Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin.

Insulin stimulates the activity of mitogen-activated protein kinase (MAPK) via its upstream activator, MAPK kinase (MEK), a dual specificity kinase that phosphorylates MAPK on threonine and tyrosine. The potential role of MAPK activation in insulin action was investigated with the specific MEK inhibitor PD98059. Insulin stimulation of MAPK activity in 3T3-L1 adipocytes (2.7-fold) and L6 myotubes (1.4-fold) was completely abolished by pretreatment of cells with the MEK inhibitor, as was the phosphorylation of MAPK and pp90Rsk, and the transcriptional activation of c-fos. Insulin receptor autophosphorylation on tyrosine residues and activation of phosphatidylinositol 3'-kinase were unaffected. Pretreatment of cells with PD98059 had no effect on basal and insulin-stimulated glucose uptake, lipogenesis, and glycogen synthesis. Glycogen synthase activity in extracts from 3T3-L1 adipocytes and L6 myotubes was increased 3-fold and 1.7-fold, respectively, by insulin. Pretreatment with 10 microM PD98059 was without effect. Similarly, the 2-fold activation of protein phosphatase 1 by insulin was insensitive to PD98059. These results indicate that stimulation of the MAPK pathway by insulin is not required for many of the metabolic activities of the hormone in cultured fat and muscle cells.

Stimulation of glycogen synthesis by insulin in human erythroleukemia cells requires the synthesis of glycosyl-phosphatidylinositol.

Although the insulin-dependent hydrolysis of glycosyl-phosphatidylinositol (GPI) may play an important role in insulin action, an absolute requirement for this glycolipid has not been demonstrated. Human K562 cells were mutated to produce a cell line (IA) incapable of the earliest step in PI glycosylation, the formation of PI-GlcNAc. Another cell line (IVD) was deficient in the deacetylation of PI-GlcNAc to form PI-GlcN and subsequent mannosylated species. Each line was transfected with wild-type human insulin receptors. Similar insulin-stimulated receptor autophosphorylation was observed in all three lines, along with a nearly identical increase in the association of phosphorylated insulin receptor substrate 1 with endogenous PI 3-kinase. Both normal and GPI-defective lines also displayed a similar 2- to 3-fold increase in phosphorylation of the Shc protein and its association with growth factor receptor-bound protein 2 in response to insulin. In contrast to these results, striking differences were noted in insulin-stimulated glycogen synthesis. In normal cells, glycogen synthesis was significantly increased by insulin, whereas no insulin stimulation was observed in GPI-deficient IA cells, and only a trace of stimulation was detected in IVD cells. These results indicate that tyrosine phosphorylation produced by insulin is not dependent on GPI synthesis, and this effect is not sufficient to elicit at least some of the metabolic effects of the hormone. In contrast, GPI synthesis is required for the stimulation of glycogen synthesis by insulin in these cells. These findings support the existence of divergent pathways in the action of insulin.

Difference between the calcium- and the phorbol ester-induced association of protein kinase C with phospholipid membrane.

1. Membrane association of protein kinase C is thought to be a prerequisite for the activation of the enzyme. 2. We studied the association of the enzyme with liposome. 3. We show here that the mechanisms for the calcium- and the phorbol ester-induced association of protein kinase C with liposome are different from each other. 4. Diacylglycerol is not crucial for the association of the enzyme with liposome.

Substitution by fatty acids for phosphatidylserine in a reconstitution of phorbol ester binding to protein kinase C.

1. Fatty acids can be substituted or phosphatidylserine in a reconstitution of phorbol ester binding to protein kinase C. 2. Phorbol ester, however, does not seem to be effectively utilized for the activation of the enzyme. 3. It is suggested that fatty acids play a role on the activation of protein kinase C in the abnormal conditions such as ischemia, while the phospholipid-dependent activation has a physiological significance in normal conditions.

Protein kinase C and simulated ischemia possible aberrations of signal transduction during ischemia.

