Kirsten B. Holven

Identification and Validation of Novel Contraction-Regulated Myokines Released from Primary Human Skeletal Muscle Cells

Proteins secreted by skeletal muscle, so called myokines, have been shown to affect muscle physiology and additionally exert systemic effects on other tissues and organs. Although recent profiling studies have identified numerous myokines, the amount of overlap from these studies indicates that the secretome of skeletal muscle is still incompletely characterized. One limitation of the models used is the lack of contraction, a central characteristic of muscle cells. Here we aimed to characterize the secretome of primary human myotubes by cytokine antibody arrays and to identify myokines regulated by contraction, which was induced by electrical pulse stimulation (EPS). In this study, we validated the regulation and release of two selected myokines, namely pigment epithelium derived factor (PEDF) and dipeptidyl peptidase 4 (DPP4), which were recently described as adipokines. This study reveals that both factors, DPP4 and PEDF, are secreted by primary human myotubes. PEDF is a contraction-regulated myokine, although PEDF serum levels from healthy young men decrease after 60 min cycling at VO2max of 70%. Most interestingly, we identified 52 novel myokines which have not been described before to be secreted by skeletal muscle cells. For 48 myokines we show that their release is regulated by contractile activity. This profiling study of the human skeletal muscle secretome expands the number of myokines, identifies novel contraction-regulated myokines and underlines the overlap between proteins which are adipokines as well as myokines.

Folic acid treatment reduces elevated plasma levels of asymmetric dimethylarginine in hyperhomocysteinaemic subjects.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, has been suggested to be a novel risk factor for endothelial dysfunction. It has previously been reported that hyperhomocysteinaemia may be associated with impaired endothelium-dependent vasodilation and reduced plasma level of NO-derived endproducts (NOx). In the present study, plasma levels of arginine and ADMA were measured in twenty-one healthy control subjects, and in twenty-one hyperhomocysteinaemic subjects before and after 6 weeks and 12 months of folic acid supplementation, and compared with previously measured plasma NOx values in the hyperhomocysteinaemic subjects. Compared with control subjects, hyperhomocysteinaemic subjects had higher plasma levels of arginine and ADMA. More importantly, folic acid therapy significantly reduced plasma levels of arginine and ADMA. Furthermore, plasma levels of arginine and ADMA were positively correlated with plasma homocysteine levels and negatively correlated with plasma folate, as well as negatively correlated with plasma NOx. Our results suggest that ADMA may be a mediator of the atherogenic effects of homocysteine.

Folic Acid Treatment Reduces Chemokine Release From Peripheral Blood Mononuclear Cells in Hyperhomocysteinemic Subjects

Elevated plasma homocysteine concentration is an independent risk factor for cardiovascular disease. However, the mechanisms by which hyperhomocysteinemia induces vascular disease are uncertain. An early step in atherogenesis involves leukocyte migration into the arterial wall, a process regulated in part by chemokines. We hypothesized that homocysteine may exert its atherogenic effect in part through chemokine-mediated mechanisms, and in the present study, we examined the effects of folic acid supplementation for 6 weeks on chemokine levels in hyperhomocysteinemic individuals. Data showed the following: (1) Compared with control subjects, hyperhomocysteinemic subjects had elevated plasma levels of the CXC chemokines, epithelial neutrophil-activating peptide (ENA)-78 (P <0.05), and growth-regulated oncogene (GRO)&agr; (P =0.088), and homocysteine was significantly correlated with ENA-78 and GRO&agr;. (2) During folic acid treatment, normalization of homocysteine levels was accompanied by a marked reduction in oxidized low density lipoprotein–stimulated release of CXC chemokines (ie, GRO&agr;, ENA-78, and interleukin-8) and CC chemokines (ie, monocyte chemoattractant peptide-1 and RANTES) in peripheral blood mononuclear cells from these individuals. (3) The oxidized low density lipoprotein–induced release of ENA-78 from peripheral blood mononuclear cells from control subjects was significantly reduced when cells were incubated in the presence of folic acid. These data may suggest that homocysteine exerts atherogenic effects in part by enhancing chemokine responses in cells involved in atherogenesis and that folic acid supplementation may downregulate these inflammatory responses.

Oxidised fish oil does not influence established markers of oxidative stress in healthy human subjects: a randomised controlled trial.

