Cathepsin-S degraded decorin are elevated in fibrotic lung disorders - development and biological validation of a new serum biomarker.

BACKGROUND: Decorin is one of the most abundant proteoglycans of the extracellular matrix and is mainly secreted and deposited in the interstitial matrix by fibroblasts where it plays an important role in collagen turnover and tissue homeostasis. Degradation of decorin might disturb normal tissue homeostasis contributing to extracellular matrix remodeling diseases. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific fragment of degraded decorin, which has potential as a novel non-invasive serum biomarker for fibrotic lung disorders. METHODS: A fragment of decorin cleaved in vitro using human articular cartilage was identified by mass-spectrometry (MS/MS). Monoclonal antibodies were raised against the neo-epitope of the cleaved decorin fragment and a competitive ELISA assay (DCN-CS) was developed. The assay was evaluated by determining the inter- and intra-assay precision, dilution recovery, accuracy, analyte stability and interference. Serum levels were assessed in lung cancer patients, patients with idiopathic pulmonary fibrosis (IPF), patients with chronic obstructive pulmonary disease (COPD) and healthy controls. RESULTS: The DCN-CS ELISA was technically robust and was specific for decorin cleaved by cathepsin-S. DCN-CS was elevated in lung cancer patients (p < 0.0001) and IPF patients (p < 0.001) when compared to healthy controls. The diagnostic power for differentiating lung cancer patients and IPF patients from healthy controls was 0.96 and 0.77, respectively. CONCLUSION: Cathepsin-S degraded decorin could be quantified in serum using the DCN-CS competitive ELISA. The clinical data indicated that degradation of decorin by cathepsin-S is an important part of the pathology of lung cancer and IPF.

Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling.

Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art tools to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C-terminus of fibronectin was developed (FBN-C). The assay was evaluated in relation to specificity, technical performance and as a marker for quantification of fibronectin in cartilage and cancer ex vivo models. The ELISA was specific and technically stable. Cleavage of tumor tissue with MMP-2 released significantly higher levels of FBN-C compared to tissue with buffer only and western blot analysis revealed that FBN-C recognizes both full length and degraded fibronectin. When ex vivo cartilage cultures were stimulated with the anabolic factor TGFß and catabolic factors TNF-α and OSM, significantly higher levels of FBN-C were found in the conditioned media. Lastly, FBN-C was released from a cancer ex vivo model. In conclusion, we were able to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. Quantification of fibronectin remodeling could be a valuable tool to understand ECM remodeling in ex vivo models of fibro-proliferative pathologies.

The complexity of allergic patients' IgE repertoire correlates with serum concentration of allergen-specific IgE.

BACKGROUND: The governing factor of both effector-cell activation and facilitated antigen presentation is IgE-repertoire complexity (IgE-clonality, -affinity and -concentration). Yet, the compositions of individual IgE repertoires and correlation between IgE-repertoire complexity and establishment of allergic sensitization remain to be determined. OBJECTIVE: In complex formation assays with recombinant IgE, allergen and CD23(+) B cells, we assess the composition of serum IgE repertoires and examine the correlation between IgE-titre and IgE-repertoire complexity. METHODS: The capacity of sera, from house dust mite-sensitized individuals, to mediate IgE-Der p 2-CD23 complex formation on CD23(+) B cells was measured. In parallel experiments, the effect of supplementing each serum with one or more Der p 2-specific monoclonal recombinant IgE antibodies on complex formation was determined. RESULTS: Only sera with the highest concentration of Der p 2-specific IgE resulted in complex formation without supplementary recombinant IgE. Intermediately titred sera supported complex formation to various degrees when supplemented with individual recombinant IgE. The degree of complex formation depended on the affinity and epitope specificity of the recombinant IgE. Complex formation by combining serum and recombinant IgEs could not be obtained with sera of relatively low titres of specific IgE. However, these sera had the capacity to dramatically enhance the low complex formation achieved with pairs of affinity-engineered recombinant IgEs. CONCLUSION AND CLINICAL RELEVANCE: Serum IgE complexity can be indirectly assessed by combining sera with defined monoclonal IgEs in IgE-allergen-CD23 complex assays. The observed differences in epitope-coverage of Der p 2-specific serum-IgE in sera of different specific IgE titres indicate that increased IgE titres correlate with increased complexity of the IgE-repertoire. A detailed knowledge of the composition and complexity of allergen-specific IgE repertoires (and the relation to IgE titre), particularly in the early phase of sensitization, may be used to improve the prediction of the persistence and severity of allergic symptoms, as well as the progression of the Allergic March.

