C-reactive protein triggers calcium signalling in human neutrophilic granulocytes via FcγRIIa in an allele-specific way.

C-reactive protein (CRP) binds to Fcγ-receptors, FcγRIIa (CD32) with high affinity and to FcγRIa (CD64) with low affinity. The binding to CD32 has been shown to be allele specific, that is, it binds to R/R131 but not to H/H131. Little is known about the cooperation of CRP and neutrophilic granulocytes (PMNs) in inflammatory reactions. The purpose of the present study was to examine CRP signalling in human PMNs, and whether this signalling is also allele specific. Cytosolic calcium of PMN was measured in a single-cell digital imaging system. Receptor expression and polymorphism were studied by real-time RT-PCR, flow cytometry and standard PCR. C-reactive protein induced cytosolic calcium signals in PMNs from homozygote R/R131 donors, but not in PMNs from heterozygote R/H131 donors. However, after the heterozygote PMNs had been incubated with IFN-γ (100 U/ml) for 2 h, both the proportion of cells responding and the size of the CRP-induced calcium signals increased. IFN-γ increased mRNA expression of CD64 about fivefold and surface protein expression of CD64 about fourfold. The calcium signal elicited by CRP was augmented by PMN adhesion to fibronectin, but almost totally abrogated by sphingosine kinase inhibitors. The signals were partly dependent on calcium influx. In conclusion, calcium signalling instigated by CRP in human PMN is FcγRIIa allele specific, as R/R131 responded to CRP, whereas R/H131 did not. However, increased expression of FcγRIa (CD64), stimulated by IFN-γ, can augment calcium signalling by CRP in low-responders. This suggests that the state of the PMNs, as well as the genetic origin, affect sensitivity for CRP.

Electrical pulse stimulation of cultured skeletal muscle cells as a model for in vitro exercise - possibilities and limitations.

The beneficial health-related effects of exercise are well recognized, and numerous studies have investigated underlying mechanism using various in vivo and in vitro models. Although electrical pulse stimulation (EPS) for the induction of muscle contraction has been used for quite some time, its application on cultured skeletal muscle cells of animal or human origin as a model of in vitro exercise is a more recent development. In this review, we compare in vivo exercise and in vitro EPS with regard to effects on signalling, expression level and metabolism. We provide a comprehensive overview of different EPS protocols and their applications, discuss technical aspects of this model including critical controls and the importance of a proper maintenance procedure and finally discuss the limitations of the EPS model.

Interferon-gamma elicits a G-protein-dependent Ca2+ signal in human neutrophils after depletion of intracellular Ca2+ stores.

Interferon-gamma (IFN-gamma) has multiple effects on Ca2+ signalling in polymorphonuclear neutrophils (PMNs), including evoked cytosolic Ca2+ transients, increased capacitative calcium influx and increased sequestration of Ca2+ in intracellular stores. The present study was conducted to elucidate the mechanism behind the Ca2+ transients. As observed before, the IFN-gamma-evoked Ca2+ signals were apparent when extracellular Ca2+ was removed. A new finding was that the proportion of responding cells and the extent of calcium release increased with increasing time in EGTA buffer. As assessed by N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated Ca2+ release, the intracellular stores were depleted during this incubation period, and the extent of depletion correlated well with the appearance of IFN-gamma-induced Ca2+ signals. This store dependence of the IFN-gamma-induced Ca2+ signals was confirmed by the appearance of IFN-gamma-evoked Ca2+ signals in the presence of extracellular Ca2+ after store depletion by thapsigargin. The appearance of IFN-gamma-mediated Ca2+-signals in the presence of EGTA indicates that IFN-gamma stimulates Ca2+ release from intracellular stores. This was confirmed by the inability of the calcium transportation blocker La3+ to abolish the IFN-gamma response and the total abrogation of the response by the phospholipase C inhibitor U73122. Although these latter results imply a role for inositol 1,4,5-trisphosphate(IP3) in IFN-gamma signalling, comparison of IFN-gamma-evoked responses with fMLP responses revealed clear differences that suggest different signal-transduction pathways. However, responses to fMLP and IFN-gamma were both depressed by pertussis toxin, and the IFN-gamma responses were, in addition, inhibited by the tyrosine kinase inhibitor genistein. Further evidence of the involvement of tyrosine kinase was a slight stimulatory effect of the protein tyrosine phosphatase inhibitor sodium orthovanadate. The PI-3K activity was of minor importance. In conclusion, we present evidence of a novel signal-transduction mechanism for IFN-gamma in PMNs, dependent on tyrosine kinase activity, a pertussis toxin-sensitive G protein and phospholipase C activity.