ATP depletion is always associated with prolonged ischemia. It was found that ATP affected calcium- and phospholipid-dependent activation of protein kinase C without hydrolysis of the nucleotide when the activation was monitored by an assay for [3H] 4-beta-phorbol-12, 13-dibutyrate binding activity in a reconstitution system having physiological concentrations of free calcium. When the ATP level was low, an increase in the free calcium concentration could not activate the enzyme. A decrease in pH exacerbated the depressed activation. The concentration of magnesium also affected the activation. On the other hand, free fatty acids, which increase during ischemia, were able to activate the enzyme at a low concentration of ATP in the absence of phorbol ester and phosphatidylserine. These results suggest that calcium- and phospholipid-dependent activation of protein kinase C is suppressed during ischemia, and that fatty acids in turn activate the enzyme. It is possible that ischemia interferes with normal signal transduction via the protein kinase C pathway and causes unusual protein phosphorylation.

In search of a second messenger for insulin.

Despite significant advances in the past few years on the chemistry and biology of insulin and its receptor, the molecular events that couple the insulin-receptor interaction to the regulation of cellular metabolism remain uncertain. Progress in this area has been complicated by the pleiotropic nature of insulin's actions. These most likely involve a complex network of pathways resulting in the coordination of mechanistically distinct cellular effects. Because the well-recognized mechanisms of signal transduction (i.e., cyclic nucleotides, ion channels) appear not to be central to insulin action, investigators have searched for a novel second messenger system. A low-molecular-weight substance has been identified that mimics certain actions of insulin on metabolic enzymes. This substance has an inositol glycan structure and is produced by the insulin-sensitive hydrolysis of a glycosyl-phosphatidylinositol in the plasma membrane. This hydrolysis reaction, which is catalyzed by a specific phospholipase C, also results in the production of a structurally distinct diacylglycerol that may selectively regulate one or more of the protein kinases C. The glycosyl-phosphatidyl-inositol precursor for the inositol glycan enzyme modulator is structurally analogous to the recently described glycosyl-phosphatidylinositol membrane protein anchor. Preliminary studies suggest that a subset of proteins anchored in this fashion might be released from cells by a similar insulin-sensitive, phospholipase-catalyzed reaction. Efforts are underway to determine the precise role of the metabolism of glycosyl-phosphatidylinositols in insulin action.

The function of glycosyl phosphoinositides in hormone action.

The molecular events involved in the cellular actions of insulin remain unexplained. Some of the acute actions of the hormone may be due to the intracellular generation of a chemical substance which modulates certain enzyme activities. Such an enzyme-modulating substance has been identified as an inositol phosphate-glycan, produced by the insulin-sensitive hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) precursor. This precursor glycolipid is structurally similar to the glycosyl-phosphoinositide membrane protein anchor. The exposure of fat, liver or muscle cells to insulin results in the hydrolysis of glycosyl-PtdIns, giving rise to the inositol phosphate glycan and diacylglycerol. This hydrolysis reaction is catalysed by a glycosyl-PtdIns-specific phospholipase C. This enzyme has been characterized and purified from a plasma membrane fraction of liver. This reaction also results in the acute release of certain glycosyl-PtdIns-anchored proteins from the cell surface. Elucidation of the functional role of glycosyl-phosphoinositides in the generation of second messengers or the release of proteins may provide further insights into the pleiotropic nature of insulin action.

Chemical identification of a tumor-derived angiogenic factor.

Neoplasms produce substances that induce blood vessel formation (angiogenesis). Fractions from ethanol extracts of the Walker 256 carcinoma were isolated by silica column chromatography and C18 reversed-phase high-performance liquid chromatography. Two of the isolated fractions induced neovascularization when tested in the rabbit corneal micropocket assay. One of the fractions was identified as nicotinamide by desorption-electron impact mass spectrometry, nuclear magnetic resonance spectroscopy, and gas chromatography-mass spectrometry. The second active fraction contained nicotinamide as part of a more complex, as yet unidentified, molecular arrangement. Microgram quantities of commercial nicotinamide induced neovascularization in the corneal micropocket assay and in the chick chorioallantoic membrane assay.

Characterization of a neuronal subtype of insulin-like growth factor I receptor.

