Increase In DAG Research Articles

Abstract 4173: Lipidome of mammographic breast density in premenopausal women

Abstract Introduction: High mammographic breast density (MBD) is a strong risk factor for breast cancer, but the biological mechanisms underlying high MBD are not well understood. We, therefore, comprehensively investigated for the first time the associations of lipid species with volumetric measures of MBD to elucidate potential biological mechanisms of high MBD in premenopausal women. Methods: We performed lipidomic profiling on 705 premenopausal women recruited during their annual screening mammogram at Washington University School of Medicine, St. Louis, MO. Lipidomic profiling for 982 lipid species was completed at Metabolon (Durham, NC®). Lipid species with greater than 300 missing values (N=125) were excluded from the analysis. Using nearest neighbor methods, we imputed the lipid species missing less than 300 values. We used Volpara 1.5 (Volpara Health®) to quantify volumetric measures of MBD - volumetric percent density (VPD), dense volume (DV), and non-dense volume (NDV). We investigated the associations of the lipid species with MBD measures using multivariable linear regression models adjusting for age, age at menarche, body shape at age 10, race/ethnicity, body fat %, family history of breast cancer, oral contraceptive use, parity/age at first birth, and alcohol use. MBD measures were log10-transformed, and lipid species were standardized. Linear coefficients were back-transformed to the original scale and considered significant if the Bonferroni corrected p<0.05. Results: In multivariable linear regression models, 34 lipid species were inversely associated with VPD. The lipid species belong to the triacylglycerol (TAG, N=26), diacylglycerol (DAG, N=6), phosphatidylcholine (PC, N=1), and cholesterol ester (N=1) pathways. DAG(16:0/18:2) and TAG54:6-FA20:4 displayed the largest inverse associations with one standard deviation increase in DAG(16:0/18:2), and TAG54:6-FA20:4 corresponding to 9.9% (p=0.002), and 9.6% (p=0.007) decrease in VPD, respectively. Eleven lipid species were significantly associated with NDV, with 10 species (all TAG) having a positive association and 1 inverse association PC(18:1/18:1). The strongest positive association was observed with TAG54:6-FA20:4. One standard deviation increase in TAG54:6-FA20:4 was associated with a 9.8% increase in NDV (p=0.01). Several of the lipid species (N=8, 23.5%) that were associated with VPD were also associated with NDV, but in opposite directions. No lipid species were significantly associated with DV. Conclusions: Our study identified many lipid species, especially in TAG and DAG pathways, that were associated with VPD and NDV and offer new insights into the biological mechanisms underlying high MBD in premenopausal women. Future studies are needed to validate our results and the translational potential. Citation Format: Kayla R. Getz, Myung S. Jeon, Chongliang Luo, Jingqin Luo, Adetunji T. Toriola. Lipidome of mammographic breast density in premenopausal women. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4173.

Ectopic expression of AtDGAT1, encoding diacylglycerol O-acyltransferase exclusively committed to TAG biosynthesis, enhances oil accumulation in seeds and leaves of Jatropha.

Background Jatropha curcas is an important biofuel crop due to the presence of high amount of oil in its seeds suitable for biodiesel production. Triacylglycerols (TAGs) are the most abundant form of storage oil in plants. Diacylglycerol O-acyltransferase (DGAT1) enzyme is responsible for the last and only committed step in seed TAG biosynthesis. Direct upregulation of TAG biosynthesis in seeds and vegetative tissues through overexpression of the DGAT1 could enhance the energy density of the biomass, making significant impact on biofuel production.ResultsThe enzyme diacylglycerol O-acyltransferase is the rate-limiting enzyme responsible for the TAG biosynthesis in seeds. We generated transgenic Jatropha ectopically expressing an Arabidopsis DGAT1 gene through Agrobacterium-mediated transformation. The resulting AtDGAT1 transgenic plants showed a dramatic increase in lipid content by 1.5- to 2 fold in leaves and 20–30 % in seeds, and an overall increase in TAG and DAG, and lower free fatty acid (FFA) levels compared to the wild-type plants. The increase in oil content in transgenic plants is accompanied with increase in average plant height, seeds per tree, average 100-seed weight, and seed length and breadth. The enhanced TAG accumulation in transgenic plants had no penalty on the growth rates, growth patterns, leaf number, and leaf size of plants.ConclusionsIn this study, we produced transgenic Jatropha ectopically expressing AtDGAT1. We successfully increased the oil content by 20–30 % in seeds and 1.5- to 2.0-fold in leaves of Jatropha through genetic engineering. Transgenic plants had reduced FFA content compared with control plants. Our strategy of increasing energy density by enhancing oil accumulation in both seeds and leaves in Jatropha would make it economically more sustainable for biofuel production.