Interferon-gamma affects protein kinase C activity in human neutrophils.

Interferon-gamma (IFN-gamma) is a priming agent of polymorphonuclear neutrophilic granulocyte (PMN) oxygen metabolism, and protein kinase C (PKC) is traditionally believed to play a central role in activation of this oxygen metabolism. In the present study, we have shown that the PKC activity in PMN is affected by IFN-gamma. After only 2 minutes exposure to IFN-gamma (100 U/ml), PKC activity was significantly increased in the noncytosolic fraction of the cells. This increase was transient, but toward the end of the priming period of 2 h, the membrane-associated PKC activity increased again to about 152% of control. In the cytosolic fraction, a small and hardly detectable decrease in PKC activity was observed. Treatment of PMN with granulocyte-macrophage colony-stimulating factor (GM-CSF), another PMN priming agent, showed no significant effects on the PKC activity. When the cells were stimulated with the bacterial peptide fMLP after a priming period with IFN-gamma or GM-CSF for 2 h, no significant difference between treated and control cells could be observed. PMN oxygen metabolism, measured by flow cytometry as an accumulation of the fluorescent compound dichlorofluorescein, was in these experiments significantly primed by IFN-gamma, both at baseline and when stimulated with fMLP. The protein kinase C inhibitors H7 and Ro31-8220 blocked the fMLP responses to some extent, but not completely. However, no significant difference between fMLP responses in control and IFN-gamma-treated cells could be detected after administration of inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

IFN-gamma induces calcium transients and increases the capacitative calcium entry in human neutrophils.

We have previously reported that long-term priming of human polymorphonuclear neutrophilic granulocytes (PMN) with interferon-gamma (IFN-gamma) increased the fMLP-stimulated calcium influx. We now show that also after short-term incubation with IFN-gamma, PMN calcium metabolism is modulated. Single adherent cells in three different calcium-containing buffers (high, normal, and low [Ca2+]) were stimulated with the bacterial peptide fMLP or the Ca-ATPase inhibitor thapsigargin (Tg) after about 5 min preincubation with IFN-gamma. The results of this protocol indicated that IFN-gamma increases both calcium influx and calcium sequestration. Store dependent Ca2+ influx, directly measured on readdition of calcium to Tg-treated cells incubated in EGTA buffer, was significantly enhanced in IFN-gamma-treated cells. This effect of IFN-gamma was enhanced by the tyrosine kinase inhibitor herbimycin A. Strikingly, in low extracellular calcium concentrations, IFN-gamma induced calcium transients in 20%-60% of the cells. The proportion of PMN responding with Ca2+ transients increased with decreasing extracellular calcium concentration. Average lagtime from addition of IFN-gamma to a response that could be measured was 7.3 sec, and average increase in [Ca2+] above the basal level was 790 nM. These IFN-gamma-induced transients could not be depressed by herbimycin A. Thus, IFN-gamma can increase capacitative calcium influx, induce calcium transients, and possibly affect calcium sequestration in human PMN.

Modulation by interferons of human neutrophilic granulocyte migration.

