The association of lipid activators with the amphipathic helical domain of CTP:phosphocholine cytidylyltransferase accelerates catalysis by increasing the affinity of the enzyme for CTP.

The biochemical mechanism for the regulation of enzyme activity by lipid modulators and the role of the amphipathic alpha-helical domain of CTP:phosphocholine cytidylyltransferase (CT) was investigated by analyzing the kinetic properties of the wild-type protein and two truncation mutants isolated from a baculovirus expression system. The CT[delta 312-367] mutant protein lacked the carboxyl-terminal phosphorylation domain and retained high catalytic activity along with both positive and negative regulation by lipid modulators. The CT[delta 257-367] deletion removed in addition the region containing three consecutive amphipathic alpha-helical repeats. The CT[delta 257-367] mutant protein exhibited a significantly lower specific activity compared to CT or CT[delta 312-367] when expressed in either insect or mammalian cells; however, CT[delta 257-367] activity was refractory to either stimulation or inhibition by lipid regulators. Lipid activators accelerated CT activity by decreasing the Km for CTP from 24.7 mM in their absence to 0.7 mM in their presence. The Km for phosphocholine was not affected by lipid activators. The activity of CT[delta 257-367] was comparable to the activity of wild-type CT in the absence of lipid activators and the CTP Km for CT[delta 257-367] was 13.9 mM. The enzymatic properties of the CT[delta 231-367] mutant were comparable to those exhibited by the CT[257-367] mutant indicating that removal of residues 231 through 257 did not have any additional influence on the lipid regulation of the enzyme. Thus, the region between residues 257 and 312 was required to confer lipid regulation on CT, and the association of activating lipids with this region of the protein stimulated catalysis by increasing the affinity of the enzyme for CTP.

Lysophosphatidylcholine attenuates the cytotoxic effects of the antineoplastic phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3- phosphocholine.

A colony-stimulating factor 1-dependent cell line was used to determine the relationship between the inhibition of phospholipid synthesis and the cytotoxic activity of the antineoplastic ether lipid, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). ET-18-OCH3 inhibited choline incorporation into phosphatidylcholine as well as total phospholipid synthesis. Exposure to ET-18-OCH3 at the G1/S boundary led to the accumulation of cells in G2, whereas the addition of ET-18-OCH3 in the G1 phase of the cell cycle prevented entry into the S phase. In both cases, ET-18-OCH3 treatment triggered DNA fragmentation and morphological changes associated with apoptosis within 10 h. The addition of lysophosphatidylcholine provided an exogenous source of cellular phospholipid and prevented ET-18-OCH3-dependent accumulation of cells in G2 and apoptosis. However, lysophosphatidylcholine did not overcome the ET-18-OCH3-dependent G1 block, although the growth-arrested cells remained viable. These data indicate that restoring phosphatidylcholine synthesis by supplementation with lysophosphatidylcholine overrides the cytotoxic but not the cytostatic activity of ET-18-OCH3.

Lysophosphatidylcholine and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine inhibit the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase step.

The regulation of the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step by lysophosphatidylcholine (LPC) and the nonhydrolyzable LPC analog, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), was investigated in a colony-stimulating factor 1-dependent murine macrophage cell line. LPC inhibited phosphatidylcholine synthesis in vivo and led to the accumulation of choline and phosphocholine coupled to the disappearance of CDP-choline pointing to CT as the intracellular target. LPC neither inhibited cell growth nor decreased the cellular content of CT or altered the distribution of CT between soluble and particulate subcellular fractions. The inhibition of phosphatidylcholine synthesis was specific for LPC since lysophospholipids lacking the choline headgroup were not inhibitors. ET-18-OCH3 was a more potent inhibitor of phosphatidylcholine synthesis than LPC and caused the translocation of CT from the soluble compartment to the particulate compartment. Both LPC and ET-18-OCH3 were inhibitors of CT activity in vitro and kinetic analysis showed competitive inhibition with respect to the lipid activator. These data point to LPC as a negative regulator of de novo phosphatidylcholine synthesis that acts at the CT step and establish the mechanism for the inhibition of phosphatidylcholine biosynthesis by antineoplastic phospholipids.

Effect of insulin on SN-1,2-diacylglycerol species and de novo synthesis in rat skeletal muscle.