Physical and Chemical Properties Study on the Groundwater in Tuz District

The present study is carried out to detect some quality properties of groundwater at Tuz,. The results showed that air temperature noted minimum at ranged between (8-35)C° , and The recorded temperatures of ground water values range between (18-25)C°, Turbidity values for all wells water at the study area ranged (0.00-29.88)NTU, It is obtained that wells near-off the mountain chain Nafed dag increase of electric conductivity comparative ranged between (734-6890)µs/cm. Groundwater in the study area was Brackish dependence to the total dissolved salts ranged (20-5860)mg/l . The pH values ranging between (4.15-8.91) . It was noted that the values of the dissolved oxygen are few and almost non-existent in some cases it ranged between (0.8 -0.30)mg/l. Analysis of biological oxygen demand refers to wells water (very clean) levels involving (0.00 -0.15)mg/l. Reason of groundwater very hardness ranging values of total hardness , calcium and magnesium hardness between (300-2440) mg/l , (160-1400)mg/l and (44-1580)mg/l respectively. The total alkalinity ranged between (74-600)mg/l , The chloride ions in ground water of the studied within the permissible limits for watering of animals and irrigation of plants, with values range (0.00-349.89)mg/l . Sulphates range between (134.5-3326)mg/l .Sodium values for all wells water at the study area ranged (112.35 -495.59)mg/l. Potassium values for all wells water at the study area ranged (1.158-51.036)mg/l. As for plant nutrients , ranged between the values of silica (9.688-25.981)mg/l.The concentration of active nitrite ions ranged between (0.000 –1.212) µg NO-NO2/L .As the values of phosphate , ranged between (0.26-0.399) µg P-PO4/L during the study period.

The pharmacokinetics of acyl, des-acyl, and total ghrelin in healthy human subjects

Ghrelin stimulates GH secretion and regulates energy and glucose metabolism. The two circulating isoforms, acyl (AG) and des-acyl (DAG) ghrelin, have distinct metabolic effects and are under active investigation for their therapeutic potentials. However, there is only limited data on the pharmacokinetics of AG and DAG. To evaluate key pharmacokinetic parameters of AG, DAG, and total ghrelin in healthy men and women. In study 1, AG (1, 3, and 5 μg/kg per h) was infused over 65 min in 12 healthy (8 F/4 M) subjects in randomized order. In study 2, AG (1 μg/kg per h), DAG (4 μg/kg per h), or both were infused over 210 min in ten healthy individuals (5 F/5 M). Plasma AG and DAG were measured using specific two-site ELISAs (study 1 and 2), and total ghrelin with a commercial RIA (study 1). Pharmacokinetic parameters were estimated by non-compartmental analysis. After the 1, 3, and 5 μg/kg per h doses of AG, there was a dose-dependent increase in the maximum concentration (C(max)) and area under the curve (AUC(0-last)) of AG and total ghrelin. Among the different AG doses, there was no difference in the elimination half-life, systemic clearance (CL), and volume of distribution. DAG had decreased CL relative to AG. The plasma DAG:AG ratio was ~2:1 during steady-state infusion of AG. Infusion of AG caused an increase in DAG, but DAG administration did not change plasma AG. Ghrelin administration did not affect plasma acylase activity. The pharmacokinetics of AG and total ghrelin appears to be linear and proportional in the dose range tested. AG and DAG have very distinct metabolic fates in the circulation. There is deacylation of AG in the plasma but no evidence of acylation.