The effect of various interferons (IFN) on neutrophilic granulocyte (PMN) random and directed migration is incompletely understood. We, therefore, investigated PMN migration with a novel micropore membrane technique. No chemotactic effect of either 10-10000 U/ml IFN-alpha or IFN-beta, or 1-1000 U/ml IFN-gamma was observed on PMN isolated from normal human venous blood. However, when present on both sides of the micropore membrane, all the IFN (1000 U/ml IFN-alpha and IFN-beta, 100 U/ml IFN-gamma) inhibited both random and directed migration toward zymosan-activated serum (ZAS). IFN-gamma was the most potent inhibitory agent and produced an inhibition of about 30%. When the bacterial peptide fMLP was used as a chemoattractant, IFN-gamma also depressed chemotaxis. Taking the reduced random migration of IFN-gamma treated cells into account, however, chemotaxis per se-toward both ZAS and fMLP-was not significantly affected. Random migration and directed migration assessed simultaneously with PMN from the same donor were clearly correlated for both control and IFN-gamma treated cells, suggesting that a general antimotility effect of IFN-gamma might explain both reduced random migration and chemotaxis. The antimotility effect of IFN-gamma was not dependent on protein synthesis or on tyrosine kinase activity. In fact, inhibition of tyrosine kinase with herbimycin A increased the ZAS-stimulated motility of both control and IFN-gamma-inhibited PMN. In conclusion, our data indicate that IFN depress both random and directed PMN migration by mechanisms that do not involve protein synthesis or protein tyrosine kinase activity.

Fibronectin promotes calcium signaling by interferon-gamma in human neutrophils via G-protein and sphingosine kinase-dependent mechanisms.

A common intracellular signal activating polymorphonuclear leukocytes (PMN) in inflammation is a change in cytosolic calcium concentration. Previously, we have shown that interferon-gamma (IFN-gamma) induces transient calcium signals in PMN, but only after intracellular calcium store depletion. Using a digital imaging system, we show that adhesion of PMN is critical for IFN-gamma-induced calcium signals, and with PMN attached to the optimal coating, the calcium signals are evoked even in presence of extracellular calcium, that is, non-depleted calcium stores. Adhesion to fibronectin, pure or extracted from plasma by gelatin, improved the IFN-gamma responses compared with serum, plasma, or vitronectin coats. In accordance with previous observations, IFN-gamma-induced calcium signals in fibronectin adherent cells were totally abolished by the G-protein inhibitor pertussis toxin and were also inhibited by the sphingosine kinase inhibitors dimethylsphingosine (DMS) and N-acetylsphingosine (N-Ac-Sp). PMN contact with fibronectin alone, measured in cells sedimenting onto a fibronectin-coated surface or by addition of fibronectin to glass-adherent cells, evoked transient calcium signals. However, PMN in suspension did not respond to the addition of fibronectin or arginine-glycine-aspartate (RGD). The fibronectin-induced calcium signals were also clearly depressed by pertussis toxin and by the sphingosine kinase inhibitors DMS, dihydrosphingosine (DHS), and N-Ac-Sp. When the product of sphingosine kinase activity, sphingosine 1-phosphate (S1-P), was added to the cells, similar calcium signals were induced, which were dependent on a pertussis toxin-sensitive G-protein activity. Finally, addition of S1-P to the cells prior to stimulation with IFN-gamma partly mimicked the priming effect of fibronectin. In conclusion, fibronectin contact evokes by itself a calcium signal in PMN and further promotes calcium signaling by IFN-gamma. We suggest that fibronectin might activate sphingosine kinase, and that the sphingosine 1-phosphate thereby generated induces a calcium signal via a G-protein-dependent mechanism. Apparently, sphingosine kinase activity is also involved in IFN-gamma induced calcium signals.

Benfotiamine increases glucose oxidation and downregulates NADPH oxidase 4 expression in cultured human myotubes exposed to both normal and high glucose concentrations.

The aim of the present work was to study the effects of benfotiamine (S-benzoylthiamine O-monophosphate) on glucose and lipid metabolism and gene expression in differentiated human skeletal muscle cells (myotubes) incubated for 4 days under normal (5.5 mM glucose) and hyperglycemic (20 mM glucose) conditions. Myotubes established from lean, healthy volunteers were treated with benfotiamine for 4 days. Glucose and lipid metabolism were studied with labeled precursors. Gene expression was measured using real-time polymerase chain reaction (qPCR) and microarray technology. Benfotiamine significantly increased glucose oxidation under normoglycemic (35 and 49% increase at 100 and 200 µM benfotiamine, respectively) as well as hyperglycemic conditions (70% increase at 200 µM benfotiamine). Benfotiamine also increased glucose uptake. In comparison, thiamine (200 µM) increased overall glucose metabolism but did not change glucose oxidation. In contrast to glucose, mitochondrial lipid oxidation and overall lipid metabolism were unchanged by benfotiamine. The expression of NADPH oxidase 4 (NOX4) was significantly downregulated by benfotiamine treatment under both normo- and hyperglycemic conditions. Gene set enrichment analysis (GSEA) showed that befotiamine increased peroxisomal lipid oxidation and organelle (mitochondrial) membrane function. In conclusion, benfotiamine increases mitochondrial glucose oxidation in myotubes and downregulates NOX4 expression. These findings may be of relevance to type 2 diabetes where reversal of reduced glucose oxidation and mitochondrial capacity is a desirable goal.

