Pivotal role of protein tyrosine phosphatase 1B (PTP1B) in the macrophage response to pro-inflammatory and anti-inflammatory challenge.

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been suggested as an attractive target to improve insulin sensitivity in different cell types. In the present work, we have investigated the effect of PTP1B deficiency on the response of human and murine macrophages. Using in vitro and in vivo approaches in mice and silencing PTP1B in human macrophages with specific siRNAs, we have demonstrated that PTP1B deficiency increases the effects of pro-inflammatory stimuli in both human and rodent macrophages at the time that decreases the response to alternative stimulation. Moreover, the absence of PTP1B induces a loss of viability in resting macrophages and mainly after activation through the classic pathway. Analysis of early gene expression in macrophages treated with pro-inflammatory stimuli confirmed this exacerbated inflammatory response in PTP1B-deficient macrophages. Microarray analysis in samples from wild-type and PTP1B-deficient macrophages obtained after 24 h of pro-inflammatory stimulation showed an activation of the p53 pathway, including the excision base repair pathway and the insulin signaling pathway in the absence of PTP1B. In animal models of lipopolysaccharide (LPS) and D-galactosamine challenge as a way to reveal in vivo inflammatory responses, animals lacking PTP1B exhibited a higher rate of death. Moreover, these animals showed an enhanced response to irradiation, in agreement with the data obtained in the microarray analysis. In summary, these results indicate that, although inhibition of PTP1B has potential benefits for the treatment of diabetes, it accentuates pro-inflammatory responses compromising at least macrophage viability.

Estrogen receptor beta-subtype selective tetrahydrofluorenones: use of a fused pyrazole as a phenol bioisostere.

Synthesis of a series of fused pyrazole tetrahydrofluorenone analogs which are potent, ERbeta subtype selective ligands is described. Analogs possessing subnanomolar ERbeta binding, greater than 100-fold ERbeta-selectivity, and oral bioavailability are reported.

Peroxisome proliferator-activated receptor-gamma-independent inhibition of macrophage activation by the non-thiazolidinedione agonist L-796,449. Comparison with the effects of 15-deoxy-delta(12,14)-prostaglandin J(2).

The effects of L-796,449 (3-chloro-4-(3-(3-phenyl-7-propylbenzofuran-6-yloxy)propylthio)phenylacetic acid; referred to henceforth as compound G), a thiazolidinedione-unrelated peroxisome proliferator activated-receptor-gamma (PPAR-gamma) agonist, on early signaling in lipopolysaccharide-activated RAW 264.7 macrophages were analyzed and compared with those elicited by 15-deoxy-Delta(12,14)-prostaglandin J(2) and the thiazolidinedione rosiglitazone. Compound G inhibited the activation of nuclear factor kappa B through the impairment of the targeting and degradation of I kappa B proteins and promoted a redistribution of I kappa B alpha and I kappa B beta in the nucleus of activated cells. Compound G inhibited I kappa B kinase (IKK) activity both in vivo and in vitro, suggesting a direct mechanism of interaction between this molecule and the IKK complex. The effect of compound G on IKK activity was independent of PPAR-gamma engagement because RAW 264.7 cells expressed negligible levels of this nuclear receptor, and rosiglitazone failed to mimic these actions. Moreover, treatment of activated macrophages with compound G enhanced the synthesis of superoxide anion, which, in combination with the NO produced under activation conditions, triggered apoptosis through the intracellular synthesis of peroxynitrite. These results suggest that compound G might contribute to the resolution of inflammation by favoring the induction of apoptosis through mechanisms independent of PPAR-gamma engagement.

Protection by nitric oxide against liver inflammatory injury in animals carrying a nitric oxide synthase-2 transgene.