Involvement of the peripheral cholinergic muscarinic system in the compensatory ovarian hypertrophy in the rat.

In the present experiments, unilateral ovariectomy (ULO) induced compensatory hypertrophy (COH) of the remaining rat ovary (60%-85% increase in ovarian weight, total proteins, and total RNA and DNA). An increased thymidine uptake preceded the organ enlargement. COH was inhibited by i.p.-administered muscarinic antagonist propantheline (dose-dependently) or botulinum toxin delivered locally to the ovary. The effects were reversed by bethanecol i.p. (a muscarinic agonist). In sham ULO animals, [3H]-scopolamine binding to ovarian membranes indicated the existence of muscarinic receptors (Kd 2.5 nM, Bmax 12 fmol/mg proteins, Hill 1.0). The ovarian 1,2-diacylglycerol (DAG) was 120-150 pmol/mg tissue and did not react to carbachol in vitro (50 microM). At 15 minutes after ULO, the [3H]-scopolamine binding was unchanged (Kd 2.6 nM, Bmax 12.6 fmol/mg tissue, Hill 1.0), but the ovarian DAG was increased (280-350 pmol/mg tissue) and increased further in response to carbachol (460-550 pmol/mg tissue). After ULO, ovarian DAG remained continuously responsive to carbachol. The ULO-induced DAG increase and enhanced susceptibility to carbachol were inhibited by the botulinum toxin or atropine pretreatments. Abdominal vagotomy done immediately before ULO also inhibited the ULO-induced DAG increase and DAG responsiveness to carbachol. However, when the vagotomy was performed 10 mins after ULO, the ovarian DAG remained responsive to carbachol in vitro. The data suggest that the peripheral cholinergic system, including the ovarian muscarinic receptors, stimulates COH. This is apparently associated with the ULO-induced coupling of the ovarian muscarinic receptors to phosphoinositide (PI) breakdown. Vagus plays a role in the occurrence of the changed muscarinic receptor-PI breakdown relationship in the remaining ovary.

An Algorithm for Task Scheduling in Heterogeneous Computing Environment

Heterogeneous computing systems are of considerable interest because a single parallel architecture may not be adequate for exploiting all the parallelism available in the program. In some cases, heterogeneous systems perform better than a single large machine and that too at a lower cost. In heterogeneous systems, task scheduling is done with a view to minimize the overall completion time of a parallel application by properly allocating the tasks to the processors. In this paper, a task-scheduling algorithm called Alternate Heterogeneous Earliest Finish Time (AHEFT) is proposed and its performance is analyzed with respect to Heterogeneous Earliest Finish Time (HEFT) algorithm. A simulation environment has been developed in C++ and both the algorithms were implemented. Different weighted Directed Acyclic Graphs (DAG's) were used to compare the two algorithms. A network of fully connected heterogeneous processors was assumed for task scheduling. The performance indices used for comparison of algorithms are Schedule Length, Speedup Factor and Efficiency. The results substantiate the fact that for higher values of CCR (Communication to Computation Ratio), AHEFT algorithm performs better than HEFT. Moreover, as the number of tasks in the DAG increase, the performance improvement of AHEFT algorithms is more as compared to HEFT.

IGF-I-induced phosphorylation of connexin 43 by PKCgamma: regulation of gap junctions in rabbit lens epithelial cells.

To determine the role of PKCgamma in insulin-like growth factor (IGF)-I-induced phosphorylation of connexin (Cx)43 and control of gap junctions in lens epithelial cells. N/N1003A rabbit lens epithelial cells were used in the experiments. PKC translocation or in vivo Cx43 phosphorylation on serine was determined by Western blot analysis. Gap junction activity was measured by scrape-loading/dye-transfer assay. The number of cell surface gap junction plaques was detected by confocal microscopy. The interaction between PKCgamma and Cx43 was determined by coimmunoprecipitation. In vitro Cx43 phosphorylation was assayed by PKC assay kit. Endogenous sn-1,2-diacylglycerol (DAG) was measured by detecting (32)P-labeled phosphatidic acid. IGF-I stimulated activation and translocation of PKCgamma in a dose- and time-dependent manner, acidic FGF (aFGF) had no effect on translocation of PKCgamma, and PKCalpha was not translocated by IGF-I at 25 ng/mL. PKCgamma translocation resulted in coimmunoprecipitation with and phosphorylation of Cx43. IGF-I- or DAG-induced activation of PKCgamma caused a decrease in gap junctions. IGF-I increased endogenous DAG. Exogenous CaCl(2) and DAG stimulated PKCgamma translocation. TMB-8, an internal calcium mobilization inhibitor, blocked CaCl(2)-induced PKCgamma translocation; however, it had no effect on IGF-I- or DAG-induced translocation of PKCgamma. PKCgamma mediated IGF-I-induced decreases in gap junctional communication through interaction with and phosphorylation of Cx43. IGF-I caused an increase in DAG, and this increased translocation of PKCgamma, whereas mobilization of calcium was not essential for IGF-I-stimulated translocation of PKCgamma.

Insulin Activates Phospholipase C-γ1 via a PI-3 Kinase Dependent Mechanism in 3T3-L1 Adipocytes

Previously we have shown that the insulin receptor and phospholipase C-γ1 physically interact in the 3T3-L1 adipocyte cell line. In this study, we investigated the ability of insulin and PDGF to stimulate PLC-γ1 enzyme activity as measured by PI-(4,5)P2 hydrolysis. Both insulin and PDGF caused a rapid (<1 min) increase in PLC activity associated with the respective receptor. PDGF treatment resulted in a higher and more sustained stimulation of PLC-γ1 activity compared to insulin (0.95 pmol/min/mg vs 0.68 pmol/min/mg). Furthermore, insulin and PDGF promoted increases in total cellular DAG, one of the products of PI-(4,5)P2 hydrolysis. Insulin-stimulated PLC activity appears to be downstream of PI-3Kinase as the DAG increase was partially blocked by Wortmannin and addition of PI-(3,4,5)P3 activated PLC-γ1 in vitro. Inhibition of PLC using U73122 or an inhibitory peptide caused a decrease in insulin-stimulated 2-deoxyglucose transport and GLUT4 translocation that was rescued by the addition of OAG, a cell-permeable synthetic DAG.

Molecular species analysis of 1,2-diacylglycerol released in response to progesterone binding to the amphibian oocyte plasma membrane

Progesterone, the physiological inducer of amphibian meiosis, acts within minutes at plasma membrane receptors of the Rana pipiens oocyte to release 1,2-diacylglycerol (DAG) from plasma and intracellular membranes. High-performance liquid chromatography (HPLC) analysis of lipid extracts of uninduced oocytes indicates the presence of at least three classes of DAG with a total DAG content of about 150 μmol/kg wet weight. Within 3–5 min after exposure to progesterone, there was a differential increase in all three DAG classes with a twofold increase in total DAG by 10 min. The fatty acid composition of the DAGs in uninduced and progesterone-stimulated oocytes was compared using thin layer chromatographic analysis of lipid extracts from oocytes double-labeled with [ 14C] or [ 3H]glycerol and [ 14C] or [ 3H]fatty acids. The ratio of labeled fatty acid/labeled glycerol was measured in phosphatidylcholine (PC), phosphatidylinositol (PI) and DAG. The linoleic (18:2) or arachidonic (20:4) acid/glycerol ratios in basal DAG were low compared to that in PC or PI. In contrast, the myristic (14:0), palmitic (16:0) or oleic (18:1) acid/glycerol ratios in basal DAG were relatively high compared to the ratio in PC and PI. A transient increase in both linoleic and palmitic acid labeling of DAG occurred within the first 1–2 min in progesterone-treated oocytes, followed by a return to or below the basal level. Arachidonic and myristic acid labeling of DAG fall within the first minute after progesterone treatment, followed by a sustained rise over the next 10 min. The [ 3H]oleic acid/[ 14C]glycerol ratio of DAG does not change significantly following exposure to progesterone. Pretreatment with a phospholipid N-methylation inhibitor (2-methylaminoethane) precluded the rise in linoleic and palmitic acid-rich DAG, whereas pretreatment with a diglyceride kinase inhibitor (D102) produced a sustained elevation of linoleic and palmitic acid-rich DAG. These results indicate that the DAG released in response to progesterone is composed of multiple new molecular species of DAG and that both the palmitate and linolate-rich forms are rapidly phosphorylated to form phosphatidic acid (PA). The newly formed DAG species differ from the basal DAG species and reflect sequential activation of sphingomyelin (SM) synthase, PC-specific phospholipase D (PLD) and PI-specific phospholipase C in response to progesterone, which we have described previously.

