Jonny Wijkander

Insulin-induced phosphorylation and activation of phosphodiesterase 3B in rat adipocytes: possible role for protein kinase B but not mitogen-activated protein kinase or p70 S6 kinase.

Insulin stimulation of adipocytes results in serine phosphorylation/activation of phosphodiesterase 3B (PDE 3B) and activation of a kinase that phosphorylates PDE 3B in vitro, key events in the antilipolytic action of this hormone. We have investigated the role for p70 S6 kinase, mitogen-activated protein kinases (MAP kinases), and protein kinase B (PKB) in the insulin signaling pathway leading to phosphorylation/activation of PDE 3B in adipocytes. Insulin stimulation of adipocytes resulted in increased activity of p70 S6 kinase, which was completely blocked by pretreatment with rapamycin. However, rapamycin had no effect on the insulin-induced phosphorylation/activation of PDE 3B or the activation of the kinase that phosphorylates PDE 3B. Stimulation of adipocytes with insulin or phorbol myristate acetate induced activation of MAP kinases. Pretreatment of adipocytes with the MAP kinase kinase inhibitor PD 98059 was without effect on the insulin-induced activation of PDE 3B. Furthermore, phorbol myristate acetate stimulation did not result in phosphorylation/activation of PDE 3B or activation of the kinase that phosphorylates PDE 3B. Using Mono Q and Superdex chromatography, the kinase that phosphorylates PDE 3B was found to co-elute with PKB, but not with p70 S6 kinase or MAP kinases. Furthermore, both PKB and the kinase that phosphorylates PDE 3B were found to translocate to membranes in response to peroxovanadate stimulation of adipocytes in a wortmannin-sensitive way. Whereas these results suggest that p70 S6 kinase and MAP kinases are not involved in the insulin-induced phosphorylation/activation of PDE 3B in rat adipocytes, they are consistent with PKB being the kinase that phosphorylates PDE 3B.

A phospholipase A2 hydrolyzing arachidonoyl-phospholipids in mouse peritonea macrophages

A calcium‐dependent phospholipase A2 with half‐maximal activity at approx. 0.7 μM free Ca2+ has been identified in the cytosolic fraction from macrophages. The enzyme eluted as a 70 kDA protein upon gel chromatography and showed increased activity after 10 min pretreatment of the cells with 10 nM phorbol myristate acetate. No significant activity could be detected in the membrane fraction. The enzyme hydrolyzed arachidonic acid‐containing phosphatidylcholine and ‐ethanolamine as well as phosphatidylinositol. The release of arachidonic acid in the vitro assay was inhibited in a dose‐dependent manner by nordihydroguaiaretic acid and quercetin that are also potent inhibitors of the mobilization of arachidonic acid in intact macrophages.

Regulation of protein kinase B in rat adipocytes by insulin, vanadate, and peroxovanadate. Membrane translocation in response to peroxovanadate

Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB.

Macrophage arachidonate-mobilizing phospholipase A2: Role of Ca2+ for membrane binding but not for catalytic activity

A recently purified Ca(2+)-dependent intracellular phospholipase A2 from spleen, kidney and macrophage cell lines is activated by Ca2+ at concentrations achieved intracellularly. Using enzyme from the murine cell line J774 we here demonstrate the formation of a ternary complex of phospholipase, 45Ca2+ and phospholipid vesicle, and provide evidence for a single Ca(2+)-binding site on the enzyme involved in its vesicle binding. Although Ca2+ binds to and functions as an activator of the enzyme, this ion does not appear to be involved in its catalytic mechanism, since enzyme brought to the phospholipid vesicle by molar concentrations of NaCl or NH4+ salts exhibited Ca(2+)-independent catalytic activity.

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.