Primary neuronal cultures from fetal rat brain were utilized to investigate the possible role of insulin-like growth factor I (IGF-I) in neuronal growth and differentiation. 125I-IGF-I binding to intact cultured neurons was specific and saturable with an apparent Kd of 7.0 +/- 1.2 nM and a Bmax of 1.8 +/- 0.3 pmol/mg protein. Binding of 125I-IGF-I to neurons was inhibited by IGF-I, followed by IGF-II and insulin. 7 S nerve growth factor, but not beta-nerve growth factor, also inhibited 125I-IGF-I binding. A similar binding site was detected on brain membranes. Affinity cross-linking of 125I-IGF-I to intact cultured neurons revealed, under reducing conditions, a major binding moiety with an Mr of 115,000 and a minor component at Mr 260,000. The former represents a neuronal type of the IGF-I receptor alpha subunit, whereas the latter probably represents an alpha dimer. The Mr = 115,000 binding component for 125I-IGF-I was also present in membranes prepared from postnatal whole brain. In contrast, the binding moiety in cultured glial cells was of Mr = 135,000, which was identical to the IGF-I receptor alpha subunit of placenta. Thus mature brain, despite its cellular heterogeneity, expresses a structural subtype of IGF-I receptor which appears to be unique to differentiated neurons. Moreover, glial and neuronal cultures secreted a polypeptide which specifically bound IGF-I; the apparent Mr of this binding protein was determined by affinity cross-linking to be approximately 35,000. The presence of neuronal IGF-I receptors and binding proteins suggested that IGF-I may exert neurotrophic effects on developing neurons. This possibility was supported by the observation that IGF-I markedly stimulated neuronal RNA synthesis.

Purification of phosphatidylinositol kinase from bovine brain myelin.

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.

Diacylglycerol-induced translocation of diacylglycerol kinase: use of affinity-purified enzyme in a reconstitution system.

Diacylglycerol-induced translocation of diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from the soluble to the membrane-bound compartments was demonstrated both in crude tissue homogenates and in a reconstituted enzyme-membrane model system. In homogenates of either rat brain or liver, incubation with diacylglycerol or phospholipase C, but not phospholipase A2 or phospholipase D, resulted in the translocation of diacylglycerol kinase activity from the soluble to the particulate fraction. This observation formed the basis for the first step in a two-step purification of diacylglycerol kinase. Enzyme extracted in 1 M salt from membranes of rat brain homogenates made in the presence of phospholipase C was purified further by affinity chromatography on a column containing phosphatidylserine, diacylglycerol, and cholesterol immobilized in polyacrylamide. This step yielded an enzyme preparation (step 2 enzyme) that was 500- to 750-fold purified (relative to the tissue homogenate) and required phosphatidylserine for stability. All other lipids tested failed to stabilize the enzyme. The properties of the enzyme preparation were similar to those of mammalian diacylglycerol kinases described by others. Reconstitution experiments showed that the soluble step 2 enzyme bound to inside-out vesicles of human erythrocytes only in the presence of diacylglycerol or phospholipase C but not phospholipase A2 or D. Redistribution of the kinase from soluble to vesicle-bound forms occurred rapidly and was dependent on the concentration of phospholipase C used to treat the vesicles. Physiological concentrations of calcium (50-1000 nM) did not enhance the phospholipase C-mediated translocation of the kinase. Thus, diacylglycerol kinase can translocate from cytosol to membranes in a manner dependent on the content of membrane-bound diacylglycerol but independent of the ambient concentration of calcium.

Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger.

The classic pathway for agonist-induced generation of diacylglycerol is via activation of a phospholipase C-mediated hydrolysis of the "phosphoinositides." We now report findings from a variety of cell types, which indicate that tumor-promoting phorbol diesters, serum, and platelet-derived growth factor activate within seconds the hydrolysis of phosphatidylcholine, as detected by the formation of diacylglycerol and phosphocholine. It is known that phorbol diesters do not stimulate hydrolysis of the phosphoinositides. Yet, in cells prelabeled with either [14C]oleate or [32P]orthophosphate, addition of the tumor promoter phorbol dibutyrate (PBt2) resulted in the rapid generation of both diacylglycerol and phosphatidate in a time- and dose-dependent manner. The fatty acid composition of the phosphatidate most resembled the fatty acid profile of phosphatidylcholine from the same cell type. Taken together, these findings suggested a role for protein kinase C in the generation of diacylglycerol (and phosphatidate) from phosphatidylcholine. To define further the pathways involved, the metabolism of cellular phosphatidylcholine was studied. In cells prelabeled with [3H]choline, addition of PBt2, but not 4 alpha-phorbol, stimulated the formation of intracellular phosphocholine within 45 sec. Furthermore, addition of platelet-derived growth factor (PDGF) or serum to "serum-starved" cells prelabeled with [3H]choline resulted in increased levels of intracellular phosphocholine within 15-30 sec. Thus, the data suggest that agonists that stimulate protein kinase C either directly (e.g., PBt2) or indirectly via activation of phosphoinositide hydrolysis (e.g., PDGF and serum) may stimulate degradation of phosphatidylcholine by phospholipase C in intact cells. However, prior down-regulation of protein kinase C by prolonged pretreatment of cells with PBt2 almost totally abolished subsequent stimulation of phosphatidylcholine degradation by PBt2 but only partially attenuated subsequent stimulation by PDGF and serum. These observations suggest that PDGF and serum act, at least partially, through a protein kinase C-independent mechanism. Lastly, the size of the cellular choline and CDP-choline pools were shown to be small and relatively insensitive to agonist addition, as compared to the size and behavior of the phosphocholine pool. Thus, the rapidly increased levels of phosphocholine (and diacylglycerol) arising in response to agonist addition appear to be derived directly from phosphatidylcholine by a phospholipase C-mediated mechanism.