The effect of pre-existent hepatic NO synthesis on liver injury induced by lipopolysaccharide was studied in animals carrying a nitric oxide synthase-2 (NOS-2) transgene under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter. These animals expressed NOS-2 in liver cells under fasting conditions. Lipopolysaccharide-induced liver injury in D-galactosamine-conditioned mice, which enhanced notably the effect of the endotoxin on the liver, was impaired in animals expressing NOS-2. This protection against inflammatory liver damage was dependent on NO synthesis and was caused by an inhibition of nuclear factor kB (NF-kB) activity and an impairment of the synthesis of the proinflammatory cytokines tumor necrosis factor a and interleukin 1b. These data indicate that intrahepatic synthesis of NO protects liver by inhibiting the release of cascades of proinflammatory mediators and suggest a beneficial role for local delivery of NO in the control of liver injury.

Resorcylic acid lactones: naturally occurring potent and selective inhibitors of MEK.

A resorcylic acid lactone, L-783,277, isolated from a Phoma sp. (ATCC 74403) which came from the fruitbody of Helvella acetabulum, is a potent and specific inhibitor of MEK (Map kinase kinase). L-783,277 inhibits MEK with an IC50 value of 4 nM. It weakly inhibits Lck and is inactive against Raf, PKA and PKC. L-783,277 is an irreversible inhibitor of MEK and is competitive with respect to ATP. L-783,290, the trans-isomer of L-783,277, was isolated from the same culture and evaluated together with several semi-synthetic resorcylic acid lactone analogs. A preliminary structure-activity relationship is presented. Several independent cell-based assays have been carried out to study the biological activities of these resorcylic acid lactone compounds and a brief result summary from these studies is presented.

A novel antiviral agent which inhibits the endonuclease of influenza viruses.

A novel anti-influenza virus compound, flutimide, was identified in extracts of a recently identified fungal species, Delitschia confertaspora (F. Pelaez, J.D. Polishook, M. Valldosera, and J.Guarro, Mycotaxon 50:115-122, 1994). The compound, a substituted 2,6-diketopiperazine, selectively inhibited the cap-dependent transcriptase of influenza A and B viruses and had no effect on the activities of other polymerases. Similar to the 4-substituted 2,4-dioxobutanoic acids, a series of transcriptase inhibitors which we described previously (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J.Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994), this inhibitor, which is a natural product, affected neither the initiation nor the elongation of influenza virus mRNA synthesis, but it specifically targeted the cap-dependent endonuclease of the transcriptase. Additionally, the compound was inhibitory to the replication of influenza A and B viruses in cell culture. The selective antiviral properties of this compound further demonstrate the utility of influenza virus endonuclease as a target of antiviral agents.

Discovery of an angiotensin II binding inhibitor from a Cytospora sp. using semi-automated screening procedures.

Cytosporin A, B and C, three antagonists of [125I]-angiotensin II binding to rat adrenal glands were discovered in fermentations of an endophytic Cytospora sp. during routine screening using semi-automated procedures. The most potent of these displayed an IC50 of 1.5-3 microM and was specific for angiotensin II AT2.

Inhibition of 6-phosphofructo-2-kinase activity by mercaptopurines.

The activity of 6-phosphofructo-2-kinase (PFK-2), the enzyme that catalyses the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), was inhibited by mercaptopurines in vitro. Inhibition was observed with the purified enzyme from rat liver and bovine heart, and in extracts from rat lymphocytes and hepatoma cells, chick embryo fibroblasts, and human HeLa and lymphoblastoid cells. Half-maximal effect was obtained with 0.1-0.2 mM mercaptopurine and maximal inhibition ranged between 50 and 90% depending on the enzyme preparation. The inhibition resulted from a decrease in Vmax with no change in Km for ATP. The inhibition was relieved by treatment of the enzyme with thiol reducing agents, suggesting that it involves the formation of a mixed disulfide between mercaptopurine and thiol group(s) essential for enzyme activity. Incubation of intact lymphocytes or lymphoblastoid cells with 2- or 6-mercaptopurine resulted in a decrease in Fru-2,6-P2 content and lactate release. A decrease in Fru-2,6-P2 content but no change in lactate release was observed in HeLa cells and fibroblasts treated with 6-mercaptopurine but not with 2-mercaptopurine. Treatment of HeLa cells with 6-mercaptopurine resulted in a decreased PFK-2 activity which could be restored by treatment of the cell extract with dithiothreitol. In isolated rat hepatocytes and perfused rat hearts mercaptopurines had little or no effect on the Fru-2,6-P2 content and lactate release. These results suggest that the effect of 6-mercaptopurine of arresting growth in lymphoid cells might involve the inhibition of glycolysis in addition to the known inhibition of de novo purine nucleotide synthesis.