Considerations for understanding protein measurements: Identification of formation, degradation and more pathological relevant epitopes.

OBJECTIVES: There is a need for precision medicine and an unspoken promise of an optimal approach for identification of the right patients for value-based medicine based on big data. However, there may be a misconception that measurement of proteins is more valuable than measurement of fewer selected biomarkers. In population-based research, variation may be somewhat eliminated by quantity. However, this fascination of numbers may limit the attention to and understanding of the single. This review highlights that protein measurements (with collagens as examples) may mean different things depending on the targeted epitope - formation or degradation of tissues, and even signaling potential of proteins. DESIGN AND METHODS: PubMed was searched for collagen, neo-epitope, biomarkers. RESULTS: Ample examples of assays with specific epitopes, either pathological such as HbA1c, or domain specific such as pro-peptides, which total protein arrays would not have identified were evident. CONCLUSIONS: We suggest that big data may be considered as the funnel of data points, in which most important parameters will be selected. If the technical precision is low or the biological accuracy is limited, and we include suboptimal quality of biomarkers, disguised as big data, we may not be able to fulfill the promise of helping patients searching for the optimal treatment. Alternatively, if the technical precision of the total protein quantification is high, but we miss the functional domains with the most considerable biological meaning, we miss the most important and valuable information of a given protein. This review highlights that measurements of the same protein in different ways may provide completely different meanings. We need to understand the pathological importance of each epitope quantified to maximize protein measurements.

Persistence of abnormal chloride conductance regulation in transformed cystic fibrosis epithelia.

An airway epithelial cell line (CF/T43) was developed by infecting cultured airway epithelial cells from patients with cystic fibrosis (CF) with the pZIPneoSV(X)1/SV40T retrovirus and selecting for G418 resistance and ion transport properties. The distinctive chloride secretory phenotypes of the CF cell line CF/T43 and a normal cell line (NL/T4) were not perturbed by SV40T-induced cell transformation. Epithelial cell lines generated from CF cells with the SV40T gene can be used to test candidate CF genes and to evaluate the molecular mechanisms responsible for the CF phenotype.

A serological marker of the N-terminal neoepitope generated during LOXL2 maturation is elevated in patients with cancer or idiopathic pulmonary fibrosis.

OBJECTIVES: Lysyl oxidase like 2 (LOXL2) is associated with poor prognosis in idiopathic pulmonary disease (IPF) and cancer. We developed an Enzyme-linked immunosorbent assay (ELISA) targeting the LOXL2 neo-epitope generated through the release of the signal peptide during LOXL2 maturation. DESIGN AND METHODS: An ELISA targeting the N-terminal site of the human LOXL2 was developed including technical optimization and validation steps. Serum LOXL2 was measured in patients with breast, colorectal, lung, ovarian, pancreatic and prostate cancer, melanoma, IPF and in healthy controls (n = 16). RESULTS: A technically robust and specific assay was developed. LOXL2 was detectable in serum from healthy controls and showed reactivity towards recombinant LOXL2. Compared to controls, LOXL2 levels were significantly (p < 0.001-0.05) elevated in serum from patients with breast, colerectal, lung, ovarian and pancreatic cancer (mean range: 49-84 ng/mL), but not in prostate cancer (mean: 36 ng/mL) and malignant melanoma patients (41 ng/mL). Serum LOXL2 was elevated in IPF patients compared to healthy controls (mean: 76.5 vs 46.8 ng/mL; p > 0.001). CONCLUSIONS: A specific ELISA towards the N-terminal neo-epitope site in LOXL2 was developed which detected significantly elevated serum levels from patients with above-mentioned cancer types or IPF compared to healthy controls.

A fragment of SPARC reflecting increased collagen affinity shows pathological relevance in lung cancer - implications of a new collagen chaperone function of SPARC.

