Free threonine in human breast milk is related to infant intestinal microbiota composition.

BACKGROUND: Accumulating evidence indicates that free amino acids (FAA) might be bioactive compounds with potential immunomodulatory capabilities. However, the FAA composition in human milk is still poorly characterized with respect to its correlation to maternal serum levels and its physiological significance for the infant. Studies addressing the relation of human milk FAA to the infants' intestinal microbiota are still missing. METHODS: As part of a pilot study, maternal serum and breast milk FAA concentrations as well as infant intestinal microbiota (16S rRNA) were determined 2 months after birth. The study cohort consisted of 41 healthy mothers and their term delivered, healthy infants with normal birthweight. The relationship between maternal serum and milk FAA was determined by correlation analyses. Associations between (highly correlated) milk FAA and infant intestinal beta diversity were tested using PERMANOVA, LefSe and multivariate regression models adjusted for common confounders. RESULTS: Seven breast milk FAA correlated significantly with serum concentrations. One of these, threonine showed a negative association with abundance of members of the class Gammaproteobacteria (R2adj = 17.1%, p = 0.006; ß= - 0.441). In addition, on the level of families and genera, threonine explained 23.2% of variation of the relative abundance of Enterobacteriaceae (R2adj; p = 0.001; ß = - 0.504) and 11.1% of variability in the abundance of Escherichia/Shigella (R2adj, p = 0.025; ß = - 0.368), when adjusted for confounders. CONCLUSION: Our study is the first to suggest potential interactions between breast milk FAA and infant gut microbiota composition during early lactation. The results might be indicative of a potential protective role of threonine against members of the Enterobacteriaceae family in breast-fed infants. Still, results are based on correlation analyses and larger cohorts are needed to support the findings and elucidate possible underlying mechanisms to assess the complex interplay between breast milk FAA and infant intestinal microbiota in detail.

Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells.

Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

Impact of endothelial lipase on cellular lipid composition.

Using mass spectrometry (MS), we examined the impact of endothelial lipase (EL) overexpression on the cellular phospholipid (PL) and triglyceride (TG) content of human aortic endothelial cells (HAEC) and of mouse plasma and liver tissue. In HAEC incubated with the major EL substrate, HDL, adenovirus (Ad)-mediated EL overexpression resulted in the generation of various lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) species in cell culture supernatants. While the cellular phosphatidylethanolamine (PE) content remained unaltered, cellular phosphatidylcholine (PC)-, LPC- and TG-contents were significantly increased upon EL overexpression. Importantly, cellular lipid composition was not altered when EL was overexpressed in the absence of HDL. [(14)C]-LPC accumulated in EL overexpressing, but not LacZ-control cells, incubated with [(14)C]-PC labeled HDL, indicating EL-mediated LPC supply. Exogenously added [(14)C]-LPC accumulated in HAEC as well. Its conversion to [(14)C]-PC was sensitive to a lysophospholipid acyltransferase (LPLAT) inhibitor, thimerosal. Incorporation of [(3)H]-Choline into cellular PC was 56% lower in EL compared with LacZ cells, indicating decreased endogenous PC synthesis. In mice, adenovirus mediated EL overexpression decreased plasma PC, PE and LPC and increased liver LPC, LPE and TG content. Based on our results, we conclude that EL not only supplies cells with FFA as found previously, but also with HDL-derived LPC and LPE species resulting in increased cellular TG and PC content as well as decreased endogenous PC synthesis.

Dose and route dependent effects of the mycotoxin deoxynivalenol in a 3D gut-on-a-chip model with flow.