Identification of an anti-A and anti-B blood group glycosyltransferase antibody after incompatible bone marrow transplant.

The occurrence of a potent antibody against plasmatic A and B glycosyltransferase activities has been characterized in a patient (blood group A1) transplanted with a bone marrow from a blood group O donor. A and B glycosyltransferases were purified to near homogeneity from plasma of A1 and B blood-group individuals. The half-maximal inhibition of both enzymes was obtained at 1 to 2 micrograms/mL of the post-transplant IgG fraction, prepared by protein A-sepharose chromatography. A and B glycosyltransferases were also recognized by the post-transplant IgG fraction after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrophoretic transfer to nitrocellulose membranes.

Oleate-induced translocation of protein kinase C to hepatic microsomal membranes.

The incubation of rat liver homogenates in the presence of oleate induces the translocation of protein kinase C from the cytosol to the endoplasmic reticulum membranes. The half-maximal effect was obtained at 0.3 mM oleate. The redistribution of this enzyme induced by oleate was also obtained with purified protein kinase C and hepatic microsomal membranes. This effect seems to be mediated by long-chain fatty acids since translocation was not obtained with esterified derivatives.

Effect of sulfonylureas on hepatic glycogen metabolism: activation of glycogen phosphorylase.

In hepatocytes isolated from fed rats, both tolbutamide and glipizide caused a dose-dependent activation of glycogen phosphorylase, possibly by a Ca2+-mediated mechanism. Maximal effects (about twofold) were already obtained when drugs were used at 0.5 mmol/L, the calculated concentrations of tolbutamide and glipizide responsible for the half-maximal effects being 60 and 30 mumol/L, respectively. The activation of glycogen phosphorylase caused the mobilization of glycogen and increased the cellular concentration of hexose 6-phosphates (glucose 6-phosphate plus fructose 6-phosphate) and that of fructose 2,6-bisphosphate. Under the influence of sulfonylureas, glucose formation was slightly stimulated while the rate of L-lactate production was more markedly incremented, indicating that sulfonylureas canalize the metabolic flux coming from glycogen mainly to the glycolytic pathway. These results suggest that a glycogenolytic action of sulfonylureas could collaborate to raise hepatic fructose 2,6-bisphosphate concentration in the fed animal.

Presence of antibody to A- and B-transferases in minor incompatible bone marrow transplant.

The contribution of the bone marrow to plasma A- or B-transferase activities has been studied in patients who underwent incompatible bone marrow transplantation (BMT). As deduced from major incompatibility (group O recipient/A donor), the contribution of the marrow to these plasma activities was c. 5-10% of the total activity. In cases of minor incompatible transplants (A recipient/O donor), normal plasma activity was present in two patients, while no activity was found in a further two in whom a potent antitransferase was detected. The antibody inhibited both A- and B-transferase activities to a high titre. The patients in whom this antibody arose exhibited severe graft-versus-host disease.

Tolbutamide enhances insulin action on gluconeogenesis and on fructose 2,6-bisphosphate levels in isolated rat hepatocytes.

In hepatocytes isolated from fed rats and incubated either under basal conditions or in the presence of glucagon, tolbutamide reduced gluconeogenesis from (U-14C) pyruvate by increasing the cellular concentration of fructose 2,6-bisphosphate. Furthermore, this sulfonylurea enhanced the inhibitory action of insulin on glucagon-stimulated gluconeogenesis; this effect was accompanied by a more marked increase of the cellular concentration of fructose 2,6-bisphosphate than that elicited by either insulin or the sulfonylurea alone. In connection with this, tolbutamide--without significant modification of cellular cyclic AMP levels--raised the proportion of 6-phosphofructo 2-kinase in active form in hepatocytes incubated either under basal conditions or in the presence of glucagon, and reinforced the action of insulin in antagonizing the glucagon-mediated inactivation of this enzyme.