Intake of fish oil reduces the risk of CHD and CHD deaths. Marine n-3 fatty acids (FA) are susceptible to oxidation, but to our knowledge, the health effects of intake of oxidised fish oil have not previously been investigated in human subjects. The aim of the present study was to investigate markers of oxidative stress, lipid peroxidation and inflammation, and the level of plasma n-3 FA after intake of oxidised fish oil. In a double-blinded randomised controlled study, healthy subjects (aged 18-50 years, n 54) were assigned into one of three groups receiving capsules containing either 8 g/d of fish oil (1.6 g/d EPA+DHA; n 17), 8 g/d of oxidised fish oil (1.6 g/d EPA+DHA; n 18) or 8 g/d of high-oleic sunflower oil (n 19). Fasting blood and morning spot urine samples were collected at weeks 0, 3 and 7. No significant changes between the different groups were observed with regard to urinary 8-iso-PGF2α; plasma levels of 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and α-tocopherol; serum high sensitive C-reactive protein; or activity of antioxidant enzymes in erythrocytes. A significant increase in plasma level of EPA+DHA was observed in both fish oil groups, but no significant difference was observed between the fish oil groups. No changes in a variety of in vivo markers of oxidative stress, lipid peroxidation or inflammation were observed after daily intake of oxidised fish oil for 3 or 7 weeks, indicating that intake of oxidised fish oil may not have unfavourable short-term effects in healthy human subjects.

Effect of marine n-3 fatty acids on circulating inflammatory markers in healthy subjects and subjects with cardiovascular risk factors

ObjectiveThe aim of the present paper was to review the literature in order to summarize the effects of marine n-3 fatty acids on circulating inflammatory markers among healthy subjects, subjects with high risk of developing cardiovascular disease (CVD) and in patients with CVD in human intervention studies.MethodsA systematic literature search in PubMed was performed. Intervention studies describing the effects of marine n-3 fatty acids on circulating inflammatory markers in healthy subjects, subjects with high risk of CVD and patients with CVD were included. The following exclusion criteria were used: (1) interventions assessing inflammatory markers with ex vivo methods (2) interventions with children (3) articles describing animal or cell culture studies. Twenty-two articles were included. Additionally, 13 papers from their literature lists were included based on the same inclusion and exclusion criteria as the literature search.Results and conclusionIntervention studies with marine n-3 fatty acids administered from either fish or fish oil demonstrate different results on inflammatory markers. No firm conclusion can be drawn about the effect of marine n-3 fatty acids on circulating inflammatory markers in healthy individuals, individuals with high risk of developing CVD or individuals with CVD related diseases.

Effect of folic acid treatment on endothelium-dependent vasodilation and nitric oxide-derived end products in hyperhomocysteinemic subjects.

PURPOSE An elevated plasma homocysteine concentration is an independent risk factor for cardiovascular diseases. In this study, we tested the hypothesis that hyperhomocysteinemia induces endothelial dysfunction mediated, at least in part, through nitric oxide-dependent mechanisms and that folic acid supplementation improves endothelial function in hyperhomocysteinemic subjects. SUBJECTS AND METHODS Endothelial function was evaluated in healthy controls and hyperhomocysteinemic subjects by measuring plasma levels of the nitric oxide-derived end products nitrite and nitrate and by assessing vasodilatory responses in the skin microcirculation and forearm vasculature. In the subjects with hyperhomocysteinemia, these measurements were repeated after 6 weeks and 12 months of folic acid supplementation. RESULTS Compared with healthy controls, hyperhomocysteinemic subjects had significantly lower median plasma levels of nitric oxide-derived end products (12.1 microM [range 4.4 to 41.8] versus 24.6 microM [13.6 to 53.2]; P <0.001), a significantly lower endothelium-dependent vasodilatory response to acetylcholine (P <0.01), hyperemic response in the microcirculation (P <0.01), and total forearm blood flow during reactive hyperemia (P = 0.01). There was no significant difference in the endothelium-independent response. Folic acid treatment for 12 months increased the plasma level of nitric oxide-derived end products by 121% (95% confidence interval [CI], 72% to 170%), the vasodilatory response to acetylcholine by 124% (95% CI, 36% to 212%), and the ischemia-mediated hyperemic responses in the microcirculation by 60% (95% CI, 25% to 96%) and in the forearm vasculature by 47% (95% CI, 21% to 73%). CONCLUSIONS Homocysteine appears to induce its atherogenic effect, at least in part, by depressing endothelial function, possibly through nitric oxide-dependent mechanisms. This effect can be reversed by folic acid supplementation.

Increased levels of C-reactive protein and interleukin-6 in hyperhomocysteinemic subjects.

Objective. Elevated plasma homocysteine concentration is considered to be an independent risk factor for cardiovascular disease. However, the mechanisms by which hyperhomocysteinemia are related to vascular disease are unclear. High‐sensitivity C‐reactive protein (CRP), a marker of inflammation, has been reported to be an independent predictor of future myocardial infarction among clinically healthy individuals. Interleukin (IL)‐6 is a regulator of CRP and has a key role in initiation of inflammation. The aim of this study was to investigate whether individuals with increased plasma homocysteine concentrations have altered levels of serum CRP and IL‐6. Material and methods. Serum concentrations of CRP and IL‐6 were measured in 39 individuals with hyperhomocysteinemia and in 39 control subjects matched for gender, age and body mass index (BMI). In addition, the inflammatory effect of IL‐6 on peripheral blood mononuclear cells was measured. Results. Compared to controls, hyperhomocysteinemic subjects have elevated serum levels of CRP and IL‐6 (p⩽0.001 and p<0.005, respectively). Importantly, this raised level of IL‐6 was also seen in hyperhomocysteinemic individuals without accompanying hypercholesterolemia or cardiovascular disease. IL‐6 increased the release of monocyte chemoattractant protein‐1 from peripheral blood mononuclear cells, with particularly enhancing effects in cells from patients with hyperhomocysteinemia. Conclusions. These data suggest that enhanced inflammation may be associated with homocysteine‐related cardiovascular disease, possibly involving IL‐6‐related mechanisms.

