indicators for Quantification of Potassium Ion Concentrations within Biological Samples.

Vital cells maintain a steep potassium ion (K+ ) gradient across the plasma membrane. Intracellular potassium ion concentrations ([K+ ]) and especially the [K+ ] within the extracellular matrix are strictly regulated, the latter within a narrow range of ∼3.5 to 5.0 mM. Alterations of the extracellular K+ homeostasis are associated with severe pathological alterations and systemic diseases including hypo- or hypertension, heart rate alterations, heart failure, neuronal damage or abnormal skeleton muscle function. In higher eukaryotic organisms, the maintenance of the extracellular [K+ ] is mainly achieved by the kidney, responsible for K+ excretion and reabsorption. Thus, renal dysfunctions are typically associated with alterations in serum- or plasma [K+ ]. Generally, [K+ ] quantifications within bodily fluids are performed using ion selective electrodes. However, tracking such alterations in experimental models such as mice features several difficulties, mainly due to the small blood volume of these animals, hampering the repetitive collection of sample volumes required for measurements using ion selective electrodes. We have recently developed highly sensitive, genetically encoded potassium ion indicators, the GEPIIs, applicable for in vitro determinations of [K+ ]. In addition to the determination of [K+ ] within bodily fluids, GEPIIs proved suitable for the real-time visualization of cell viability over time and the exact determination of the number of dead cells. © 2019 The Authors.

A sensitive chemiluminescence method to measure the lipoxygenase catalyzed oxygenation of complex substrates.

Oxidative modification of low-density lipoprotein (LDL) has been implicated as a patho-physiological process in early atherogenesis and 15-lipoxygenases (15-LOX) may be involved. While studying the in vitro kinetics of the 15-LOX/LDL interaction, we found that the conventional spectrophotometric assays failed in the range of substrate saturation owing to the high optical density of concentrated LDL solutions. Therefore, we developed a much more sensitive assay system which was based on peroxide induced isoluminol enhanced chemiluminescence. With this method reliable kinetic data were obtained at LDL concentrations of up to 1 mg/ml. To validate this luminometric method the kinetic parameters of 15-LOX catalyzed oxygenation of linoleic acid (Km=3.7 microM, kcat=17 s-1) were determined and we observed a good agreement with previously published data obtained with a spectrophotometric assay. Moreover, we found that the kinetic constants of 15-LOX catalyzed LDL oxidation (Km=0.64 microM, kcat=0.15 s-1) are quite different from those of free fatty acid oxygenation and that the cholesterol esters are preferentially oxidized during 15-LOX/LDL interaction. Vitamin E depletion does not reduce the rate of LDL oxidation and analysis of the structure of the oxygenation products suggests that the majority of the products were formed via direct LOX catalyzed oxidation of LDL ester lipids. The luminometric method described here is not restricted to the measurement of LOX catalyzed LDL oxidation, but may also be used to determine kinetic constants for the oxidation of other complex substrates such as biomembranes or liposomes.

In muscle-specific lipoprotein lipase-overexpressing mice, muscle triglyceride content is increased without inhibition of insulin-stimulated whole-body and muscle-specific glucose uptake.

In patients with type 2 diabetes, a strong correlation between accumulation of intramuscular triclycerides (TGs) and insulin resistance has been found. The aim of the present study was to determine whether there is a causal relation between intramuscular TG accumulation and insulin sensitivity. Therefore, in mice with muscle-specific overexpression of human lipoprotein lipase (LPL) and control mice, muscle TG content was measured in combination with glucose uptake in vivo, under hyperinsulinemic-euglycemic conditions. Overexpression of LPL in muscle resulted in accumulation of TGs in skeletal muscle (85.5 +/- 33.3 vs. 25.7 +/- 23.1 micromol/g tissue in LPL and control mice, respectively; P < 0.05). During the hyperinsulinemic clamp study, there were no differences in plasma glucose, insulin, and FFA concentrations between the two groups. Moreover, whole-body, as well as skeletal muscle, insulin-mediated glucose uptake did not differ between LPL-overexpressing and wild-type mice. Surprisingly, whole-body glucose oxidation was decreased by approximately 60% (P < 0.05), whereas nonoxidative glucose disposal was increased by approximately 50% (P < 0.05) in LPL-overexpressing versus control mice. In conclusion, overexpression of human LPL in muscle increases intramuscular TG accumulation, but does not affect whole-body or muscle-specific insulin-mediated uptake, findings that argue against a simple causal relation between intramuscular TG content and insulin resistance.

C16 ceramide is crucial for triacylglycerol-induced apoptosis in macrophages.

Triacylglycerol (TG) accumulation caused by adipose triglyceride lipase (ATGL) deficiency or very low-density lipoprotein (VLDL) loading of wild-type (Wt) macrophages results in mitochondrial-mediated apoptosis. This phenotype is correlated to depletion of Ca(2+) from the endoplasmic reticulum (ER), an event known to induce the unfolded protein response (UPR). Here, we show that ER stress in TG-rich macrophages activates the UPR, resulting in increased abundance of the chaperone GRP78/BiP, the induction of pancreatic ER kinase-like ER kinase, phosphorylation and activation of eukaryotic translation initiation factor 2A, the translocation of activating transcription factor (ATF)4 and ATF6 to the nucleus and the induction of the cell death executor CCAAT/enhancer-binding protein homologous protein. C16:0 ceramide concentrations were increased in Atgl-/- and VLDL-loaded Wt macrophages. Overexpression of ceramide synthases was sufficient to induce mitochondrial apoptosis in Wt macrophages. In accordance, inhibition of ceramide synthases in Atgl-/- macrophages by fumonisin B1 (FB1) resulted in specific inhibition of C16:0 ceramide, whereas intracellular TG concentrations remained high. Although the UPR was still activated in Atgl-/- macrophages, FB1 treatment rescued Atgl-/- macrophages from mitochondrial dysfunction and programmed cell death. We conclude that C16:0 ceramide elicits apoptosis in Atgl-/- macrophages by activation of the mitochondrial apoptosis pathway.