The matricellular protein SPARC (secreted proteome acidic and rich in cysteine) is known to bind collagens and regulate fibrillogenesis. Cleavage of SPARC at a single peptide bond, increases the affinity for collagens up to 20-fold. To investigate if this specific cleavage has pathological relevance in fibrotic disorders, we developed a competitive ELISA targeting the generated neo-epitope on the released fragment and quantified it in serum from patients with lung cancer, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and healthy subjects. Furthermore, the ability of SPARC to protect fibrillar collagens from proteolytic degradation was investigated in vitro, potentially adding a new collagen chaperone function to SPARC. The fragment was significantly elevated in lung cancer patients when compared to healthy subjects measured in a discovery cohort (p = 0.0005) and a validation cohort (p < 0.0001). No significant difference was observed for IPF and COPD patients compared to healthy subjects. When recombinant SPARC was incubated with type I or type III collagen and matrix metalloproteinase-9, collagen degradation was completely inhibited. Together, these data suggest that cleavage of SPARC at a specific site, which modulates collagen binding, is a physiological mechanism increased during pathogenesis of lung cancer. Furthermore, inhibition of fibrillar collagen degradation by SPARC adds a new chaperone function to SPARC which may play additional roles in the contribution to increased collagen deposition leading to a pro-fibrotic and tumorigenic environment.

Selective response of human airway epithelia to luminal but not serosal solution hypertonicity. Possible role for proximal airway epithelia as an osmolality transducer.

The response of cultured human nasal epithelia to hypertonic bathing solutions was tested using ion-selective microelectrode and quantitative microscopy. Raised luminal, but not serosal, osmolality (+/- 150 mM mannitol) decreased Na+ absorption but did not induce Cl- secretion. Raised luminal osmolality increased cell Cl- activity, Na+ activity, and transepithelial resistance and decreased both apical and basolateral membrane potentials and the fractional resistance of the apical membrane; equivalent circuit analysis revealed increases in apical, basolateral, and shunt resistances. Prolonged exposure (10 min) to 430 mosM luminal solution elicited no regulation of any parameter. Optical measurements revealed a reduction in the thickness of preparations only in response to luminal hypertonic solutions. We conclude that (a) airway epithelial cells exhibit asymmetric water transport properties, with the apical membrane water permeability exceeding that of the basolateral membrane; (b) the cellular response to volume loss is a deactivation of the basolateral membrane K+ conductance and the apical membrane Cl- conductance; (c) luminal hypertonicity slows the rate of Na+ absorption but does not induce Cl- secretion; and (d) cell volume loss increases the resistance of the paracellular path. We speculate that these properties configure human nasal epithelium to behave as an osmotic sensor, transducing information about luminal solutions to the airway wall.

Eicosapentaenoic acid causes transient accumulation of lipids in rat myocardium.

Rats were given eicosapentaenoic acid (EPA) or palmitic acid (PALM) up to 15 days, and control animals were given carboxymethylcellulose. All suspensions which were given by gastric intubation contained tocopherol. Heart triacylglycerols, heart cholesterol and heart phospholipids significantly increased after one day of EPA treatment, but they were normalized within 15 days. Both after 2 and 10 days of treatment with palmitic acid the heart triacylglycerols were significantly greater than control. The heart cholesterol and heart phospholipids were significantly greater than control after 10 days of treatment with palmitic acid. Total carnitine palmitoyltransferase (CPT) activity in heart was significantly greater in rats treated with EPA for 15 days compared to control, but treatment with palmitic acid had no effect. The fatty acyl-CoA oxidase activity was greater in rats treated with EPA for 15 days and palmitic acid for 10 days compared to control. The fractional volume of lipid droplets in myocardial cells was calculated from electronmicrographs and was 0.112 +/- 0.016% after 1 day of EPA treatment compared to 0.035 +/- 0.016% in the control group. After 5 and 15 days the fractional volume was the same as control. The fractional volume of lipid droplets in rats treated with palmitic acid for 10 days was 0.120 +/- 0.023%. Treatment with EPA caused an immediate accumulation of lipids and lipid droplets in the rat heart which after few days normalized in parallel with an increased activity of total CPT in the heart.

On the effect of 2-deuterium- and 2-methyl-eicosapentaenoic acid derivatives on triglycerides, peroxisomal beta-oxidation and platelet aggregation in rats.