Phorbol 12,13-dibutyrate and mitogens increase fructose 2,6-bisphosphate in lymphocytes. Comparison of lymphocyte and rat-liver 6-phosphofructo-2-kinase.

The influence of tumour promoters and growth factors on glycolysis and on fructose-2,6-bisphosphate concentration was studied in isolated mouse spleen lymphocytes and in purified B-cells. The intracellular concentration of fructose 2,6-bisphosphate and the rate of lactate release were increased 2-3-fold in spleen lymphocytes exposed to active phorbol esters, mitogenic lectins, interleukin 4 or lipopolysaccharide. The maximal effect was observed after 1 h of exposure. In these cells hexose 6-phosphates increased 2-fold and 6-phosphofructo-2-kinase activity remained unchanged after treatment with phorbol 12,13-dibutyrate or with lectins. Exposure of B-cells to phorbol 12,13-dibutyrate, interleukin 4 or lipopolysaccharide increased the glycolytic flux and the concentration of fructose 2,6-bisphosphate without relation to their mitogenic activity. Lymphocytes and rat liver 6-phosphofructo-2-kinase were partially purified using the same procedure. The lymphocyte enzyme was not inhibited by sn-glycerol 3-phosphate in contrast to the potent inhibition observed in liver. Treatment of both enzymes with the catalytic subunit of the cyclic-AMP-dependent protein kinase failed to inactivate 6-phosphofructo-2-kinase from lymphocytes. These differences suggest that lymphocytes and liver contain different forms of this enzyme.

Presence of an inhibitor of glycosyltransferase activity in a patient following an ABO incompatible liver transplant.

The contribution of the liver to plasma ABO glycosyltransferase activity has been studied in a group O individual transplanted with a liver from a group B donor. The B transferase activity present in the post-transplantation plasma was negligible. However, a potent B transferase inhibitor, absent from the pretransplantation plasma, was present after transplantation. The inhibitor was present in the excluded fraction following Sephadex G-25 gel filtration, but was retained by a protein A-Sepharose column, suggesting that it was an IgG antibody. This inhibitor was also effective in reducing A transferase activity.

Expression of the v-src or v-fps oncogene increases fructose 2,6-bisphosphate in chick-embryo fibroblasts. Novel mechanism for the stimulation of glycolysis by retroviruses.

The concentration of fructose 2,6-bisphosphate and the activity of 6-phosphofructo-2-kinase are increased after infection of chick-embryo fibroblasts with the Rous sarcoma virus, or with a temperature-sensitive mutant of this virus at the permissive, but not at the non-permissive, temperature. This is observed after transformation by retroviruses carrying either the v-src or v-fps, but not the v-mil and/or v-myc, oncogenes. Comparison of the effects of the Rous sarcoma virus with those of phorbol myristate acetate on fructose 2,6-bisphosphate suggests that both result from the stimulation of a step which is rate-limiting for 6-phosphofructo-2-kinase activation and which is also controlled by protein kinase C.

Chlorpropamide raises fructose-2,6-bisphosphate concentration and inhibits gluconeogenesis in isolated rat hepatocytes.

The addition of chlorpropamide to hepatocytes isolated from fed rats raised the cellular concentration of fructose-2,6-bisphosphate (F-2,6-P2), a regulatory metabolite that plays a relevant role in the control of hepatic glucose metabolism. The effect of chlorpropamide was dose dependent; a statistically significant increase was already seen at 0.2 mM of the sulfonylurea. The accumulation of F-2,6-P2 caused by chlorpropamide (1 mM) was parallel to the stimulation of L-lactate production (36.6 +/- 4.8 versus 26.1 +/- 2.6 mumol of lactate/g of cells X 20 min; N = 5, P less than 0.05) and to the inhibition of gluconeogenesis (0.57 +/- 0.1 versus 0.94 +/- 0.09 mumol of [U-14C]pyruvate converted to glucose/g of cells X 20 min; N = 5, P less than 0.05). In addition, chlorpropamide enhanced the inhibitory action evoked by insulin on glucagon-stimulated gluconeogenesis. This combined effect of chlorpropamide and insulin seems to be correlated with the synergistic accumulation of F-2,6-P2 provoked by the simultaneous action of these two agents on glucagon-treated hepatocytes. Finally, neither 6-phosphofructo-2-kinase activity nor hepatocyte cyclic AMP levels were significantly changed by the presence of the sulfonylurea in the incubation medium. Our results support the concept that chlorpropamide, by a cyclic AMP-independent mechanism, increases the hepatic content of F-2,6-P2 and, in this way, enhances the glycolytic flux and inhibits glucose output by the liver.

Stimulation of pyruvate kinase phosphatase activity by insulin in isolated rat hepatocytes.

Addition of insulin (10(-8)M) to hepatocytes, incubated either in the absence or in the presence of a suboptimal concentration of glucagon, caused the reactivation of pyruvate kinase and simultaneously provoked a transient stimulation of pyruvate kinase phosphatase activity (40-70% over control values). The stimulatory effect of insulin on pyruvate kinase phosphatase activity was dose-dependent (ED50 = 1 to 2 X 10(-11)M) and persisted after Sephadex G-25 filtration or ammonium sulfate precipitation of hepatocytes extracts. Our results demonstrate that insulin exerts a short-term regulation on hepatic pyruvate kinase phosphatase activity.

Effect of glutamine on fructose 2,6-bisphosphate and on glucose metabolism in HeLa cells and in chick-embryo fibroblasts.

Glutamine caused a dose-dependent decrease in fructose 2,6-bisphosphate concentration in both HeLa cells and chick-embryo fibroblasts. The effect was complete within 15 min in HeLa cells, but required more than 9 h in the fibroblasts. Half-maximal effects were obtained with 0.1-0.3 mM-glutamine. In chick-embryo fibroblasts, but not in HeLa cells, glutamine induced a time-dependent decrease in the activity of phosphofructokinase-2, which correlated with the decrease in fructose 2,6-bisphosphate. Glutamine decreased the glycolytic flux by about 25% only in chick-embryo fibroblasts. The difference in glycolytic response between the two types of cells might correspond to a difference in the sensitivity of phosphofructokinase-1 for fructose 2,6-bisphosphate. In HeLa cells, glutamine caused a 2-3-fold stimulation of the synthesis of glycogen, a 50% decrease in the concentration of fructose 1,6-bisphosphate and a more than 80% decrease in the concentration of 5-phosphoribosyl pyrophosphate; the concentrations of hexose 6-phosphates and ATP were not affected.

Stimulatory effect of vasoactive intestinal peptide on glycogenolysis and gluconeogenesis in isolated rat hepatocytes: antagonism by insulin.

We have studied the effect of Vasoactive Intestinal Peptide (VIP) on glycogenolysis and gluconeogenesis (as measured by the conversion of [U-14C]pyruvate into glucose) in hepatocytes isolated from fed rats. The influence of VIP on glycogen phosphorylase alpha and pyruvate kinase activities, as well as on cAMP levels, was also evaluated. In addition, the possible antagonism of insulin on these VIP-mediated effects was investigated. VIP enhanced both glycogenolysis and gluconeogenesis in a dose-dependent manner. At 10(-6) M VIP, both processes were increased 2-fold as compared to the basal values; the calculated half-maximal stimulatory concentrations were 2.5 x 10(-8) M and 4 x 10(-8) M, respectively. VIP also caused a dose-dependent activation of glycogen phosphorylase and inactivation of pyruvate kinase. At 10(-6) M VIP, glycogen phosphorylase a was increased 3-fold and pyruvate kinase activity was reduced by 46%. The addition of 10(-7) M VIP to the incubation medium caused a 2-fold increase of basal cAMP levels. All these VIP-mediated effects were markedly blocked by the presence of 10(-8) M insulin. As compared to glucagon (10(-7) M) the potency of an equimolar concentration of VIP, in terms of stimulation of gluconeogenesis, inactivation of pyruvate kinase, and activation of glycogen phosphorylase ranged from 35-45%. Our results indicate that VIP increases hepatic glucose output through the stimulation of both glycogenolysis and gluconeogenesis. These effects seem to be mediated by a cAMP-dependent mechanism.