Cellular proliferation and second messenger formation altered by dialysis membranes.

In this study we investigated the effect of different dialysis membranes on the clonal murine haematopoietic cell line 32D cells transmembrane signalling machinary, monitored by 1,2-diacylglycerol (DAG) formation, and on their ability to respond to the physiological growth factor interleukin-3 (IL-3). Cells were exposed to dialysers (cuprophane, CU; polysulphone, PS; cellulose diacetate, CA; polyacrylonitrile, AN69; polymethylmethacrylate, PMMA; cellulose triacetate, CT; polyamide, PA; and polycarbonate, PC); they were collected, counted, and treated with physiological amount of IL-3. Cell proliferation was monitored as incorporation of radioactivity in duplicating DNA. DAG was measured by thin-layer chromatography in cell labelled with tritiated glycerol overnight. CU and PA stimulated cell proliferation in comparison with resting cells. PS and TC did not significantly affect thymidine incorporation either in IL-3-stimulated, or in resting cells. Cells exposed to AN69, PC, and CA showed depressed basal incorporation of thymidine (70, 54 and 56% of controls respectively) but retained the ability to respond to IL-3 in a manner not statistically different from controls. PMMA reduced thymidine incorporation both in basal condition and after IL-3 stimulation CU and PC activated early cell signalling (1.95 x and 1.31 x respectively, DAG increase over control), while PA and TC depressed DAG generation (0.38 x and 0.47 x respectively, DAG increase over control). PS, CA, AN69, and PMMA did not stimulate DAG generation. Alternations of intracellular mitogenic signalling appear to correlate with the ability to make a cell competent for function. These results might help to elucidate the effect of different dialysers, at the molecular level, on the blood cell behaviour in vivo.

Molecular Species Analysis of 1,2‐Diglycerides on Phorbol Ester Stimulation of LA‐N‐1 Neuroblastoma Cells During Proliferation and Differentiation

1,2-Diacyl-sn-glycerol (DAG) is a product of cell activation that has emerged as an important intracellular messenger whose primary function appears to be the activation of protein kinase C. They originate by the activation of phospholipases, which hydrolyze different phospholipids depending on the external stimulus and the nature of the cells, leading to the production of different molecular species. In the present study the quantitative changes in the total mass and the molecular species of DAG formed on phorbol ester (12-O-tetradecanoyl-phorbol 13-acetate) stimulation were investigated in proliferating and retinoic acid (RA)-differentiated human LA-N-1 cells. The TPA treatment of both cell types elicited an increase in the total amount of DAG. The increase was biphasic; i.e., an initial peak at 2-5 min was followed by a sustained increase that persisted for > 30 min. The analysis of the molecular species of DAG and phospholipids showed that in proliferating LA-N-1 cells, the DAG increase corresponds to the production of mainly saturated/monounsaturated (16:0-18:1, 18:0-18:1) and saturated/saturated (16:0-16:0, 16:0-18:0) species, suggesting that they originate essentially from the hydrolysis of phosphatidylcholine. In contrast, RA-differentiated cells responded to TPA treatment by increasing the level of saturated/polyunsaturated (16:1-22:6, 18:0-22:6, 16:0-20:4, 18:0-20:4) and monounsaturated/monounsaturated (18:1-18:1) species, suggesting mainly a phosphatidylethanolamine origin. These findings indicate that the treatment of LA-N-1 cells with TPA generates different molecular species of DAG depending on their physiological state. These observations suggest in turn that different phospholipases are activated by TPA in proliferating and RA-differentiated cells.