Specific postsynaptic density proteins bind tubulin and calmodulin-dependent protein kinase type II.

Cytoskeletal interactions which contribute to the assembly of the postsynaptic density (PSD) were investigated. PSDs bound 125I-tubulin specifically with an apparent Km of 2 X 10(-7) M and a Bmax of about 1 nmol/mg of protein. 125I-Tubulin blots revealed that a group of polypeptides between Mr 135,000 and 147,000 (P-140) was a major tubulin-binding PSD component. The P-140 polypeptides were highly enriched in the PSD fraction of purified synaptosomes and could not be detected in crude brain cytoplasm preparations. These polypeptides were subject to phosphorylation by endogenous calmodulin-dependent protein kinase type II, with a concomitant reduction in 125I-tubulin binding. The tubulin-binding polypeptides could also associate with the radiolabeled alpha- and beta-subunits of the calmodulin-dependent protein kinase. These observations are consistent with a role for the P-140 polypeptides in organizing the molecular structure of the PSD. The data also suggest that this structure may be modified by Ca2+-sensitive phosphorylation, thus permitting neuronal activity to modulate the cytoskeletal interactions of the PSD.

Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid.

Insulin binding to plasma membrane receptors results in the generation of substances that acutely mimic the actions of the hormone on certain target enzymes. Two such substances, which modulate the activity of the high-affinity cAMP phosphodiesterase (EC 3.1.4.17), have been purified from hepatic plasma membranes. The two have similar properties and activities but can be resolved by ion-exchange chromatography and high-voltage electrophoresis. They exhibit a net negative charge, even at pH 1.9, and an apparent molecular weight of approximately 1400. The generation of these substances from membranes by insulin can be reproduced by addition of a phosphatidylinositol-specific phospholipase C purified from Staphylococcus aureus. This enzyme is known to selectively hydrolyze phosphatidylinositol and release from membranes several proteins that are covalently linked to phosphatidylinositol by a glycan anchor. Both enzyme-modulating substances appear to be generated by the phosphodiesterase cleavage of a phosphatidylinositol-containing glycolipid precursor that has been characterized by thin-layer chromatography. Some of the chemical properties of these substances have been examined. They appear to be related complex carbohydrate-phosphate substances containing glucosamine and inositol. These findings suggest that insulin may activate a selective phospholipase activity that hydrolyzes a membrane phospholipid, releasing a carbohydrate-containing molecule that regulates cAMP phosphodiesterase and perhaps other insulin-sensitive enzymes.

Insulin-stimulated hydrolysis of a novel glycolipid generates modulators of cAMP phosphodiesterase.

Insulin action may involve the intracellular generation of low molecular weight substances that modulate certain key enzymes. The production of two substances that regulate the activity of adenosine 3',5'-monophosphate phosphodiesterase was evaluated in cultured myocytes by incorporation of radiolabeled precursors. Insulin caused the rapid hydrolysis of a chemically undefined membrane glycolipid, resulting in the production of two related complex carbohydrates as well as diacylglycerol. Both the glycolipid precursor and the aqueous products were monitored by labeling with radioactive inositol and glucosamine. Depletion of the labeled precursor and the appearance of labeled water-soluble products and diacylglycerol occurred within 30 seconds after hormone treatment and was followed by rapid resynthesis of the precursor. The aqueous products that were radioactively labeled appeared chromatographically and electrophoretically identical to phosphodiesterase modulating activities produced by insulin from the same cells. The purified radiolabeled and bioactive substances had similar chemical properties. Hydrolysis of the glycolipid precursor and subsequent generation of products could be reproduced by incubation of extracted lipids with a phosphatidylinositol-specific phospholipase C. These studies suggest that insulin stimulates an endogenous, selective phospholipase C activity that hydrolyzes a novel glycolipid, resulting in the generation of a complex carbohydrate-phosphate substance containing inositol and glucosamine that may mediate some of the actions of the hormone.