Deoxynivalenol (DON) is the most prevalent mycotoxin in human food and is ubiquitously detected in human bodyfluids. DON leads to intestinal barrier dysfunction, as observed from animal- and cell culture models with the known disadvantages. Here we present the effects of DON in a gut-on-a-chip model, as the first study incorporating the effects of intestinal flow. Using the OrganoPlate 3-lane, Caco-2 cells were seeded against an extracellular matrix (ECM) and formed leak tight tubules. DON was then applied in different concentrations (3 µM to 300 µM) via the apical or the basolateral channel. Permeability was assessed using continuous TEER and barrier integrity assays (BIA). Zonulin-1, toxicity (LDH) and proinflammatory status (IL-8) was analyzed. DON exposure led to a dose dependent decrease in para-and transcellular barrier integrity, which was more sensitive to basal than apical application (route). Timelaps/Continuous TEER measurements however revealed bidirectional effects, with even TEER-inducing effects of lower concentrations (until 10 µM). IL-8 secretion into luminal supernatants was only induced by apical DON. Attributed to the flow, the barrier-disintegrating effects of DON start at higher concentrations than in other culture models. The barrier was more sensitive to basolateral DON, even though DON had to pass the ECM; and IL-8 secretion was independent of TEER-alterations. Thus, the gut-on-a chip model might be a good alternative to further characterize the bidirectional effects of DON with reasonable throughput incorporating flow.

Distinct maternal amino acids and oxylipins predict infant fat mass and fat-free mass indices.

Interested in maternal determinants of infant fat mass index (FMI) and fat-free mass index (FFMI), considered as predictors for later development of obesity, we analysed amino acids (AA) and oxylipins in maternal serum and breast milk (BM). FMI and FFMI were calculated in 47 term infants aged 4 months (T4). Serum AA were analysed in pregnancy (T1, T2) and 6-8 weeks postpartum (T3). At T3, AA and oxylipins were analysed in BM. Biomarker-index-associations were identified by regression analysis. Infant FMI (4.1 ± 1.31 kg/m2; MW ± SD) was predicted by T2 proline (R2 adj.: 7.6%, p = .036) and T3 BM 11-hydroxy-eicosatetraenoic-acid (11-HETE) and 13-hydroxy-docosahexaenoic-acid (13-HDHA; together:35.5% R2 adj., p < .001). Maternal peripartum antibiotics (AB) emerged as confounders (+AB: 23.5% higher FMI; p = .025). Infant FFMI (12.1 ± 1.19 kg/m2; MW ± SD) was predicted by histidine (R2 adj.: 14.5%, p < .001) and 17-HDHA (BM, R2 adj.:19.3%, p < .001), determined at T3. Confirmed in a larger cohort, the parameters could elucidate connections between maternal metabolic status, nutrition, and infant body development.

Investigating New Sensory Methods Related to Taste Sensitivity, Preferences, and Diet of Mother-Infant Pairs and Their Relationship With Body Composition and Biomarkers: Protocol for an Explorative Study.

BACKGROUND: Early experiences with different flavors play an important role in infant development, including food and taste acceptance. Flavors are already perceived in utero with the development of the taste and olfactory system and are passed on to the child through breast and bottle feeding. Therefore, the first 1000 days of life are considered a critical window for infant developmental programming. OBJECTIVE: The objective of our study is to investigate, both in the prenatal and postnatal period, taste sensitivity, preferences, and dietary diversity of mother-infant pairs. The explorative study design will also report on the impact of these variables on body composition (BC) and biomarkers. In contrast to conventional methods, this study involves long-term follow-up data collection from mother-infant pairs; moreover, the integration of audiovisual tools for recording infants' expressions pertaining to taste stimuli is a novelty of this study. Considering these new methodological approaches, the study aims to assess taste-related data in conjunction with BC parameters like fat-free mass or fat mass, biomarkers, and nutritional intake in infants and children. METHODS: Healthy pregnant women aged between 18 and 50 years (BMI≥18.5 kg/m2 to ≤30 kg/m2; <28 weeks of gestation) were recruited from January 2014 to October 2014. The explorative design implies 2 center visits during pregnancy (24-28 weeks of gestation and 32-34 weeks of gestation) and 2 center visits after delivery (6-8 weeks postpartum and 14-16 weeks postpartum) as well as follow-up visits at 1, 3-3.5, and 6 years after delivery. Data collection encompasses anthropometric and biochemical measurements as well as BC analyses with air displacement plethysmography, taste perception assessments, and multicomponent questionnaires on demographics, feeding practices, and nutritional and lifestyle behaviors. Audiovisual data from infants' reactions to sensory stimuli are collected and coded by trained staff using Baby Facial Action Coding and the Body Action Posture System. Birth outcomes and weight development are obtained from medical records, and additional qualitative data are gathered from 24 semistructured interviews. RESULTS: Our cohort represents a homogenous group of healthy women with stringent exclusion criteria. A total of 54 women met the eligibility criteria, whereas 47 mother-child pairs completed data collection at 4 center visits during and after pregnancy. Follow-up phases, data analyses, and dissemination of the findings are scheduled for the end of 2023. The study was approved by the ethics committee of the Medical University of Graz (EC No 26-066 ex 13/14), and all participants provided informed consent. CONCLUSIONS: The results of this study could be useful for elucidating the connections between maternal and infant statuses regarding diet, taste, biomarkers, and prenatal and postnatal weight development. This study may also be relevant to the establishment of further diagnostic and interventional strategies targeting childhood obesity and early body fat development. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/37279.

Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome.

Adipose triglyceride lipase (ATGL) was recently identified as an important triacylglycerol (TG) hydrolase promoting the catabolism of stored fat in adipose and nonadipose tissues. We now demonstrate that efficient ATGL enzyme activity requires activation by CGI-58. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman Syndrome (CDS), a rare genetic disease where TG accumulates excessively in multiple tissues. CGI-58 interacts with ATGL, stimulating its TG hydrolase activity up to 20-fold. Alleles of CGI-58 carrying point mutations associated with CDS fail to activate ATGL. Moreover, CGI-58/ATGL coexpression attenuates lipid accumulation in COS-7 cells. Antisense RNA-mediated reduction of CGI-58 expression in 3T3-L1 adipocytes inhibits TG mobilization. Finally, expression of functional CGI-58 in CDS fibroblasts restores lipolysis and reverses the abnormal TG accumulation typical for CDS. These data establish an important biochemical function for CGI-58 in the lipolytic degradation of fat, implicating this lipolysis activator in the pathogenesis of CDS.

Acyl chain-dependent effect of lysophosphatidylcholine on endothelial prostacyclin production.

Previously we identified palmitoyl-lysophosphatidylcholine (16:0 LPC), linoleoyl-LPC (18:2 LPC), arachidonoyl-LPC (20:4 LPC), and oleoyl-LPC (18:1 LPC) as the most prominent LPC species generated by the action of endothelial lipase (EL) on high-density lipoprotein. In the present study, the impact of those LPC on prostacyclin (PGI(2)) production was examined in vitro in primary human aortic endothelial cells (HAEC) and in vivo in mice. Although 18:2 LPC was inactive, 16:0, 18:1, and 20:4 LPC induced PGI(2) production in HAEC by 1.4-, 3-, and 8.3-fold, respectively. LPC-elicited 6-keto PGF1α formation depended on both cyclooxygenase (COX)-1 and COX-2 and on the activity of cytosolic phospholipase type IVA (cPLA2). The LPC-induced, cPLA2-dependent (14)C-arachidonic acid (AA) release was increased 4.5-fold with 16:0, 2-fold with 18:1, and 2.7-fold with 20:4 LPC, respectively, and related to the ability of LPC to increase cytosolic Ca(2+) concentration. In vivo, LPC increased 6-keto PGF(1α) concentration in mouse plasma with a similar order of potency as found in HAEC. Our results indicate that the tested LPC species are capable of eliciting production of PGI(2), whereby the efficacy and the relative contribution of underlying mechanisms are strongly related to acyl-chain length and degree of saturation.

Cloning and characterisation of GIRK1 variants resulting from alternative RNA editing of the KCNJ3 gene transcript in a human breast cancer cell line.

The aim of this study was to investigate the impact of increased mRNA levels encoding GIRK1 in breast tumours on GIRK protein expression. mRNA levels encoding hGIRK1 and hGIRK4 in the MCF7, MCF10A and MDA-MB-453 breast cancer cell lines were assessed and the corresponding proteins detected using Western blots. cDNAs encoding for four hGIRK1 splice variants (hGIRK1a, 1c, 1d and 1e) were cloned from the MCF7 cell line. Subcellular localisation of fluorescence labelled hGIRK1a-e and hGIRK4 and of endogenous GIRK1 and GIRK4 subunits was monitored in the MCF7 cell line. All hGIRK1 splice variants and hGIRK4 were predominantly located within the endoplasmic reticulum. Heterologous expression in Xenopus laevis oocytes and two electrode voltage clamp experiments together with confocal microscopy were performed. Only the hGIRK1a subunit was able to form functional GIRK channels in connection with hGIRK4. The other splice variants are expressed, but exert a dominant negative effect on heterooligomeric channel function. Hence, alternative splicing of the KCNJ3 gene transcript in the MCF7 cell line leads to a family of mRNA's, encoding truncated versions of the hGIRK1 protein. The very high abundance of mRNA's encoding GIRK1 together with the presence of GIRK1 protein suggests a pathophysiological role in breast cancer.

Optimization of diagnostic ELISA-based tests for the detection of auto-antibodies against tumor antigens in human serum.

Colorectal cancer is one of the most common cancer types worldwide and it continues to be a serious public health problem. Early detection and diagnosis are of great importance in cancer management. At present, diagnostic blood tests are based on the detection of tumor-associated markers such as carcino-embryonic antigen (CEA), the cancer antigen CA19-9 for gastrointestinal cancer, CA15-3 for breast cancer or CA125 for ovarian cancer. The lack of sensitivity and specificity of these markers prevents their general use in cancer screening of an average risk population. Therefore, new cancer biomarkers or better screening methods are necessary to improve the diagnostics of the disease. This study was directed to the optimization of a diagnostic, enzyme linked immunosorbent assay (ELISA) based test to identify and validate new serum markers, such as extracellular Protein Kinase A (ecPKA) and Nicotinamide N-Methyltransferase (NNMT). In this type of assay, the cancer antigens are quantified indirectly - by detecting the presence of auto-antibodies against tumor proteins in human serum. The result of the optimization and validation process was in the case of ecPKA a reproducible and stable assay. In case of NNMT the assay was probably not sensitive enough.

Reduced expression of adipose triglyceride lipase decreases arachidonic acid release and prostacyclin secretion in human aortic endothelial cells.

BACKGROUND: Vascular endothelial cells represent an important source of arachidonic acid (AA)-derived mediators involved in the generation of anti- or proatherogenic environments. Evidence emerged (in mast cells), that in addition to phospholipases, neutral lipid hydrolases as adipose triglyceride lipase (ATGL) also participate in this process. OBJECTIVE: To examine the impact of ATGL on AA-release from cellular phospholipids (PL) and on prostacyclin secretion in human aortic endothelial cells (HAEC). METHODS AND RESULTS: siRNA-mediated silencing of ATGL promoted lipid droplet formation and TG accumulation in HAEC (nile red stain). ATGL knockdown decreased the basal and A23187 (calcium ionophore)-induced release of 14C-AA from (14C-AA-labeled) HAEC. In A23187-stimulated ATGL silenced cells, this was accompanied by a decreased content of 14C-AA in cellular PL and a decreased secretion of prostacyclin (determined by 6-keto PGF1α EIA). CONCLUSIONS: In vascular endothelial cells, the efficiency of stimulus-induced AA release and prostacyclin secretion is dependent on ATGL.

Acyl chain-dependent effect of lysophosphatidylcholine on endothelium-dependent vasorelaxation.

Previously we identified palmitoyl-, oleoyl-, linoleoyl-, and arachidonoyl-lysophosphatidylcholine (LPC 16:0, 18:1, 18:2 and 20:4) as the most prominent LPC species generated by endothelial lipase (EL). In the present study, we examined the impact of those LPC on acetylcholine (ACh)- induced vascular relaxation. All tested LPC attenuated ACh-induced relaxation, measured ex vivo, using mouse aortic rings and wire myography. The rank order of potency was as follows: 18:2>20:4>16:0>18:1. The attenuating effect of LPC 16:0 on relaxation was augmented by indomethacin-mediated cyclooxygenase (COX)-inhibition and CAY10441, a prostacyclin (PGI2)- receptor (IP) antagonist. Relaxation attenuated by LPC 20:4 and 18:2 was improved by indomethacin and SQ29548, a thromboxane A2 (TXA2)- receptor antagonist. The effect of LPC 20:4 could also be improved by TXA2- and PGI2-synthase inhibitors. As determined by EIA assays, the tested LPC promoted secretion of PGI2, TXA2, PGF2α, and PGE2, however, with markedly different potencies. LPC 16:0 was the most potent inducer of superoxide anion production by mouse aortic rings, followed by LPC 18:2, 20:4 and 18:1, respectively. The strong antioxidant tempol recovered relaxation impairment caused by LPC 18:2, 18:1 and 20:4, but not by LPC 16:0. The tested LPC attenuate ACh-induced relaxation through induction of proconstricting prostanoids and superoxide anions. The potency of attenuating relaxation and the relative contribution of underlying mechanisms are strongly related to LPC acyl-chain length and degree of saturation.

Endothelial Lipase Plasma Levels are Increased in Patients With Significant Carotid Artery Stenosis and History of Neurological Impairment.

BACKGROUND: Endothelial lipase (EL) is a phospholipase expressed predominantly by vascular endothelial cells. The goal of the present study was to examine whether EL plasma levels in patients with carotid artery stenosis differ between those with previous history of neurological impairment and those without neurological symptoms. METHODS: EL plasma levels were measured by a competitive ELISA assay. RESULTS: EL plasma levels were significantly higher in the symptomatic, compared with the asymptomatic group (mean 489.61 ± 145 ng/ml (n = 31) vs. 388.39 ± 133 ng/ml (n = 24), t-test, P = 0.011). CONCLUSION: We concluded that increased EL plasma levels reflect the patients' overall susceptibility for cerebrovascular events. KEYWORDS: Atherosclerosis; Carotid artery stenosis; Endothelial lipase; Neurological impairment; Carotid endarterectomy.

Acyl chain-dependent effect of lysophosphatidylcholine on cyclooxygenase (COX)-2 expression in endothelial cells.

OBJECTIVE: Previously we identified palmitoyl-, oleoyl- linoleoyl-, and arachidonoyl-lysophosph-atidylcholine (LPC 16:0, 18:1, 18:2 and 20:4) as the most prominent LPC species generated by endothelial lipase (EL). In the present study, we examined the capacity of those LPC to modulate expression of cyclooxygenase (COX)-2 in vascular endothelial cells. METHODS & RESULTS: LPC 16:0 and 20:4 promoted both COX-2 mRNA- and protein synthesis with different potencies and kinetics. While LPC 18:1 induced a weak and transient increase in COX-2 mRNA, but not protein, LPC 18:2 increased COX-2 protein, without impacting mRNA. Chelation of intracellular Ca(2+) and inhibition of p38 MAPK markedly attenuated 16:0 LPC- and 20:4 LPC- elicited induction of COX-2 expression, whereas inhibition of phospholipase C (PLC) attenuated only the effect of 16:0 LPC. LPC 16:0 and 20:4 differed markedly in their potencies to increase cytosolic Ca(2+) concentration and in the kinetics of p38 MAPK activation. While the effects of 16:0 and 20:4 LPC on COX-2 expression were profoundly sensitive to silencing of either c-Jun or p65 (NF-κB), respectively, silencing of cyclic AMP responsive element binding protein (CREB) attenuated markedly the effect of both LPC. CONCLUSION: Our results indicate that the tested LPC species are capable of inducing COX-2 expression, whereby the efficacy and the relative contribution of underlying signaling mechanisms markedly differ, due to the length and degree of saturation of LPC acyl chains.

Increased expression of endothelial lipase in symptomatic and unstable carotid plaques.

The aim of this study was to evaluate endothelial lipase (EL) protein expression in advanced human carotid artery plaques (HCAP) with regard to plaque (in)stability and the incidence of symptoms. HCAP were collected from 66 patients undergoing carotid endarterectomy (CEA). The degree of plaque (in)stability was estimated by ultrasound and histology. In HCAP sections, EL expression was determined by immunostaining and the intensity was assessed on a semi-quantitative scale (low: <25%, high: >25% positive cells). Monocytes and macrophages in adjacent HCAP sections were stained with a CD163 specific antibody. High EL staining was more prevalent in histologically unstable plaques (in 33.3% of fibrous plaques, 50% of ulcerated non-complicated plaques and 79.2% of ulcerated complicated plaques; χ(2) test, p = 0.004) and in the symptomatic group (70.8 vs. 42.9% in the asymptomatic group; χ(2) test, p = 0.028). The majority of EL immunostaining was found in those HCAP regions exhibiting a strong CD163 immunostaining. EL in HCAP might be a marker and/or promoter of plaque instability and HCAP-related symptomatology.

Docosahexaenoic acid-induced unfolded protein response, cell cycle arrest, and apoptosis in vascular smooth muscle cells are triggered by Ca²âº-dependent induction of oxidative stress.

Proliferation of vascular smooth muscle cells is a characteristic of pathological vascular remodeling and represents a significant therapeutic challenge in several cardiovascular diseases. Docosahexaenoic acid (DHA), a member of the n-3 polyunsaturated fatty acids, was shown to inhibit proliferation of numerous cell types, implicating several different mechanisms. In this study we examined the molecular events underlying the inhibitory effects of DHA on proliferation of primary human smooth muscle cells isolated from small pulmonary artery (hPASMCs). DHA concentration-dependently inhibited hPASMC proliferation, induced G1 cell cycle arrest, and decreased cyclin D1 protein expression. DHA activated the unfolded protein response (UPR), evidenced by increased mRNA expression of HSPA5, increased phosphorylation of eukaryotic initiation factor 2α, and splicing of X-box binding protein 1. DHA altered cellular lipid composition and led to increased reactive oxygen species (ROS) production. DHA-induced ROS were dependent on both intracellular Ca(2+) release and entry of extracellular Ca(2+). Overall cellular ROS and mitochondrial ROS were decreased by RU360, a specific inhibitor of mitochondrial Ca(2+) uptake. DHA-induced mitochondrial dysfunction was evidenced by decreased mitochondrial membrane potential and decreased cellular ATP content. DHA triggered apoptosis as found by increased numbers of cleaved caspase-3- and TUNEL-positive cells. The free radical scavenger Tempol counteracted DHA-induced ROS, cell cycle arrest, induction of UPR, and apoptosis. We conclude that Ca(2+)-dependent oxidative stress is the central and initial event responsible for induction of UPR, cell cycle arrest, and apoptosis in DHA-treated hPASMCs.

Endothelial lipase (EL) and EL-generated lysophosphatidylcholines promote IL-8 expression in endothelial cells.

OBJECTIVE: Previously we identified palmitoyl-lysophosphatidylcholine (LPC 16:0), as well as linoleoyl-, arachidonoyl- and oleoyl-LPC (LPC 18:2, 20:4 and 18:1) as the most prominent LPC species generated by the action of endothelial lipase (EL) on high-density lipoprotein (HDL). In the present study, the impact of EL and EL-generated LPC on interleukin-8 (IL-8) synthesis was examined in vitro in primary human aortic endothelial cells (HAEC) and in mice. METHODS AND RESULTS: Adenovirus-mediated overexpression of the catalytically active EL, but not its inactive mutant, increased endothelial synthesis of IL-8 mRNA and protein in a time- and HDL-concentration-dependent manner. While LPC 18:2 was inactive, LPC 16:0, 18:1 and 20:4 promoted IL-8 mRNA- and protein-synthesis, differing in potencies and kinetics. The effects of all tested LPC on IL-8 synthesis were completely abrogated by addition of BSA and chelation of intracellular Ca(2+). Underlying signaling pathways also included NFkB, p38-MAPK, ERK, PKC and PKA. In mice, adenovirus-mediated overexpression of EL caused an elevation in the plasma levels of MIP-2 (murine IL-8 analogue) accompanied by a markedly increased plasma LPC/PC ratio. Intravenously injected LPC also raised MIP-2 plasma concentration, however to a lesser extent than EL overexpression. CONCLUSION: Our results indicate that EL and EL-generated LPC, except of LPC 18:2, promote endothelial IL-8 synthesis, with different efficacy and kinetics, related to acyl-chain length and degree of saturation. Accordingly, due to its capacity to modulate the availability of the pro-inflammatory and pro-adhesive chemokine IL-8, EL should be considered an important player in the development of atherosclerosis.

Adipose tissue as a source of nicotinamide N-methyltransferase and homocysteine.

Nicotinamide N-methyltransferase (NNMT) catalyses the conversion of nicotinamide to 1-methylnicotinamide and plays an important role in hepatic detoxification reactions. Here we show that, in addition to the liver, 3T3-L1 adipocytes as well as human and murine adipose tissue explants express high amounts of enzymatically active NNMT. NNMT mRNA levels and enzyme activity increased in 3T3-L1 cells in a differentiation-dependent manner. Homocysteine, the atherogenic product of the NNMT-catalyzed reaction, was secreted from 3T3-L1 cells or adipose tissue cultures. Homocysteine release increased during 3T3-L1 differentiation and was reduced when adipose tissue was treated with the NNMT inhibitor 1-methylnicotinamide. Nicotinic acid (NA), a widely used drug to lower elevated plasma lipid levels, induced NNMT enzyme activity in white adipose tissue of mice. In tissue culture nicotinamide treatment led to an increase in adipose tissue homocysteine secretion. These data support the concept that adipose tissue NNMT contributes to the increased plasma homocysteine levels in patients treated with NA.

Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle.

Hormone-sensitive lipase (HSL) is believed to play an important role in the mobilization of fatty acids from triglycerides (TG), diglycerides, and cholesteryl esters in various tissues. Because HSL-mediated lipolysis of TG in adipose tissue (AT) directly feeds non-esterified fatty acids (NEFA) into the vascular system, the enzyme is expected to affect many metabolic processes including the metabolism of plasma lipids and lipoproteins. In the present study we examined these metabolic changes in induced mutant mouse lines that lack HSL expression (HSL-ko mice). During fasting, when HSL is normally strongly induced in AT, HSL-ko animals exhibited markedly decreased plasma concentrations of NEFA (-40%) and TG (-63%), whereas total cholesterol and HDL cholesterol levels were increased (+34%). Except for the increased HDL cholesterol concentrations, these differences were not observed in fed animals, in which HSL activity is generally low. Decreased plasma TG levels in fasted HSL-ko mice were mainly caused by decreased hepatic very low density lipid lipoprotein (VLDL) synthesis as a result of decreased NEFA transport from the periphery to the liver. Reduced NEFA transport was also indicated by a depletion of hepatic TG stores (-90%) and strongly decreased ketone body concentrations in plasma (-80%). Decreased plasma NEFA and TG levels in fasted HSL-ko mice were associated with increased fractional catabolic rates of VLDL-TG and an induction of the tissue-specific lipoprotein lipase (LPL) activity in cardiac muscle, skeletal muscle, and white AT. In brown AT, LPL activity was decreased. Both increased VLDL fractional catabolic rates and increased LPL activity in muscle were unable to provide the heart with sufficient NEFA, which led to decreased tissue TG levels in cardiac muscle. Our results demonstrate that HSL deficiency markedly affects the metabolism of TG-rich lipoproteins by the coordinate down-regulation of VLDL synthesis and up-regulation of LPL in muscle and white adipose tissue. These changes result in an "anti-atherogenic" lipoprotein profile.

Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.

Mobilization of fatty acids from triglyceride stores in adipose tissue requires lipolytic enzymes. Dysfunctional lipolysis affects energy homeostasis and may contribute to the pathogenesis of obesity and insulin resistance. Until now, hormone-sensitive lipase (HSL) was the only enzyme known to hydrolyze triglycerides in mammalian adipose tissue. Here, we report that a second enzyme, adipose triglyceride lipase (ATGL), catalyzes the initial step in triglyceride hydrolysis. It is interesting that ATGL contains a "patatin domain" common to plant acyl-hydrolases. ATGL is highly expressed in adipose tissue of mice and humans. It exhibits high substrate specificity for triacylglycerol and is associated with lipid droplets. Inhibition of ATGL markedly decreases total adipose acyl-hydrolase activity. Thus, ATGL and HSL coordinately catabolize stored triglycerides in adipose tissue of mammals.