Insulin treatment increases the SN-1,2-diacylglycerol (DAG) concentration in skeletal muscle. Because DAG may participate in transmission or modulation of the insulin receptor signal, we examined the effect of insulin on total DAG and on different DAG species in isolated rat hemidiaphragms incubated with 5 mmol/L glucose. Five DAG species (16:0-18:1 omega 9, 16:0-18:1 omega 7, 18:0-18:1 omega 9, 18:0-18:2 omega 6, and 18:1-18:2) were identified and quantified. After a 5-minute incubation with 60 nmol/L insulin, neither total DAG nor a DAG species increased; exposure to insulin for 10 or 20 minutes increased the concentration of total DAG and of several DAG species. Insulin did not increase DAG in muscles incubated without glucose. Two sources for the insulin-mediated DAG increase were considered: phosphatidylcholine (PC) hydrolysis and de novo DAG synthesis from glucose. Concentrations of choline and phosphocholine in muscle were not increased after 10-minute incubations with insulin. However, insulin increased 14C incorporation from [U-14C]glucose into DAG, triacylglycerol (TAG), and total lipids approximately threefold. Okadaic acid (OKA), an inhibitor of phosphoprotein phosphatases 1 and 2A, increased muscle DAG content and synthesis from glucose, similar to the effect of insulin. Doses of OKA or insulin that increased DAG mass greatly exceeded those required for stimulation of glucose transport. The insulin-mediated, relatively slow increase in muscle DAG observed here likely reflects primarily de novo synthesis from glucose. This effect would be downstream of insulin stimulation of glucose transport. However, a possible insulin-mediated, rapid transient increase in muscle DAG content and PC hydrolysis cannot be ruled out by our studies.

Okadaic acid, insulin, and denervation effects on glucose and amino acid transport and glycogen synthesis in muscle.

Effects of okadaic acid (OKA) and calyculin A, cell-permeating specific inhibitors of phosphoprotein phosphatases-1 and -2A, were studied in isolated rat hemidiaphragms. OKA stimulated glucose transport (half-maximum = approximately 0.1 microM; maximum = approximately 1 microM) but was less effective than 6 nM insulin. Insulin and OKA effects were not additive. OKA diminished or abolished glucose transport-stimulation by insulin. System A amino acid transport was also stimulated by OKA, insulin was more effective, and preexposure to OKA inhibited insulin stimulation. Calyculin A affected both transport systems similarly to OKA. OKA did not affect basal glycogen synthesis but abolished its stimulation by insulin. Denervated muscles develop post-receptor insulin resistance. Glucose transport and glycogen synthesis were essentially unresponsive to insulin 3 days postdenervation; however, glucose transport was stimulated by OKA similarly to controls. OKA did not affect glycogen synthesis in denervated muscle except for abolishing a small insulin effect. The data suggest similar acute regulation of glucose and system A amino acid transport in muscle. Enhanced Ser/Thr phosphorylation of unidentified protein(s) stimulates both processes but inhibits their full stimulation by insulin. Postdenervation insulin resistance likely reflects impaired signal transduction.

Purification from human pregnancy serum of a low molecular weight mitogen similar to placental lactogen and growth hormone.

Recently, we isolated from the serum of pregnant women a factor that induced rapid proliferation of a lactogen-dependent rat lymphoma cell line (Nb2). This mitogenic factor is reasonably specific to pregnancy, since it was present in serum samples from second trimester as well as term-pregnant women, but not in those of adult men or cycling females. It is unlikely that this mitogenic activity (referred to as pregnancy mitogen [PM]) is due to contamination by classical lactogens, since acetone fractionation of serum yielded a preparation devoid of placental lactogen and prolactin, as determined by radioimmunoassays. Further purification of acetone precipitates from term-pregnant serum by ion exchange chromatography and gel filtration yielded a mitogenic activity with a relative mol wt of approximately 10,000. PM activity in the NB2 cell bioassay was not affected by the presence of prolactin antiserum. However, its activity was immunoneutralized by coincubation with anti-placental lactogen serum and, to a lesser extent, anti-growth hormone serum. It appears that PM was not generated by our extraction procedure, since gel filtration of whole serum also yielded a bioactive fraction of approximately 10 kDa. PM was further purified to homogeneity by high-performance liquid chromatography. Examination of the preliminary amino acid composition of PM revealed differences from that of a bioactive fragment of growth hormone and a corresponding portion of placental lactogen, suggesting that PM could be either a molecular variant of these hormones or a novel protein.

Effects of insulin and phospholipase C in control and denervated rat skeletal muscle.

Phospholipase C (PLC), an enzyme that increases endogenous 1,2-diacylglycerol (DAG), caused dose-dependent stimulation of 2-deoxy-D-glucose (2-DG) uptake in rat soleus muscles; the maximal effect was less than that of insulin. In denervated muscles the effect of insulin on 2-DG uptake was markedly reduced, whereas the response to PLC was identical to that of control muscles. Both PLC and insulin stimulated glucose incorporation into glycogen in control but not in denervated solei. Amino acid transport was unaffected by PLC; however, the enzyme completely inhibited the stimulation of amino acid transport by insulin. PLC did not activate the insulin receptor tyrosine kinase but decreased activation of the receptor by insulin in vivo. Basal muscle DAG content increased after denervation. Incubation with PLC markedly increased DAG in control and in denervated muscle. Insulin increased total DAG mass less than PLC in control muscles and did not affect DAG in denervated muscles. In media without added Ca2+, PLC stimulation of DAG production was impaired, and 2-DG uptake was unresponsive to PLC. The data are consistent with, but do not prove, that a subpopulation of DAGs may participate in insulin-mediated stimulation of glucose transport. They also suggest that the denervation-induced insulin resistance of glucose transport may reflect impaired generation of certain DAGs involved in the signaling cascade.

Insulin administration in vivo increases 1,2-diacylglycerol in rat skeletal muscle.

Based on in vitro studies, an insulin-mediated increase in muscle 1,2-diacylglycerol (DAG) content has been proposed as a signal for the insulin induced stimulation of glucose transport. A recent study [Turinsky, J., Bayly, B.P. and O'Sullivan, D.M. (1990) J. Biol. Chem. 265, 7933-7938] challenged this hypothesis because no increase in muscle 1,2-diacylglycerol was observed after in vivo infusions of insulin at doses which markedly stimulated muscle glucose transport. We observed a 30-45% increase in DAG in rat gastrocnemius and diaphragm muscles, 5-15 min after intramuscular or intravenous injections of 1-3 U of insulin per rat, doses which would be expected to activate insulin receptors more fully. The effects on DAG were similar whether or not hypoglycemia was prevented by co-injection of glucose.

Synergy of monocytes and lymphocytes from idiopathic minimal lesion nephrotic patients in relapse in the production of the supernatant factor that increases rat glomerular basement membrane sulfate uptake.

We have previously shown a significant increase in 35sulfate uptake in rat glomerular basement membrane (GBM) when glomeruli were cocultured with peripheral blood mononuclear cells (PBMC) from idiopathic minimal lesion nephrotic syndrome (IMLNS) patients in relapse or with the supernatants of the same PBMC cultures. The purpose of this study was to determine the cell source of the supernatant factor. Rat glomeruli were cocultured with PBMC, with monocytes or with lymphocytes from 11 patients with IMLNS in relapse. Monocytes and lymphocytes were separated using adherence to plastic technique. Rat glomeruli cultured without PBMC served as controls. There was a significant increase in 35sulfate uptake in the GBM when glomeruli were cocultured with PBMC (geometric mean [GM]: 794 cpm/mg dry glomerular weight) as compared to glomeruli cultured with monocytes (GM: 316) (p less than 0.0005), glomeruli cultured with lymphocytes (GM: 309) (p less than 0.05), and glomeruli cultured alone (GM: 302) (p less than 0.00005). No significant differences in 35sulfate uptake were seen between monocytes, lymphocytes and glomeruli alone. These data showed that monocytes and lymphocytes are needed for the production of the supernatant factor that increases rat GBM 35SO4 uptake. Based on these and previous data (Concanavalin A stimulation of IMLNS PBMC results in an increased GBM uptake of 35sulfate, Pediatric Research 20: 321, 1986), we postulate that monocytes could trigger or amplify the production of the supernatant factor by lymphocytes.

Effect of concanavalin A on nephrotic peripheral blood mononuclear cells mediated increased 35sulfate uptake in rat glomerular basement membrane.

We have previously shown a significant increase in 35sulfate uptake in rat glomerular basement membrane (GBM) when glomeruli were cocultured with peripheral blood mononuclear cells (PBMC) from patients with idiopathic minimal lesion nephrotic syndrome (IMLNS) in relapse, but an uptake not different than normal controls if glomeruli were incubated with PBMC of patients in remission. In the present study we examined 35sulfate uptake by GBM after PBMC from 12 IMLNS patients in remission were stimulated with Concanavalin A (Con A) (10 micrograms/ml of culture media). There was a significant increase in 35sulfate GBM uptake when glomeruli were cocultured with Con A-stimulated IMLNS PBMC (geometric mean), 331 cpm/mg dry glomerular weight) as compared to glomeruli cocultured with IMLNS PBMC (geometric mean, 200) (p = 0.048); glomeruli alone stimulated with Con A (geometric mean, 182) (p = 0.008) or glomeruli alone (geometric mean, 146) (p = 0.002). No significant differences were seen between the groups when glomeruli were cocultured with PBMC from 12 normal adults. These data show that Con A stimulated PBMC from IMLNS patients in remission alter the sulfate metabolism of rat GBM. The stimulation of PBMC with Con A reproduces the increase in 35sulfate uptake observed when glomeruli are cocultured with PBMC from IMLNS in relapse. Sulfated compounds in the GBM may play a role in glomerular permeability. Since stimulated nephrotic PBMC alter the metabolism of GBM sulfated compounds, these findings may have pathogenic significance.

Effect of supernatants from nephrotic peripheral blood mononuclear cells on 35sulfate incorporation in rat glomerular basement membrane.

In previous research, we showed that when peripheral blood mononuclear cells (PBMC) from patients with idiopathic minimal lesion nephrotic syndrome (IMLNS) in relapse were cocultured with rat glomeruli, there was an increased glomerular basement membrane (GBM) uptake of 35sulfate. This study was done to determine whether the increased uptake was due to substances secreted into the nephrotic PBMC culture supernatants. 35sulfate uptake in rat GBM was significantly higher when glomeruli were cocultured with PBMC from 12 IMLNS patients in relapse (geometric mean [GM] = 513 cpm/mg) than when simultaneous assays were done using either PBMC from eight control subjects (GM = 158) (p less than 0.05) or glomeruli incubated without PBMC (GM = 275 cpm/mg) (p less than 0.01). 35sulfate uptake did not increase when glomeruli were cocultured with PBMCs from 11 MLNS patients in remission. Rat GBM 35sulfate uptake was significantly higher when glomeruli were incubated in the supernatants of the PBMC cultures from 16 IMLNS patients in relapse (GM = 234 cpm/mg) than it was when glomeruli were cultured in the supernatants from normal control PBMC (GM = 141 cpm/mg; p less than 0.002) or from glomeruli cultured alone (GM = 141 cpm/mg) (p less than 0.04). Supernatants from PBMC cultures of 11 IMLNS patients in remission did not increase rat GBM 35sulfate uptake. These data suggest that PBMC from IMLNS patients in relapse secrete a factor(s) released into supernatants that increases the 35sulfate rat GBM uptake. Since sulfated compounds in the GBM play a role in glomerular permeability, this finding may have pathogenic significance.

Effect of peripheral blood mononuclear cells from patients with nephrotic syndrome on uptake of 35sulfate by glomerular basement membrane.

We have studied the effect of peripheral blood mononuclear cells (PBMC) from nephrotic patients on the incorporation of 35Sulfate in rat glomerular basement membrane. 35Sulfate uptake increased when glomeruli were incubated with PBMC from 10 patients with idiopathic minimal lesion nephrotic syndrome (IMLNS) in relapse compared to simultaneous assays on 5 controls (2295 +/- 643 cpm/mg dry glomerular weight, mean +/- SEM, and 962 +/- 248 cpm/mg, respectively) (P less than 0.02). 35Sulfate incorporation did not increase when glomeruli were incubated with PBMC from seven nephrotic patients with other glomerulopathies (1283 +/- 642 cpm/mg) as compared to controls (1016 +/- 503 cpm/mg). No significant differences in 35Sulfate uptake were seen between glomerular cultures with and without control PBMC. These data show that PBMC from patients with IMLNS in relapse alter, at least in the rat GBM, the metabolism of sulfated compounds. Since these compounds have a role in glomerular permeability, this finding may have pathogenic significance.

Incorporation of 35sulfate into the glomerular basement membrane in puromycin aminonucleoside nephrosis.

The in vitro incorporation of 35sulfate into the glomerular basement membrane (GBM) of rats with puromycin aminonucleoside was compared with that of normal rats. 35Sulfate incorporation into the GBM of glomeruli from nephrotic rats did not differ significantly from that observed in normal rats. These data suggest that 35sulfate anionic charges in the GBM play no important role in the pathogenesis of the proteinuria in this experimental model.