Purification of insulin-like growth factor I receptor from human placental membranes.

Insulin-like growth factor (IGF) I receptor was purified from Triton X-100-solubilized human placental membranes by wheat germ agglutinin-Sepharose chromatography followed by immunoaffinity chromatography using alpha IR-3, a monoclonal antibody directed against the IGF-I receptor. Purification of 3200-fold and 2800-fold was achieved from wheat germ agglutinin-Sepharose eluates with regard to IGF-I binding and kinase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed two major protein bands corresponding to the alpha and beta subunits of the receptor, which accounted for at least 90% of the protein content. The purified receptor bound 10-20 micrograms of IGF-I/mg of protein and was more than 95% free of contamination by insulin receptor. It sedimented in glycerol gradients as a single species with a sedimentation coefficient of 13.7 S and gave three protein bands with Mr = approximately 300,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that alpha 2 beta 2 is an intact form of the IGF-I receptor. The purified receptor, when incubated with [gamma-32P] ATP, became phosphorylated at tyrosine residues of its beta subunit. This was stimulated 3-fold by IGF-I. It also had IGF-I-stimulated tyrosine kinase activity (5264 pmol of 32P incorporated/min/mg of protein) toward a synthetic peptide corresponding to the autophosphorylation site of pp60src. These data strongly suggest that it is a tyrosine-specific protein kinase.

Binding of calmodulin to the neuronal cytoskeletal protein kinase type II cooperatively stimulates autophosphorylation.

The kinetics of autophosphorylation of the cytoskeletal form of the neuronal calmodulin-dependent protein kinase type II were studied as a function of calmodulin binding under the same conditions. Whereas calmodulin binding was noncooperative with respect to calmodulin concentration (Hill coefficient = 1), the activation of autophosphorylation and the phosphorylation of exogenous substrates showed marked positive cooperativity (Hill coefficient greater than or equal to 1.6). Reduction of the active calmodulin concentration by the addition of the calmodulin antagonist trifluoperazine confirmed the cooperative nature of enzyme activation, because autophosphorylation was more sensitive to the drug than was binding at high concentrations of calmodulin. At intracellular levels of calmodulin the binding and activation of autophosphorylation were cooperative functions of magnesium and calcium concentration. The calmodulin-dependent cooperative activation seems to be a unique feature of the cytoskeletal, but not the soluble, form of the protein kinase and may result from the supramolecular organization of the cytoskeletal enzyme. These observations suggest that interactions among the subunits of the oligomeric cytoskeletal calmodulin-dependent protein kinase regulate enzyme activation, enhancing the sensitivity of the enzyme to small changes in the intracellular calcium levels that may be particularly relevant to signaling at the synapse.

Intracellular activation of protein kinase C and regulation of the surface transferrin receptor by diacylglycerol is a spontaneously reversible process that is associated with rapid formation of phosphatidic acid.

The effect of the synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (diC8), on the expression of the surface transferrin receptor reveals that exogenous diC8 can act as an intracellular activator of protein kinase C and stimulate both down-regulation and increased receptor phosphorylation in a manner similar to that induced by the active tumor promotor, 4 beta-phorbol 12,13-dibutyrate. Unlike the spontaneously irreversible effect noted when 4 beta-phorbol 12, 13-dibutyrate is added, this same effect mediated by diC8 is brief, lasting only minutes, and is spontaneously reversible. The rate of reversibility is dependent on the concentration of diC8 added, and it is associated with rapid formation of a newly detected intracellular phospholipid that corresponds to sn-1,2-dioctanoyl phosphatidic acid. These data, in conjunction with findings that demonstrate that exogenous diacylglycerols (including diC8) when added to cells do not stimulate cellular phospholipase A2 or C, argue that protein kinase C is activated only briefly in this system since exogenous diC8 is subject to rapid intracellular metabolism to phosphatidic acid.

Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells.

DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 4 degrees C as well as at 30 degrees C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase II, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase II by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA.