Selective decrease of membrane-associated PKC-α and PKC-ε in response to elevated intracellular O-GlcNAc levels in transformed human glial cells

Increased flux through the hexosamine biosynthetic pathway (HBP) has been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. It has been suggested that this effect is due to increases in the β-O-linked N-acetylglucosamine (O-GlcNAc) modification. Herein, we demonstrate the effect of increasing the O-GlcNAc modification on the translocation of select PKC isozymes in a human astroglial cell line. Treating cells with either 8 mM d-glucosamine (GlcN), 5 mM streptozotocin (STZ), or 80 μM O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) produced a significant increase in the O-GlcNAc modification on both cytosolic and membrane proteins; however, both the level and rate of O-GlcNAc increase varied with the compound. GlcN treatment resulted in a rapid, transient translocation of PKC-βII that was maximal after 3 h (73±8%) and also produced a 48±15% decrease in membrane-associated PKC-ε after 9 h of treatment. Similar to GlcN treatment, STZ and PUGNAc treatment also resulted in decreased levels of PKC-ε in the membrane fraction. Significant decreases were seen as early as 5 h and, by 9 h of treatment, had decreased by 87±6% with STZ and 73±7% with PUGNAc. Unlike GlcN, both STZ and PUGNAc produced a decrease in PKC-α membrane levels by 9 h posttreatment (78±10% with STZ and 66±8% with PUGNAc) while neither compound produced any changes in PKC-βII translocation. In addition, none of the three compounds affected membrane levels of PKC-ι. Altogether, these results demonstrate a novel link between increased levels of the O-GlcNAc modification and the regulation of specific PKC isoforms.     

Increased protein O-GlcNAc modification inhibits inflammatory and neointimal responses to acute endoluminal arterial injury

Inflammation plays a major role in vascular disease. We have shown that leukocyte infiltration and inflammatory mediator expression contribute to vascular remodeling after endoluminal injury. This study tested whether increasing protein O-linked-N-acetylglucosamine (O-GlcNAc) levels with glucosamine (GlcN) and O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) inhibits acute inflammatory and neointimal responses to endoluminal arterial injury. Ovariectomized rats were treated with a single injection of GlcN (0.3 mg/g ip), PUGNAc (7 nmol/g ip) or vehicle (V) 2 h before balloon injury of the right carotid artery. O-GlcNAc-modified protein levels decreased markedly in injured arteries of V-treated rats at 30 min, 2 h, and 24 h after injury but returned to control (contralateral uninjured) levels after 14 days. Both GlcN and PUGNAc increased O-GlcNAc-modified protein levels in injured arteries compared with V controls at 30 min postinjury; the GlcN-mediated increase persisted at 24 h but was not evident at 14 days. Proinflammatory mediator expression increased markedly after injury and was reduced significantly (30-50%) by GlcN and PUGNAc. GlcN and PUGNAc also inhibited infiltration of neutrophils and monocytes in injured arteries. Chronic (14 days) treatment with GlcN reduced neointima formation in injured arteries by 50% compared with V controls. Acute GlcN and PUGNAc treatment increases O-GlcNAc-modified protein levels and inhibits acute inflammatory responses in balloon-injured rat carotid arteries; 14 day GlcN treatment inhibits neointima formation in these vessels. Augmenting O-GlcNAc modification of proteins in the vasculature may represent a novel anti-inflammatory and vasoprotective mechanism.     