Mechanisms of inhibition of lipolysis by insulin, vanadate and peroxovanadate in rat adipocytes

Vanadate and peroxovanadate (pV), potent inhibitors of tyrosine phosphatases, mimic several of the metabolic actions of insulin. Here we compare the mechanisms for the anti-lipolytic action of insulin, vanadate and pV in rat adipocytes. Vanadate (5 mM) and pV (0.01 mM) inhibited lipolysis induced by 0.01-1 microM isoprenaline, vanadate being more and pV less efficient than insulin (1 nM). A loss of anti-lipolytic effect of pV was observed by increasing the concentration of isoprenaline and/or pV. pV induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 to a greater extent than insulin, whereas vanadate affected these components little if at all. In addition, only a higher concentration (0.1 mM) of pV induced the tyrosine phosphorylation of p85, the 85 kDa regulatory subunit of phosphoinositide 3-kinase (PI-3K). Vanadate activated PI-3K-independent (in the presence of 10 nM isoprenaline) and PI-3K-dependent (in the presence of 100 nM isoprenaline) anti-lipolytic pathways, both of which were found to be independent of phosphodiesterase type 3B (PDE3B). pV (0.01 mM), like insulin, activated PI-3K- and PDE3B-dependent pathways. However, the anti-lipolytic pathway of 0.1 mM pV did not seem to require insulin receptor substrate-1-associated PI-3K and was found to be partly independent of PDE3B. Vanadate and pV (only at 0.01 mM), like insulin, decreased the isoprenaline-induced activation of cAMP-dependent protein kinase. Overall, these results underline the complexity and the diversity in the mechanisms that regulate lipolysis.

Protein-mediated intermembrane contact specifically enhances Ca2+-induced fusion of phosphatidate-containing membranes

Ca2+-induced fusion of glycolipid-phospholipid vesicles containing several different anionic phospholipids was investigated, with and without lectin-mediated intervesicle contact. In vesicles containing phosphatidylserine, phosphatidylinositol or its mono- or diphosphate as the anionic phospholipid fusion was induced only at 1-10 mM Ca2+ both in the absence and presence of lectin. In contrast, the Ca2+-threshold for fusion of phosphatidate-containing vesicles was reduced to less than or equal to 0.1 mM Ca2+ by lectin-mediated intermembrane contact.

Proton-induced membrane fusion role of phospholipid composition and protein-mediated intermembrane contact

Glycolipid-phospholipid vesicles containing phosphatidate and phosphatidylethanolamine were found to undergo proton-induced fusion upon acidification of the suspending medium from pH 7.4 to pH 6.5 or lower, as determined by an assay for lipid intermixing based on fluorescence resonance energy transfer. Lectin-mediated contact between the vesicles was required for fusion. Incorporation of phosphatidylcholine in the vesicles inhibited proton-induced fusion. Vesicles in which phosphatidate was replaced by phosphatidylserine underwent fusion only when pH was reduced below 4.5, while no significant fusion occurred (pH greater than or equal to 3.5) when the anionic phospholipid was phosphatidylinositol. It is suggested that partial protonation of the polar headgroup of phosphatidate and phosphatidylserine, respectively, causes a sufficient reduction in the polarity and hydration of the vesicle surface to trigger fusion at sites of intermembrane contact.

Phosphatidylinositol 3-kinase in zymosan- and bacteria-induced signalling to mobilisation of arachidonic acid in macrophages

Stimulation of mouse peritoneal macrophages with zymosan or bacteria results in activation of 85-kDa cytosolic phospholipase A(2) (cPLA(2)) and release of arachidonate. We have investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in the signalling leading to activation of cPLA(2) and release of arachidonate in response to zymosan and the bacterium Prevotella intermedia. The specific PtdIns 3-kinase inhibitor wortmannin completely inhibited zymosan- and bacteria-induced release of arachidonate with an IC(50) value of 10-20 nM. Wortmannin also completely inhibited the zymosan-induced activation of cPLA(2), while the cPLA(2) activation by bacteria was partially inhibited by about 50%. Further experiments showed that zymosan-induced activation of extracellular signal-regulated kinase was inhibited, and bacteria-induced activation of the kinase strongly reduced, in the presence of wortmannin. Also zymosan-induced activation of p38 mitogen-activated protein kinase was inhibited by wortmannin, while p38 activation induced by bacteria was not. The zymosan- and bacteria-induced activation of phospholipase C, as determined by the generation of inositol phosphates, was also inhibited by wortmannin. Moreover, zymosan caused activation of PtdIns 3-kinase, which was totally inhibited by wortmannin. In contrast to zymosan and bacteria, arachidonate release induced by calcium ionophore alone, or further amplified by phorbol ester, was not sensitive to wortmannin. These results suggest that PtdIns 3-kinase constitutes a critical component in the zymosan- and bacteria-induced signalling leading to release of arachidonate and that PtdIns 3-kinase is positioned upstream of phospholipase C in this pathway.