A series of 2-substituted eicosapentaenoic acid (EPA) derivatives (as ethyl esters) have been synthesized and evaluated as hypolipidemic and antithrombotic agents in feeding experiments in rats. Repeated administration of purified 2-methyl-eicosapentaenoic acid and its deuterium analogues (all as ethyl esters) to rats resulted in a decrease in plasma triglycerides and high density lipoprotein cholesterol. The 2-methyl-EPA analogues were, apparently, four times more potent than EPA in inducing the triglyceride lowering effect. The 2-deuterium-2-methyl-EPA decreased plasma cholesterol level to approximately 40%. A moderate enlargement of the liver was observed in 2-methyl-EPA treated rats. This was accompanied with an acute reduction in the liver content of triglycerides and a stimulation of peroxisomal beta-oxidation and fatty acyl-CoA oxidase activity. The results suggest that the triglyceride-lowering effect of 2-methyl-EPA may be due to a reduced supply of fatty acids for hepatic triglyceride biosynthesis because of increased fatty acid oxidation. Platelet aggregation with ADP and A23187 was performed ex vivo in platelet-rich plasma, after administration of different doses of the EPA-derivatives for five days. EPA and 2,2-dideuterium EPA had no effect on ADP-induced aggregation, while 2-deuterium-, 2-methyl- and 2-deuterium-2-methyl EPA produced a biphasic effect, i.e. potentiation and inhibition at low (250 mg/day kg body weight) and higher doses (600-1300 mg/day kg body weight), respectively. A23187-induced platelet aggregation was affected in a similar way by feeding the 2-substituted EPA derivatives, except that 2-deuterium-2-methyl EPA had no effect relative to EPA itself and that the inhibition was far greater than that for ADP-induced aggregation (approximately 100% inhibition with 600 mg 2-methyl-EPA/day kg body weight). The ranking order of the EPA-derivatives to affect platelet aggregation and to cause hypolipidemia was different, suggesting different mechanisms. Our observations suggest that the effects of the EPA derivatives on platelet aggregation could be related to the degree of bulkiness around C2 and that an asymmetric substitution at C2 caused inhibition of platelet aggregation while a symmetric substitution did not. It is suggested that the bulky, asymmetric derivatives inhibit platelet aggregation by altering platelet membrane phospholipid packing.

Early effects on mitochondrial and peroxisomal beta-oxidation by the hypolipidemic 3-thia-fatty acids in rat livers.

A single administration of 3-thiadicarboxylic and tetradecylthioacetic acids stimulates both mitochondrial and peroxisomal beta-oxidation and lowers plasma triacylglycerol levels. An increased rate of mitochondrial beta-oxidation and carnitine palmitoyl-transferase activity was established after 3 h and this was accompanied by a lowering of plasma triacylglycerol. Peroxisomal beta-oxidation, however, remained unchanged up to 8 h and was significantly increased after 12 h. These results suggest that after a single administration of 3-thia fatty acids mitochondrial beta-oxidation precedes peroxisomal beta-oxidation. Furthermore, they show that the observed tricylglycerol-lowering effect, which is established early (3-4 h) after the administration of 3-thia fatty acids, is initially due to an increased mitochondrial beta-oxidation.

Proton pump activity of mitochondria-rich cells. The interpretation of external proton-concentration gradients.

We have hypothesized that a major role of the apical H(+)-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl- via an apical Cl-/HCO3(-)-exchanger. The activity of the H+ pump was studied by monitoring mucosal [H+]-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 +/- 0.07 (mean +/- SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 +/- 18 microns, corresponding to an estimated "unstirred layer" thickness of 329 +/- 29 microns. Mucosal acidification was dependent on serosal pCO2, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H+. The [H+] was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate [H+]-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H+ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace's equation. Model calculations predicted that with a physiological density of MR cells, the [H+] profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing [H+]-gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external [H+]-gradient depended on the mucosal anion such as to indicate that base (HCO3-) is excreted in exchange not only for Cl-, but also for Br- and I-, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.

Cholesterol induced lipid accumulation in myocardial cells of rats.