O-GlcNAc modification of proteins affects volume regulation in Jurkat cells

An increasing amount of recent research has demonstrated that the hexosamine biosynthesis pathway (HBP) plays a significant role in the modulation of intracellular signaling transduction pathways, and affects cellular processes via modification of protein by O-linked β-N-acetylglucosamine (O-GlcNAc). Besides the many known and postulated effects of protein O-GlcNAc modifications, there is little available data on the role of O-GlcNAc in cellular volume regulation. Our objective was to test the effect of increased O-GlcNAc levels on hypotonia-induced volume changes in Jurkat cells. We pretreated Jurkat cells for 1 h with glucosamine (GlcN), PUGNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino-N-phenylcarbamate) an inhibitor of O-GlcNAcase, or a high level of glucose to induce elevated levels of O-GlcNAc. We found that the response of Jurkat cells to hypotonic stress was significantly altered. The hypotonia induced cell-swelling was augmented in both GlcN and PUGNAc-treated cells and, to a lesser extent, in high glucose concentration-treated cells. Evaluated by NMR measurements, GlcN and PUGNAc treatment also significantly reduced intracellular water diffusion. Taken together, increased cell swelling and reduced water diffusion caused by elevated O-GlcNAc show notable analogy to the regulatory volume changes seen by magnetic resonance methods in nervous and other tissues in different pathological states. In conclusion, we demonstrate for the first time that protein O-GlcNAc could modulate cell volume regulation.     

Insulin stimulates the activity of a novel protein kinase C, PKC-epsilon, in cultured fetal chick neurons

In this study we examined the effects of insulin on protein kinase C (PKC) activity in cultured fetal chick neurons. PKC activity, measured as 32P incorporation into histone H1 in the presence of calcium (500 microM), phosphatidylserine (100 micrograms/ml), and diolein (3.3 micrograms/ml) minus the incorporation in the presence of calcium alone, was detected in neuronal cytosolic (207 +/- 33 pmol/min/mg) and membrane (33 +/- 8 pmol/min/mg) fractions. Insulin added to intact neurons increased the activity of PKC in both cytosolic and membrane fractions by about 40%. Neurons preincubated with cycloheximide (10 micrograms/ml) 30 min prior to insulin treatment showed the same degree of stimulation of PKC activity by insulin. The activation of PKC was maximal within 5-10 min of insulin exposure and was sustained for at least 60 min. Insulin stimulated PKC in a dose-dependent manner, with a maximal response obtained at 100 ng/ml. Addition of phosphatidylserine and diolein to neuronal cell extracts resulted in the phosphorylation of four major cytosolic proteins (70, 57, 18, and 16 kDa) and one major membrane protein (75 kDa). Phosphorylation of all five proteins was increased 2-fold in extracts from insulin-treated neurons. Immunoblot analysis of whole cell extracts using antibodies against PKC-alpha, PKC-beta, PKC-gamma, PKC-delta, and PKC-epsilon revealed that cultured fetal chick neurons contained only one of these PKC isoforms, the epsilon-isoform. The enzyme was mostly cytosolic. Insulin had no effect on either the amount of distribution of PKC-epsilon in cultured neurons but induced a small change in the mobility of PKC-epsilon on sodium dodecyl sulfate-polyacrylamide gels. When assay conditions were designed to measure specifically the activity of PKC-epsilon, using a synthetic peptide substrate in the absence of calcium, activity was 50 +/- 12% higher in insulin-treated cells (p less than 0.005). PKC activity in control and insulin treated-neurons was almost completely inhibited when assays included a peptide identical to the pseudo-substrate binding site of PKC-epsilon. We conclude that PKC-epsilon is the major PKC isoform present in cultured fetal chick neurons. Insulin stimulates PKC-epsilon activity by a mechanism that does not involve translocation of the enzyme from cytosol to membrane.     

Insulin resistance of glycogen synthase mediated by o-linked N-acetylglucosamine

We have investigated the mechanism by which high concentrations of glucose inhibit insulin stimulation of glycogen synthase. In NIH-3T3-L1 adipocytes cultured in low glucose (LG; 2.5 mm), the half-maximal activation concentration (A(0.5)) of glucose 6-phosphate was 162 +/- 15 microm. Exposure to either high glucose (HG; 20 mm) or glucosamine (GlcN; 10 mm) increased the A(0.5) to 558 +/- 61 or 612 +/- 34 microm. Insulin treatment with LG reduced the A(0.5) to 96 +/- 10 microm, but cells cultured with HG or GlcN were insulin-resistant (A(0.5) = 287 +/- 27 or 561 +/- 77 microm). Insulin resistance was not explained by increased phosphorylation of synthase. In fact, culture with GlcN decreased phosphorylation to 61% of the levels seen in cells cultured in LG. Hexosamine flux and subsequent enzymatic protein O-glycosylation have been postulated to mediate nutrient sensing and insulin resistance. Glycogen synthase is modified by O-linked N-acetylglucosamine, and the level of glycosylation increased in cells treated with HG or GlcN. Treatment of synthase in vitro with protein phosphatase 1 increased basal synthase activity from cells cultured in LG to 54% of total activity but was less effective with synthase from cells cultured in HG or GlcN, increasing basal activity to only 13 or 16%. After enzymatic removal of O-GlcNAc, however, subsequent digestion with phosphatase increased basal activity to over 73% for LG, HG, and GlcN. We conclude that O-GlcNAc modification of glycogen synthase results in the retention of the enzyme in a glucose 6-phosphate-dependent state and contributes to the reduced activation of the enzyme in insulin resistance.     

Glucosamine-induced increase in Akt phosphorylation corresponds to increased endoplasmic reticulum stress in astroglial cells

Increased glucose flux through the hexosamine biosynthetic pathway (HBP) is known to affect the activity of a number of signal transduction pathways and lead to insulin resistance. Although widely studied in insulin responsive tissues, the effect of increased HBP activity on largely insulin unresponsive tissues, such as the brain, remains relatively unknown. Herein, we investigate the effects of increased HBP flux on Akt activation in a human astroglial cells line using glucosamine, a compound commonly used to mimic hyperglycemic conditions by increasing HBP flux. Cellular treatment with 8 mM glucosamine resulted in a 96.8% ± 24.6 increase in Akt phosphorylation after 5 h of treatment that remained elevated throughout the 9-h time course. Glucosamine treatment also resulted in modest increases in global levels of the O-GlcNAc protein modification. Increasing O-GlcNAc levels using the O-GlcNAcase inhibitor streptozotocin (STZ) also increased Akt phosphorylation by 96.8% ± 11.0 after only 3 h although for a shorter duration than glucosamine; however, the more potent O-GlcNAcase inhibitors O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2′-propyl-α-d-glucopyranoso-[2,1-d]-Δ2′-thiazoline (NAGBT) failed to mimic the increases in phospho-Akt indicating that the Akt phosphorylation is not a result of increased O-GlcNAc protein modification. Further analysis indicated that this increased phosphorylation was also not due to increased osmotic stress and was not attenuated by N-acetylcysteine eliminating the potential role of oxidative stress in the observed phospho-Akt increases. Glucosamine treatment, but not STZ treatment, did correlate with a large increase in the expression of the endoplasmic reticulum (ER) stress marker GRP 78. Altogether, these results indicate that increased HBP flux in human astroglial cells results in a rapid, short-term phosphorylation of Akt that is likely a result of increased ER stress. The mechanism by which STZ increases Akt phosphorylation, however, remains unknown.     

Protein kinase C (PKC) isoforms translocate to Triton-insoluble fractions in stimulated human neutrophils: correlation of conventional PKC with activation of NADPH oxidase.

The responses of human neutrophils (PMN) involve reorganization and phosphorylation of cytoskeletal components. We investigated the translocation of protein kinase C (PKC) isoforms to PMN cytoskeletal (Triton-insoluble) fractions, in conjunction with activation of the respiratory burst enzyme NADPH oxidase. In resting PMN, PKC-delta (29%) and small amounts of PKC-alpha (0.6%), but not PKC-betaII, were present in cytoskeletal fractions. Upon stimulation with the PKC agonist PMA, the levels of PKC-alpha, PKC-betaII, and PKC-delta increased in the cytoskeletal fraction, concomitant with a decrease in the noncytoskeletal (Triton-soluble) fractions. PKC-delta maximally associated with cytoskeletal fractions at 160 nM PMA and then declined, while PKC-alpha and PKC-betaII plateaued at 300 nM PMA. Translocation of PKC-delta was maximal by 2 min and sustained for at least 10 min. Translocation of PKC-alpha and PKC-betaII was biphasic, plateauing at 2-3 min and then increasing up to 10 min. Under maximal stimulation conditions, PKC isoforms were entirely cytoskeletal associated. Translocation of the NADPH oxidase component p47phox to the cytoskeletal fraction correlated with translocation of PKC-alpha and PKC-betaII, but not with translocation of PKC-delta. Oxidase activity in cytoskeletal fractions paralleled translocation of PKC-alpha, PKC-betaII, and p47phox. Stimulation with 1,2-dioctanoylglycerol resulted in little translocation of PKC isoforms or p47phox, and in minimal oxidase activity. We conclude that conventional PKC isoforms (PKC-alpha and/or PKC-betaII) may regulate PMA-stimulated cytoskeletal association and activation of NADPH oxidase. PKC-delta may modulate other PMN responses that involve cytoskeletal components.     

Opening of mitochondrial K(ATP) channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning

We examined whether the mitochondrial ATP-sensitive K channel (K(ATP)) is an effector downstream of protein kinase C-epsilon (PKC-epsilon) in the mechanism of preconditioning (PC) in isolated rabbit hearts. PC with two cycles of 5-min ischemia/5-min reperfusion before 30-min global ischemia reduced infarction from 50.3 +/- 6.8% of the left ventricle to 20.3 +/- 3.7%. PC significantly increased PKC-epsilon protein in the particulate fraction from 51 +/- 4% of the total to 60 +/- 4%, whereas no translocation was observed for PKC-delta and PKC-alpha. In mitochondria separated from the other particulate fractions, PC increased the PKC-epsilon level by 50%. Infusion of 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) blocker, after PC abolished the cardioprotection of PC, whereas PKC-epsilon translocation by PC was not interfered with 5-HD. Diazoxide, a mitochondrial K(ATP) opener, infused 10 min before ischemia limited infarct size to 5.2 +/- 1.4%, but this agent neither translocated PKC-epsilon by itself nor accelerated PKC-epsilon translocation after ischemia. Together with the results of earlier studies showing mitochondrial K(ATP) opening by PKC, the present results suggest that mitochondrial K(ATP)-mediated cardioprotection occurs subsequent to PKC-epsilon activation by PC.     

PUGNAc treatment leads to an unusual accumulation of free oligosaccharides in CHO cells

Free oligosaccharides (fOS) are generated as the result of N-glycoproteins catabolism that occurs in two distinct principal pathways: the endoplasmic reticulum-associated degradation (ERAD) of misfolded newly synthesized N-glycoproteins and the mature N-glycoproteins turnover pathway. The O-(2-acetamidO-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) is a potent inhibitor of the O-GlcNAcase (OGA) catalysing the cleavage of β-O-linked 2-acetamido-2-deoxy-β-D-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationaly O-GlcNAc modified proteins. In order to estimate the impact of O-GlcNAc modification on N-glycoproteins catabolism, fOS were analysed by mass spectrometry (MS). MS analysis revealed the appearance of an unusual population of fOS after PUGNAc treatment. The structures representing this population have been identified as containing non-reducing end GlcNAc residues resulting from incomplete lysosomal fOS degradation. Only observed after PUGNAc treatment, the NButGt, another OGA inhibitor, did not lead to the appearance of this population. These abnormal fOS structures have clearly been shown to accumulate in membrane fractions as the consequence of lysosomal β-hexosaminidases inhibition by PUGNAc. As lysosomal storage disorders (LSD) are characterized by the accumulation of storage material as fOS in lysosomes, our study evokes that the use of PUGNAc could mimic a LSD. This study clearly points out another off target effects of PUGNAc that need to be taken into account in the use of this drug.     

Inhibition of phospholipase C-beta1-mediated signaling by O-GlcNAc modification

Here we report inhibition of phospholipase C-beta1 (PLC-beta1)-mediated signaling by post-translational glycosylation with beta-N-acetylglucosamine (O-GlcNAc modification). In C2C12 myoblasts, isoform-specific knock-down experiments using siRNA showed that activation of bradykinin (BK) receptor led to stimulation of PLC-beta1 and subsequent intracellular Ca2+ mobilization. In C2C12 myotubes, O-GlcNAc modification of PLC-beta1 was markedly enhanced in response to treatment with glucosamine (GlcNH2), an inhibitor of O-GlcNAase (PUGNAc) and hyperglycemia. This was associated with more than 50% inhibition of intracellular production of IP3 and Ca2+ mobilization in response to BK. Since the abundance of PLC-beta1 remained unchanged, these data suggest that O-GlcNAc modification of PLC-beta1 led to inhibition of its activity. Moreover, glucose uptake stimulated by BK was significantly blunted by treatment with PUGNAc. These data support the notion that O-GlcNAc modification negatively modulates the activity of PLC-beta1.     

Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance

The O-GlcNAc post-translational modification is considered to act as a sensor of nutrient flux through the hexosamine biosynthetic pathway. A cornerstone of this hypothesis is that global elevation of protein O-GlcNAc levels, typically induced with the non-selective O-GlcNAcase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-D-glycopyranosylidene) amino-N-phenylcarbamate), causes insulin resistance in adipocytes. Here we address the potential link between elevated O-GlcNAc and insulin resistance by using a potent and selective inhibitor of O-GlcNAcase (NButGT (1,2-dideoxy-2'-propyl-alpha-D-glucopyranoso-[2,1-D]-Delta 2'-thiazoline), 1200-fold selectivity). A comparison of the structures of a bacterial homologue of O-GlcNAcase in complex with PUGNAc or NButGT reveals that these inhibitors bind to the same region of the active site, underscoring the competitive nature of their inhibition of O-GlcNAcase and the molecular basis of selectivity. Treating 3T3-L1 adipocytes with NButGT induces rapid increases in global O-GlcNAc levels, but strikingly, NButGT treatment does not replicate the insulin desensitizing effects of the non-selective O-GlcNAcase inhibitor PUGNAc. Consistent with these observations, NButGT also does not recapitulate the impaired insulin-mediated phosphorylation of Akt that is induced by treatment with PUGNAc. Collectively, these results suggest that increases in global levels of O-GlcNAc-modified proteins of cultured adipocytes do not, on their own, cause insulin resistance.     

Differential localization of conventional protein kinase C isoforms during mouse oocyte development

Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, plays a central role in signal transduction pathways. In many biological systems where Ca(2+) serves as a second messenger, regulatory control is mediated by PKC. The activation of PKC depends on its binding to RACK1 receptor, which is an intracellular protein anchor for activated PKC. We demonstrate that the conventional PKC (cPKC) isoforms, PKC-alpha, PKC-betaI, and PKC-betaII, as well as RACK1, are expressed in mouse oocytes (germinal vesicle [GV]) and mature eggs (metaphase II [MII]). In GV oocytes, PKC-alpha, PKC-betaII, and RACK1 were uniformly distributed in the cytoplasm, while PKC-betaI was localized in the cytoplasm and in the plasma membrane as well. Treatment of GV oocytes with the biologically active phorbol ester, 12-o-tetradecanoyl phorbol-13-acetate (TPA), resulted in a rapid translocation of the cytosolic PKC-alpha, but not PKC-betaI, PKC-betaII, or RACK1, to the plasma membrane. This was associated with inhibition of GV breakdown. In MII eggs (17 h post-hCG), PKC-alpha was uniformly distributed in the cytoplasm while PKC-betaI and -betaII were distributed in the cytoplasm and in the plasma membrane as well. Treatment with TPA resulted in a rapid translocation of PKC-alpha from the cytoplasm to the plasma membrane and a significant decrease of PKC-betaI throughout the cytoplasm, while it also remained in the cell periphery. No change in the distribution of PKC-betaII or RACK1 was observed. TPA also induced pronucleus formation. Physiological activation of MII eggs by sperm induced cortical granule exocytosis associated with significant translocation of PKC-alpha and -betaI, but not -betaII, to the plasma membrane. Overall, these results suggest a possible involvement of cPKC isoforms in the mechanism of mouse oocyte maturation and egg activation.     