Tissue-specific activity of lipoprotein lipase in skeletal muscle regulates the expression of uncoupling protein 3 in transgenic mouse models.

Uncoupling protein (UCP)-2 and UCP-3 are two recently discovered proteins similar to UCP-1, which regulates thermogenesis in brown adipose tissue (BAT). Whereas UCP-1 expression is restricted to BAT, UCP-2 is widely expressed. UCP-3 is found mainly in skeletal muscle and BAT. A large body of evidence exists that the expression of UCP-2 and UCP-3 in skeletal muscle of mice is regulated by feeding/fasting, and some studies have suggested that this effect might be caused by the changing concentration of plasma non-esterified fatty acids (NEFAs). In an attempt to determine whether the increased import of triacylglycerol-derived NEFAs can also affect UCP expression, we determined the mRNA levels of UCP-1, UCP-2 and UCP-3 in BAT and muscle of induced mutant mouse lines that overexpressed or lacked lipoprotein lipase (LPL) in these tissues. The expression levels of UCP-1 and UCP-2 in BAT and in skeletal and cardiac muscle respectively were not affected by variations in tissue LPL activities. In contrast, UCP-3 mRNA levels were induced 3.4-fold in mice with high levels of LPL in skeletal muscle, and down-regulated in mice that lacked LPL in skeletal muscle. The presence or absence of LPL in BAT had no effect on UCP-3 expression levels. The response of UCP-3 mRNA expression to variations in LPL activity in skeletal muscle was independent of the feeding status or of plasma NEFA concentrations. These findings indicated that NEFAs as lipolytic products of LPL-mediated triacylglycerol hydrolysis markedly affect UCP-3 expression and that increased LPL activities occurring during fasting in skeletal muscle contribute to the induction of UCP-3 expression by promoting the increased uptake of NEFAs. In addition, our results demonstrate that UCP-2 and UCP-3 are differentially regulated in response to LPL-mediated NEFA uptake in skeletal muscle of mice.

The role of lipoprotein lipase in adipose tissue development and metabolism.

Lipoprotein lipase (LPL) is essential for the hydrolysis and distribution of triglyceride-rich lipoprotein-associated fatty acids among extrahepatic tissues. Additionally, the enzyme facilitates several non-lipolysis associated functions including the cellular uptake of whole lipoprotein particles and lipophilic vitamins. The tissue-specific variations of LPL expression have been implicated in the pathogenesis of various lipid disorders, obesity and atherosclerosis. Transgenic technology provided the means to study the physiological response to the overexpression or absence of the enzyme in adipose tissue, muscle and macrophages. The effects of varying LPL expression in adipose tissue and muscle are summarized in this article.

Adenovirus-mediated rescue of lipoprotein lipase-deficient mice. Lipolysis of triglyceride-rich lipoproteins is essential for high density lipoprotein maturation in mice.

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides and the subsequent uptake of free fatty acids in extrahepatic tissues. Deficiency of LPL in humans (Type I hyperlipoproteinemia) is associated with massive chylomicronemia, low high density lipoprotein (HDL) cholesterol levels, and recurrent attacks of pancreatitis when not controlled by a strict diet. In contrast to humans, homozygous LPL knock-out mice (L0) do not survive suckling and die between 18 and 24 h after birth. In this study, an adenovirus-based protocol was utilized for the transient expression of LPL during the suckling period in an effort to rescue L0 mice. After a single intraperitoneal injection of 5x10(9) plaque-forming units of LPL-expressing virus immediately after birth, more than 90% of L0 mice survived the first days of life. 3% of L0 mice survived the entire suckling period and lived for up to 20 months, although LPL activity in mouse tissues and postheparin plasma was undetectable in all animals after 6 weeks of age. Adult LPL-deficient mice were smaller than their littermates until 2-3 months of age and exhibited very high triglyceride levels in the fed (4997 +/- 1102 versus 113.4 +/- 18.7 mg/dl) and fasted state (2007 +/- 375 versus 65.5 +/- 7.4 mg/dl). Plasma total cholesterol levels, free fatty acids, and ketone bodies were elevated in L0 mice, whereas plasma glucose was normal. Most strikingly, L0 mice lacked apoA-I-containing prebeta-HDL particles as well as mature HDL resulting in undetectable HDL cholesterol and HDL-apoA-I levels. HDL deficiency in plasma was evident despite normal apoA-I mRNA levels in the liver and normal apoA-I protein levels in plasma, which were predominantly found in the chylomicron fraction. The absence of prebeta-HDL and mature HDL particles supports the concept that the lipolysis of triglyceride-rich lipoproteins is an essential step for HDL maturation.