OBJECTIVE: The aim was to investigate whether cholesterol feeding of rats for only 10 d would result in lipid accumulation in myocardial cells and a change in the peroxisomal beta oxidation in the heart compared to the hearts of rats on standard feeds. METHODS: Eight rats received a cholesterol diet (2%) and eight rats received a standard diet for 10 d. Lipids were measured in serum, liver, and heart. Palmitoyl-CoA hydrolase and fatty acyl-CoA oxidase were determined in total homogenate of hearts. The fractional volume of lipid droplets in myocardial cells was calculated from micrographs at a magnification of x9600 obtained by electron microscopy. RESULTS: The fractional volume of lipid droplets in the cardiomyocytes increased from 0.109(SEM 0.019)% to 0.259(0.037)%, (p < 0.003), as a result of cholesterol feeding. Cholesterol and triglycerides in the heart measured by biochemical methods increased by 13% and 24%, respectively. There was no difference in palmitoyl-CoA hydrolase or fatty acyl-CoA oxidase in the hearts of the cholesterol fed group compared to the control group, suggesting an unaltered peroxisomal beta oxidation of fatty acids in the myocardium. The serum triglycerides and serum phospholipids were reduced in the cholesterol fed rats (p < 0.05 and p < 0.001, respectively). Rats fed cholesterol diet showed a reduced ratio of HDL cholesterol to total cholesterol in serum, together with an increased liver weight and relative liver weight compared to the control rats. The addition of cholesterol to the diet increased liver cholesterol to 21.0(1.6) mumol.g-1 wet weight compared to 5.87(0.93) mumol.g-1 in the controls on standard diet (p < 0.0001). Cholesterol feeding had no effect on the hepatic level of phospholipids, but the liver triglycerides tended to increase from 23.3(9.5) mumol.g-1 wet weight to 53.3(6.3) mumol.g-1. CONCLUSIONS: Lipid accumulation in the myocardial cells was shown by morphometry after 10 d of cholesterol feeding, without any visible damage to the tissue.

Collagen degradation products measured in serum can separate ovarian and breast cancer patients from healthy controls: A preliminary study.

BACKGROUND: During cancer the otherwise tightly controlled homeostasis of the extracellular matrix (ECM) is disturbed. The protein composition changes, the ECM stiffens and increased levels of proteases are secreted. The combination of these processes result in release of specific protein fragments (e.g. collagens) to the circulation, which when measured may reflect disease pathogenesis. OBJECTIVE: To investigate if biomarkers of protease-degraded collagen could differentiate ovarian and breast cancer patients from healthy controls when measured in serum. METHODS: The levels of markers reflecting MMP-degradation of type I (C1M), type III (C3M) and type IV (C4M, C4M12) collagen were assessed in serum from ovarian cancer patients (n= 10), breast cancer patients (n= 14) and healthy controls (n= 49) using validated ELISAs. The markers were compared using one way ANOVA and AUC was calculated. RESULTS: All markers were significantly elevated in serum from ovarian cancer patients (p< 0.0001) and breast cancer patients (p< 0.04-0.0001) compared to healthy controls. Furthermore, diagnostically the markers were able to differentiate ovarian (AUROC 90%-93%) and breast cancer patients (AUROC 76%-93%) from healthy controls, with C1M being the strongest differentiator of disease vs. CONCLUSION: Four serum biomarkers reflecting altered MMP-mediated collagen turnover were able to differentiate ovarian and breast cancer patients from healthy controls.

Levels of circulating MMP-7 degraded elastin are elevated in pulmonary disorders.

OBJECTIVES: Elastin is a signature protein of the lungs. Matrix metalloproteinase-7 (MMP-7) is important in lung defence mechanisms and degrades elastin. However, MMP-7 activity in regard to elastin degradation has never been quantified serologically in patients with lung diseases. An assay for the quantification of MMP-7 generated elastin fragments (ELM7) was therefore developed to investigate MMP-7 derived elastin degradation in pulmonary disorders such as idiopathic pulmonary fibrosis (IPF) and lung cancer. DESIGN AND METHODS: Monoclonal antibodies (mABs) were raised against eight carefully selected MMP-7 cleavage sites on elastin. After characterisation and validation of the mABs, one mAB targeting the ELM7 fragment was chosen. ELM7 fragment levels were assessed in serum samples from patients diagnosed with IPF (n=123, baseline samples, CTgov reg. NCT00786201), and lung cancer (n=40) and compared with age- and sex-matched controls. RESULTS: The ELM7 assay was specific towards in vitro MMP-7 degraded elastin and the ELM7 neoepitope but not towards other MMP-7 derived elastin fragments. Serum ELM7 levels were significantly increased in IPF (113%, p<0.0001) and lung cancer (96%, p<0.0001) compared to matched controls. CONCLUSIONS: MMP-7-generated elastin fragments can be quantified in serum and may reflect pathological lung tissue turnover in several important lung diseases.

Eicosapentaenoic acid at hypotriglyceridemic dose enhances the hepatic antioxidant defense in mice.

The effect of oral administration of purified (95%) eicosapentaenoic acid on serum lipids, hepatic peroxisomal enzymes, antioxidant enzymes and lipid peroxidation was compared with that of palmitic acid fed mice and corresponding controls. After 10 d, a dose of 1000 mg eicosapentaenoic acid per day/kg body weight lowered serum triglycerides by 45%, while no significant change in serum cholesterol level was noted in comparison to palmitic acid fed mice and controls. Hepatic acyl-CoA oxidase and catalase activities increased by 50% and 30%, respectively, in the eicosapentaenoic acid fed group. In addition, the hepatic reduced glutathione content and the activities of glutathione transferase, glutathione peroxidase and glutathione reductase, increased significantly during eicosapentaenoic acid treatment. The levels of hepatic lipid peroxides were lower after eicosapentaenoic acid feeding, while no significant change was noted in the palmitic acid fed mice when compared to the controls. Taken together, the present data demonstrate for the first time that at hypolipidemic doses eicosapentaenoic acid feeding i) enhances the hepatic antioxidant defense, and ii) does not cause a significant differential induction of the two peroxisomal enzymes, acyl-CoA oxidase and catalase, as was noted after administration of hypolipidemic peroxisome proliferating compounds, such as clofibrate in rodents.

Relationship between translocation of long-chain acyl-CoA hydrolase, phosphatidate phosphohydrolase and CTP:phosphocholine cytidylyltransferase and the synthesis of triglycerides and phosphatidylcholine in rat liver.

Translocation of long-chain acyl-coenzyme A hydrolase from the microsomal fraction to the cytosolic fraction was promoted in cell-free extracts of rat liver by palmitic acid, oleic acid, tetradecylthioacetic acid, and tetradecylthiopropionic acid, and by their CoA esters. The CoA esters were more effective than the non-esterified acids in the translocation of the enzyme. Treatment of normolipidemic rats with sulfur-substituted non-beta-oxidizable fatty acid analogues resulted in a transitory increase in hepatic concentration of long-chain acyl-CoA. Longer feeding times almost normalized the hepatic long-chain acyl-CoA content. Microsomal long-chain acyl-CoA hydrolase activity was inhibited, whereas the activity of the cytosolic form was stimulated. The rise in enzyme activity coincided with a reduction in liver content of triglyceride and an increase in hepatic phospholipid content. The results suggest that the activity of long-chain acyl-CoA hydrolase in the cytosol may control the amount of acyl-CoA thioesters in the liver. Esterified and non-esterified fatty acids caused in vitro translocation of phosphatidate phosphohydrolase and cytidine 5'-triphosphate (CTP):phosphocholine cytidylyltransferase from the cytosolic fraction to the microsomal fraction. However, the translocation of these two enzyme systems was not obtained in vivo. The activity of phosphatidate phosphohydrolase decreased in microsomal and cytosolic fractions while the activity of cytidylyltransferase in these fractions increased. The activities of soluble phosphatidate phosphohydrolase and long-chain acyl-CoA hydrolase appeared to be inversely correlated.(ABSTRACT TRUNCATED AT 250 WORDS)

The hypotriglyceridemic effect of eicosapentaenoic acid in rats is reflected in increased mitochondrial fatty acid oxidation followed by diminished lipogenesis.

The effect of eicosapentaenoic acid (EPA) on fatty acid oxidation and on key enzymes of triglyceride metabolism and lipogenesis was investigated in the liver of rats. Repeated administration of EPA to normolipidemic rats resulted in a time-dependent decrease in plasma triglycerides, phospholipids and cholesterol. The triglyceride-lowering effect was observed after one day of feeding whereas lowering of plasma cholesterol and phospholipids was observed after five days of treatment. The triglyceride content of liver was reduced after two-day treatment. At that time, increased mitochondrial fatty acid oxidation occurred whereas mitochondrial and microsomal glycerophosphate acyltransferase was inhibited. The phosphatidate phosphohydrolase activity was unchanged. Adenosine triphosphate:citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase were inhibited during the 15 d of EPA treatment whereas peroxisomal beta-oxidation was increased. At one day of feeding, however, when the hypotriglyceridemic effect was established, the lipogenic enzyme activities were reduced to the same extent in palmitic acid-treated animals as in EPA-treated rats. In cultured rat hepatocytes, the oxidation of [14C]palmitic acid to carbon dioxide and acid-soluble products was stimulated in the presence of EPA. These results suggest that the instant hypolipidemia in rats given EPA could be explained at least in part by a sudden increase in mitochondrial fatty acid oxidation, thereby reducing the availability of fatty acids for lipid synthesis in the liver for export, e.g., in the form of very low density lipoproteins, even before EPA induced peroxisomal fatty acid oxidation, reduced triglyceride biosynthesis and diminished lipogenesis.