Localization and kinetics of protein kinase C-epsilon anchoring in cardiac myocytes

Protein kinase C-epsilon (PKC-epsilon) plays a central role in cardiac cell signaling, but mechanisms of translocation and anchoring upon activation are poorly understood. Conventional PKC isoforms rely on a rapid Ca2+-mediated recruitment to cell membranes, but this mechanism cannot be employed by PKC-epsilon or other PKC isoforms lacking a Ca2+-binding domain. In this study, we used recombinant green fluorescent protein (GFP) fusion constructs and confocal microscopy to examine the localization, kinetics, and reversibility of PKC-epsilon anchoring in permeabilized rat cardiac myocytes. PKC-epsilon-GFP bound with a striated pattern that co-localized with alpha-actinin, a marker of the Z-line of the sarcomere. Binding required activation of PKC and occurred slowly but reversibly with apparent rate constants of k(on) = 4.6 +/- 1.2 x 10(3) M(-1) x s(-1) and k(off) = 1.4 +/- 0.5 x 10(-3) s(-1) (t1/2 = 8 min) as determined by fluorescence recovery after photobleaching and by perfusion experiments. A truncated construct composed of the N-terminal 144-amino-acid variable region of PKC-epsilon (epsilonV1-GFP), but not an analogous N-terminal domain of PKC-delta, mimicked the Z-line decoration and slow binding rate of the full-length enzyme. These findings suggest that the epsilonV1 domain is important in determining PKC-epsilon localization and translocation kinetics in cardiac muscle. Moreover, PKC-epsilon translocation is not a diffusion-controlled binding process but instead may be limited by intramolecular conformational changes within the V1 domain. The k(off) for epsilonV1-GFP was two- to threefold faster than for full-length enzyme, indicating that other domains in PKC-epsilon contribute to anchoring by prolonging the bound state.     

Isoform-specific induction of PKC-epsilon by high glucose protects heart-derived H9c2 cells against hypoxic injury

We investigated which PKC isoforms are involved in high glucose-induced protection against hypoxic injury. Treatment for 48 h with high glucose (22 mM) markedly increased the expression of PKC- epsilon in the particulate fraction (213+/-22.1% of the control) but had no effect on other types of PKC isoforms, suggesting that the high glucose-induced increase in PKC expression is isoform-specific. The mRNA level for PKC- epsilon was also substantially increased, reaching its peak after 4h of high glucose treatment. The high glucose increased PKC-epsilon activity in the particulate fraction up to 183+/-32.2% of the control. During hypoxia, the amount of PKC-epsilon in the particulate fraction was remarkably diminished in the low glucose-treated cells, but remained at a higher level in high glucose-treated cells. The treatment with epsilon V1-2 (10 microM), a specific inhibitor of PKC epsilon, abolished the protective effect of high glucose against hypoxia. These results suggest that isoform-specific induction of PKC-epsilon is involved in high glucose-induced protection against hypoxic injury in heart-derived H9c2 cells.     

Protein O-GlcNAc modulates motility-associated signaling intermediates in neutrophils