CXC-chemokines in coronary artery disease: possible pathogenic role of interactions between oxidized low-density lipoprotein, platelets and peripheral blood mononuclear cells

Summary.  CXC‐chemokines may be involved in atherogenesis. Herein we examined the possible role of CXC‐chemokines in the inflammatory interactions between oxidized (ox‐) low‐density lipoprotein (LDL), platelets and peripheral blood mononuclear cells (PBMC) in 15 patients with coronary artery disease (CAD) without ‘traditional’ risk factors and 15 carefully matched controls. Our main findings were: (a) ox‐LDL stimulated the release of the CXC‐chemokines interleukin (IL)‐8, ENA‐78 and GRO‐α from PBMC, particularly in CAD. (b) In platelets, ox‐LDL induced release of ENA‐78 and, when combined with SFLLRN, also of GRO‐α, with significantly higher response in CAD. (c) Platelet‐rich plasma, especially when costimulated with ox‐LDL, enhanced the release of IL‐8 from PBMC, particularly in CAD patients. (d) Freshly isolated PBMC showed markedly increased IL‐8 mRNA expression in CAD patients. Our findings suggest enhanced inflammatory interactions between ox‐LDL, platelets and PBMC in CAD patients involving CXC‐chemokine related mechanisms, possible contributing to atherogenesis in these and other CAD patients.

Fish Oil Supplementation Alters the Plasma Lipidomic Profile and Increases Long-Chain PUFAs of Phospholipids and Triglycerides in Healthy Subjects

Background While beneficial health effects of fish and fish oil consumption are well documented, the incorporation of n-3 polyunsaturated fatty acids in plasma lipid classes is not completely understood. The aim of this study was to investigate the effect of fish oil supplementation on the plasma lipidomic profile in healthy subjects. Methodology/Principal Findings In a double-blinded randomized controlled parallel-group study, healthy subjects received capsules containing either 8 g/d of fish oil (FO) (1.6 g/d EPA+DHA) (n = 16) or 8 g/d of high oleic sunflower oil (HOSO) (n = 17) for seven weeks. During the first three weeks of intervention, the subjects completed a fully controlled diet period. BMI and total serum triglycerides, total-, LDL- and HDL-cholesterol were unchanged during the intervention period. Lipidomic analyses were performed using Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (QTOFMS), where 568 lipids were detected and 260 identified. Both t-tests and Multi-Block Partial Least Square Regression (MBPLSR) analysis were performed for analysing differences between the intervention groups. The intervention groups were well separated by the lipidomic data after three weeks of intervention. Several lipid classes such as phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylglycerol, and triglycerides contributed strongly to this separation. Twenty-three lipids were significantly decreased (FDR<0.05) in the FO group after three weeks compared with the HOSO group, whereas fifty-one were increased including selected phospholipids and triglycerides of long-chain polyunsaturated fatty acids. After seven weeks of intervention the two intervention groups showed similar grouping. Conclusions/Significance In healthy subjects, fish oil supplementation alters lipid metabolism and increases the proportion of phospholipids and triglycerides containing long-chain polyunsaturated fatty acids. Whether the beneficial effects of fish oil supplementation may be explained by a remodeling of the plasma lipids into phospholipids and triglycerides of long-chain polyunsaturated fatty acids needs to be further investigated. Trial Registration ClinicalTrials.gov NCT01034423

Expression of retinoic acid receptor and retinoid X receptor subtypes in rat liver cells: implications for retinoid signalling in parenchymal, endothelial, Kupffer and stellate cells.

In the present study, a systematic examination of the relative expression pattern of the nuclear retinoid receptors (RAR and RXR) in various liver cells was performed. Our data demonstrate that RXRalpha is the dominant receptor in all liver cells, and that RARbeta is also expressed at a high level in most cells. More specifically, RARbeta and RARalpha were the most predominant of the RAR subtypes in parenchymal cells, while all three RAR subtypes were equally expressed in endothelial and Kupffer cells. The total level of expression of all the RXR subtypes were larger than the total level of expression of all the RAR subtypes in all liver cells. This is in agreement with the observation that RXR is a heterodimer partner not only for RAR, but also for other members in the steroid/thyroid receptor superfamily of ligand-dependent transcription factors.