Eicosapentaenoic acid, but not docosahexaenoic acid, increases mitochondrial fatty acid oxidation and upregulates 2,4-dienoyl-CoA reductase gene expression in rats.

The aim of the present study was to investigate whether eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) was responsible for the triglyceride-lowering effect of fish oil. In rats fed a single dose of EPA as ethyl ester (EPA-EE), the plasma concentration of triglycerides was decreased at 8 h after acute administration. This was accompanied by an increased hepatic fatty acid oxidation and mitochondrial 2,4-dienoyl-CoA reductase activity. The steady-state level of 2,4-dienoyl-CoA reductase mRNA increased in parallel with the enzyme activity. An increased hepatic long-chain acyl-CoA content, but a reduced amount of hepatic malonyl-CoA, was obtained at 8 h after acute EPA-EE treatment. On EPA-EE supplementation, both EPA (20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3) increased in the liver, whereas the hepatic DHA (22:6n-3) concentration was unchanged. On DHA-EE supplementation retroconversion to EPA occurred. No statistically significant differences were found, however, for mitochondrial enzyme activities, malonyl-CoA, long-chain acyl-CoA, plasma lipid levels, and the amount of cellular fatty acids between DHA-EE treated rats and their controls at any time point studied. In cultured rat hepatocytes, the oxidation of [1-14C]palmitic acid was reduced by DHA, whereas it was stimulated by EPA. In the in vivo studies, the activities of phosphatidate phosphohydrolase and acetyl-CoA carboxylase were unaffected after acute EPA-EE and DHA-EE administration, but the fatty acyl-CoA oxidase, the rate-limiting enzyme in peroxisomal fatty acid oxidation, was increased after feeding these n-3 fatty acids. The hypocholesterolemic properties of EPA-EE may be due to decreased 3-hydroxy-3-methylglutaryl-CoA reductase activity. Furthermore, replacement of the ordinary fatty acids, i.e., the monoenes (16:1n-7, 18:1n-7, and 18:1n-9) with EPA and some conversion to DPA concomitant with increased fatty acid oxidation is probably the mechanism leading to changed fatty acid composition. In contrast, DHA does not stimulate fatty acid oxidation and, consequently, no such displacement mechanism operates. In conclusion, we have obtained evidence that EPA, and not DHA, is the fatty acid primarily responsible for the triglyceride-lowering effect of fish oil in rats.

Purinergic signalling - a possible mechanism for KCNQ1 channel response to cell volume challenges.

AIM: A number of K(+) channels are regulated by small, fast changes in cell volume. The mechanisms underlying cell volume sensitivity are not known, but one possible mechanism could be purinergic signalling. Volume activated ATP release could trigger signalling pathways that subsequently lead to ion channel stimulation and cell volume back-regulation. Our aim was to investigate whether volume sensitivity of the voltage-gated K(+) channel, KCNQ1, is dependent on ATP release and regulation by purinergic signalling. METHODS: We used Xenopus oocytes heterologously expressing human KCNQ1, KCNE1, water channels (AQP1) and P2Y2 receptors. ATP release was monitored by a luciferin-luciferase assay and ion channel conductance was recorded by two-electrode voltage clamp. RESULTS: The luminescence assay showed that oocytes released ATP in response to mechanical, hypoosmotic stimuli and hyperosmotic stimuli. Basal ATP release was approx. three times higher in the KCNQ1 + AQP1 and KCNQ1 injected oocytes compared to the non-injected ones. Exogenously added ATP (0.1 mm) did not have any substantial effect on volume-induced KCNQ1 currents. Nevertheless, apyrase decreased all currents by about 50%. Suramin inhibited about 23% of the KCNQ1 volume sensitivity. Expression of P2Y2 receptors stimulated endogenous Cl(-) channels, but it also led to 68% inhibition of the KCNQ1 currents. Adenosine (0.1 mm) also inhibited the KCNQ1 currents by about 56%. CONCLUSION: Xenopus oocytes release ATP in response to mechanical stimuli and cell volume changes. Purinergic P2 and P1 receptors confer some of the KCNQ1 channel volume sensitivity, although endogenous adenosine receptors and expressed P2Y2 receptors do so in the negative direction.