Metabolic switching of human skeletal muscle cells in vitro.

In this review we will focus on external factors that may modify energy metabolism in human skeletal muscle cells (myotubes) and the ability of the myotubes to switch between lipid and glucose oxidation. We describe the metabolic parameters suppressibility, adaptability and substrate-regulated flexibility, and show the influence of nutrients such as fatty acids and glucose (chronic hyperglycemia), and some pharmacological agents modifying nuclear receptors (PPAR and LXR), on these parameters in human myotubes. Possible cellular mechanisms for changes in these parameters will also be highlighted.

Leukaemia inhibitory factor stimulates glucose transport in isolated cardiomyocytes and induces insulin resistance after chronic exposure.

AIMS/HYPOTHESIS: Hypertrophic and failing hearts have increased utilisation of glucose, but also develop insulin resistance and reduced ability to produce ATP. Increased levels of the IL-6-related cytokine leukaemia inhibitory factor (LIF) are found in failing hearts, and we have recently shown that LIF reduces ATP production in isolated cardiomyocytes. In the present study we investigated effects of LIF on glucose metabolism, and how LIF-treated cells respond to insulin stimulation. METHODS: Cardiomyocytes were isolated from adult Wistar rats by collagen digestion, maintained in culture for 48 h, and then treated with 1 nmol/l LIF. RESULTS: Acute LIF treatment increased deoxyglucose uptake compared with controls, but no additive effect was observed in cardiomyocytes treated with LIF and insulin. The phosphatidylinositol 3-kinase inhibitor wortmannin did not affect LIF-induced glucose uptake. LIF had no effect on AMP-activated protein kinase phosphorylation. Cardiomyocytes treated with LIF for 48 h did not respond to insulin by increasing deoxyglucose uptake and showed a reduced insulin-mediated uptake of oleic acid and formation of complex lipids compared with control cells. Chronic LIF treatment increased gene expression of the suppressor of cytokine signalling (Socs) 3 and reduced expression of solute carrier family 2, member 4 (Slc2a4, previously known as glucose transporter 4 [Glut4]). In line with these observations, chronic LIF treatment reduced insulin-mediated phosphorylation of both Akt/protein kinase B (PKB) and glycogen synthase kinase (GSK)-3. CONCLUSIONS/INTERPRETATION: Acute LIF treatment increased glucose uptake in isolated cardiomyocytes by a pathway different from that of insulin. Chronic LIF treatment induced insulin resistance, possibly mediated by altered expression of Socs3 and Slc2a4, and impaired insulin-mediated phosphorylation of GSK-3 and Akt/PKB.

Lipid metabolism in human skeletal muscle cells: effects of palmitate and chronic hyperglycaemia.

This review focuses on the effect of exogenous factors known to be of importance for the development of insulin resistance in differentiated human myotubes. Recent data from our laboratory on the effects of fatty acid pre-treatment and chronic glucose oversupply on fatty acid and glucose metabolism, without and with acute insulin are presented, and discussed in the context of other recent publications in the field. Pre-treatment of myotubes with palmitate, chronic hyperglycaemia, and acute high concentrations of insulin changed fatty acid metabolism in favour of accumulation of intracellular lipids. Acute insulin exposure increased (14)C-oleate uptake and levels of free fatty acids (FFA) and triacylglycerol (TAG). Palmitate pre-treatment further increased oleate uptake, both under basal conditions and in the presence of insulin, with a marked increase in the phospholipid (PL) fraction, with a concomitant reduction in oleate oxidation. Chronic hyperglycaemia also promoted increased lipogenesis and elevated levels of cellular lipids. Changes in fatty acid metabolism in human muscle, in particular fatty acid oxidation, are probably crucial for the molecular mechanism behind skeletal muscle insulin resistance and impaired glucose metabolism. Differentiated human skeletal muscle cells may be an ideal system to further explore the mechanisms regulating lipid metabolism.