The modification of serine/threonine residues on cytoplasmic and nuclear proteins by N-acetylglucosamine (O-GlcNAc) is suggested to play a role in the regulation of a variety of signal transduction pathways. We have previously shown that glucosamine (GlcNH(2)), a metabolic precursor of O-GlcNAcylation, increases (2)O-GlcNAc and enhances motility in neutrophils. Here, we extend this correlation by showing that a mechanistically distinct means of increasing O-GlcNAc, achieved by inhibition of O-GlcNAc removal with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), increases basal cellular motility and directional migration induced by the chemoattractant formyl-methionine-leucine-phenylalanine (fMLP). Furthermore, we demonstrate that O-GlcNAc modulates the activities of signaling intermediates known to regulate neutrophil movement. GlcNH(2) and PUGNAc increase both the basal and fMLP-induced activity of a central mediator of cellular motility, the small GTPase Rac. Phosphoinositide 3-kinase, an important regulator of Rac activity and neutrophil motility, is shown to regulate the signaling pathway on which GlcNH(2) and PUGNAc act. Rac is an important upstream regulatory element in p38 and p44/42 mitogen-activated protein kinase (MAPK) signaling in neutrophils, and these MAPKs are implicated in chemotactic signal transduction. We show that GlcNH(2) and PUGNAc treatment increases p42/44 and p38 MAPK activities and that these increases are associated with activation of upstream MAPK kinases. These data indicate that O-GlcNAcylation is an important signaling element in neutrophils that modulates the activities of several critical signaling intermediates involved in the regulation of cellular movement.     

Possible molecular targets of halogenated aromatic hydrocarbons in neuronal cells

Halogenated aromatic hydrocarbon including polychlorinated biphenyls (PCBs) are persistent and bioaccumulative environmental toxicants. Although health effects associated with exposure to these chemicals, including motor dysfunction and impairment in memory and learning, have been identified, their molecular site of action is unknown. Previous study from this laboratory demonstrated that, while ortho PCBs perturbed intracellular signaling mechanisms including Ca2+ homeostasis, receptor-mediated inositol phosphate production and translocation of PKC, non-ortho PCBs did not. Since PKC signaling pathway is implicated in the modulation of motor behavior, as well as learning and memory, and the roles of PKC are isoform-specific, we have now studied the effects of two structurally distinct PCBs on isoforms of PKC in cerebellar granule cell culture model. Cells were exposed to 2,2'-dichlorobiphenyl (ortho PCB; 2,2'-DCB) or 4,4'-dichlorobiphenyl (non-ortho PCB; 4,4'-DCB) for 15 min, respectively, and subsequently fractionated and immunoblotted against the selected PKC monoclonal antibodies (alpha, gamma, delta, epsilon, lambda, iota). While 2,2'-DCB induced a translocation of PKC-alpha [cytosol (% control): 54 +/- 12 at 25 microM and 66 +/- 10 at 50 microM; membrane (% control): 186 +/- 37 at 25 microM and 200 +/- 48 at 50 microM] and -epsilon [cytosol (% control): 92 +/- 12 at 25 microM and 97 +/- 15 at 50 microM; membrane (% control): 143 +/- 23 at 25 microM and 192 +/- 24 at 50 microM] from cytosol to membrane fraction in a concentration-dependent manner, 4,4'-DCB had no effects. 2,2'-DCB induced translocation of PKC-alpha was blocked by pretreatment with sphingosine, suggesting a possible role of sphingolipid pathway. Although reports on implication of PKC-gamma with learning and memory are relatively extensive, the expression of this particular isoform in the primary cerebellar granule cells was below the detectable level. PKC-delta, -lambda and -iota were present in these cells, but were not altered by PCB exposure. These results suggest that the effects of 2,2'-DCB on PKC is isoform-dependent and PKC-alpha as well as PKC-epsilon may be target molecules for ortho-PCBs in neuronal cells.     

Hyperglycemia and inhibition of glycogen synthase in streptozotocin-treated mice: role of O-linked N-acetylglucosamine

Glycogen synthase is post-translationally modified by both phosphate and O-linked N-acetylglucosamine (O-GlcNAc). In 3T3-L1 adipocytes exposed to high concentrations of glucose, O-GlcNAc contributes to insulin resistance of glycogen synthase. We sought to determine whether O-GlcNAc also regulates glycogen synthase in vivo. Glycogen synthase activity in fat pad extracts was inhibited in streptozotocin (STZ)-treated diabetic mice. The half-maximal activation concentration for glucose 6-phosphate (A(0.5)) was increased to 830 +/- 120 microm compared with 240 +/- 20 microm in control mice (C, p < 0.01), while the basal glycogen synthase activity (%I-form) was decreased to 2.4 +/- 1.4% compared with 10.1 +/- 1.8% in controls (p < 0.01). Glycogen synthase activity remained inhibited after compensatory insulin treatment. After insulin treatment kinetic parameters of glycogen synthase were more closely correlated with blood glucose (A(0.5), r(2) = 0.70; %I-form, r(2) = 0.59) than insulin levels (A(0.5), r(2) = 0.04; %I-form, r(2) = 0.09). Hyperglycemia also resulted in an increase in the level of O-GlcNAc on glycogen synthase (16.1 +/- 1.8 compared with 7.0 +/- 0.9 arbitrary intensity units for controls, p < 0.01), even though the level of phosphorylation was identical in diabetic and control mice either with (STZ: 2.9 +/- 1.0 and C: 3.2 +/- 0.8) or without (STZ: 12.2 +/- 2.8 and C: 13.8 +/- 3.0 arbitrary intensity units) insulin treatment. In all mice the percent activation of glycogen synthase that could be achieved in vitro by recombinant protein phosphatase 1 (230 +/- 30%) was significantly greater in the presence of beta-d-N-acetylglucosaminidase (410 +/- 60%, p < 0.01). This synergistic stimulation of glycogen synthase due to codigestion by protein phosphatase 1 and beta-d-N-acetylglucosaminidase was more pronounced in STZ-diabetic mice (310 +/- 70%) compared with control mice (100 +/- 10%, p < 0.05). The findings demonstrate that O-GlcNAc has a role in the regulation of glycogen synthase both in normoglycemia and diabetes.     

High glucose-induced Nox1-derived superoxides downregulate PKC-betaII, which subsequently decreases ACE2 expression and ANG(1-7) formation in rat VSMCs

In rat diabetic animal models, ANG(1-7) treatment prevents the development of cardiovascular complications. Angiotensin-converting enzyme (ACE)2 is a major ANG(1-7)-generating enzyme in vascular smooth muscle cells (VSMCs), and its expression is decreased by a prolonged exposure to high glucose (HG), which is reflected by lower ANG(1-7) levels. However, the underlying mechanism of its downregulation is unknown and was the subject of this study. Rat aortic VSMCs were maintained in normal glucose (NG) or HG ( approximately 4.1 and approximately 23.1 mmol/l, respectively) for up to 72 h. Several PKC and NADPH oxidase inhibitors and short interfering (si)RNAs were used to determine the mechanism of HG-induced ACE2 downregulation. Cell lysates were subjected to Western blot analysis, real-time quantitative PCR, and ANG(1-7) radioimmunodetection. At 72 h of HG exposure, ACE2 mRNA, protein, and ANG(1-7) levels were decreased (0.17 +/- 0.01-, 0.47 +/- 0.03-, and 0.16 +/- 0.01-fold, respectively), and the expression of NADPH oxidase subunit Nox1 was increased (1.70 +/- 0.2-fold). The HG-induced ACE2 decrease was reversed by antioxidants and Nox1 siRNA as well as by inhibitors of glycotoxin formation. ACE2 expression was PKC-betaII dependent, and PKC-betaII protein levels were reduced in the presence of HG (0.32 +/- 0.03-fold); however, the PKC-betaII inhibitor CG-53353 prevented the HG-induced ACE2 loss and Nox1 induction, suggesting a nonspecific effect of the inhibitor. Our data suggest that glycotoxin-induced Nox1 expression is regulated by conventional PKCs. ACE2 expression is PKC-betaII dependent. Nox1-derived superoxides reduce PKC-betaII expression, which lowers ACE2 mRNA and protein levels and consequently decreases ANG(1-7) formation.     

Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation

The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-betaII, PKC-gamma, PKC-eta, PKC-mu, and PKC-lambda also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-epsilon and PKC-zeta were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 microM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-gamma and PKC-theta in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.