Interferon-gamma modulates cytosolic free calcium in human neutrophilic granulocytes.

To investigate the role of cytosolic free calcium ([Ca2+]i in interferon-gamma (IFN-gamma) pre-activation (priming) of human neutrophilic granulocytes (PMN) we used three different fluorescence methods, i.e. digital imaging of single, adherent, Fura-2 loaded cells, flow cytometric measurements of single, non-adherent, Fluo-3 loaded cells, and spectrofluorometry of Indo-1 loaded PMN in suspension. IFN-gamma increased the [Ca2+]i level in single, adherent PMN during the second phase of the fMLP response. The bacterial peptide fMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) is a known stimulant of the calcium/inositol phosphate system. The [Ca2+]i increase was abolished in Ca(2+)-free test buffer. Furthermore, the baseline [Ca2+]i level was found to be slightly increased in IFN-gamma primed PMN as analysed with flow cytometry. On the other hand, these [Ca2+]i responses were not detectable with the other methods used. We suggest that IFN-gamma increases the plasma membrane permeability for calcium in PMN, and substantiate this by demonstrating compliance with a capacitative model for intracellular calcium regulation. Mathematical modeling also suggested that IFN-gamma primed human PMN may sequester 13% more Ca2+ than unprimed cells in fMLP-insensitive intracellular stores. Thus, the Ca2+ responses to IFN-gamma are modest and not easily detectable with some of the methods currently in use. They nevertheless explain why fMLP elicits brisker responses from PMN after IFN-gamma priming.

Chronic hyperglycaemia promotes lipogenesis and triacylglycerol accumulation in human skeletal muscle cells.

AIMS/HYPOTHESIS: The present study was conducted to evaluate the effect of hyperglycaemia in itself on glucose and lipid metabolism in human skeletal muscle cells. METHODS: Satellite cells were isolated from biopsy samples from the vastus lateralis muscle and differentiated into multinucleated myotubes in cultures. Metabolism studies were performed using isotopes ([3H]deoxyglucose, [14C]glucose, [14C]oleic acid and [14C]palmitic acid), and mRNA and protein levels were analysed by real-time RT-PCR and western blotting respectively. RESULTS: Exposure of myotubes to 20 mmol/l glucose for 4 days reduced insulin-stimulated glucose uptake and glycogen synthesis to 57+/-5% (p<0.0001) and 56+/-5% (p<0.0001) of normoglycaemic (5.5 mmol/l glucose) controls respectively. Basal glucose uptake and glycogen synthesis were both reduced, whereas glucose oxidation was unaltered. Total cell content of glycogen and expression of GLUT1 and GLUT4 mRNA were not affected. There was a significant increase in the incorporation of glucose into cellular NEFA (88+/-17% increase, p=0.006), triacylglycerol (44+/-21% increase, p=0.04) and cholesterol ester (89+/-36% increase, p=0.02) in hyperglycaemic myotubes compared with controls. Diacylglycerol tended to be increased though not significantly, and phospholipid formation were unchanged. Relative to controls, total cell content of triacylglycerol was increased by 25+/-7% (p=0.02) and acyl-CoA:1,2-diacylglycerol acyltransferase 1 activity was increased by 34+/-4% (p=0.004), whereas acyl-CoA:1,2-diacylglycerol acyltransferase 1 mRNA expression was unchanged. Total cellular uptake of palmitic acid was reduced by 18+/-3% (p=0.006) in hyperglycaemic cells compared with controls, while uptake of oleic acid was unchanged. Oxidation of palmitic acid or oleic acid was not affected by hyperglycaemia. CONCLUSIONS/INTERPRETATION: Chronic hyperglycaemia increased triacylglycerol accumulation and the incorporation of carbohydrate into triacylglycerol (i.e. de novo lipogenesis) concomitantly with a reduced insulin-stimulated glucose uptake and glycogen synthesis. Enhanced acyl-CoA:1,2-diacylglycerol acyltransferase 1 activity supported the increased triacylglycerol synthesis during hyperglycaemia.