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 (16:0–18:1ω9, 16:0–18:1ω7, 118:0–18:11ω9, 18:0–118:2ω6, 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.

Pressurization of isolated renal arteries increases inositol trisphosphate and diacylglycerol.

Inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) concentrations were measured in isolated, cannulated dog renal arteries under control conditions (0 mmHg) and in response to step elevations in transmural pressure. There was a pressure-dependent increase in IP3 at 60 and 120 mmHg, reaching significance at 120 mmHg (P < 0.05) and a significant increase in DAG at both 60 and 120 mmHg measured after maintaining pressure for 15 min. Similarly, IP3 measurements made 90 s after a step increase in transmural pressure also exhibited a pressure-dependent profile, again reaching significance at 120 mmHg. Calculation of active tension demonstrated these renal arteries developed pressure-dependent myogenic tone. To assess the role of the endothelium in this regard, IP3 was measured before and after endothelial removal at 0 and 60 mmHg. Pressure-dependent myogenic tone was still present upon endothelial removal. In the absence of the endothelium, we observed a significant increase in total IP3 at 60 compared with 0 mmHg; furthermore, the increase in IP3 in the absence of the endothelium was significantly greater than that observed when the endothelium was intact. Given that the primary source of IP3 is via the actions of phospholipase C (PLC) on phosphatidylinositol 4,5-bisphosphate, these biochemical data directly demonstrate that elevation of transmural pressure in dog renal arteries activates PLC.

Temporal regulation of the IgE-dependent 1,2-diacylglycerol production by tyrosine kinase activation in a rat (RBL 2H3) mast-cell line.

We explored the possible role of tyrosine kinases in the IgE-dependent regulation of 1,2-diacylglycerol (DAG) production in RBL 2H3 cells. When triggered via their high-affinity IgE receptors (Fc epsilon RI), there was a rapid phosphorylation of tyrosine residues on a number of proteins. The phosphorylation of these proteins and ultimately histamine release were inhibited in a concentration-dependent manner by the tyrosine kinase inhibitor, tyrphostin. In cells labelled with [3H]myristic acid, we observed a characteristic biphasic increase in [3H]DAG production. In the presence of tyrosine kinase inhibitor, the initial increase in DAG was still observed, but the secondary increase, which was dependent on phosphatidylcholine-specific phospholipase D (PC-PLD) activation, was completely abolished. Tyrphostin significantly inhibited IgE-dependent activation of PC-PLD, suggesting that PC-PLD activation was regulated by tyrosine phosphorylation. Furthermore, when proteins from RBL 2H3 cells were immunoprecipitated with an anti-phosphotyrosine antibody, PC-PLD activity was recovered from the immunoprecipitated fraction. These results demonstrate that the secondary, but not the initial, phase of 1,2-DAG production in response to Fc epsilon RI aggregation is regulated by the initial activation of tyrosine kinases and that PC-PLD may be regulated directly by this mechanism.

Alkalosis- and ATP-induced increases in the diacyglycerol pool in alveolar type II cells are derived from phosphatidylcholine and phosphatidylinositol.

Alkalosis and ATP increase surfactant secretion in alveolar type II cells, possibly via non-receptor- and receptor-mediated mechanisms respectively. We compared the effects of these two agonists on phosphatidylinositol (PI) and 1,2-diacylglycerol (DAG) pools and on phosphatidylcholine (PC) hydrolysis in alveolar type II cells. Alkalosis, caused by transfer of cells from 5% (control) to 0% CO2 in air, and ATP increased the secretion of surfactant compared with the controls. The stimulated secretion was inhibited by staurosporine, a protein kinase C inhibitor. DAG and PI contents of control cells were 50 +/- 1.1 (mean +/- S.E.M., n = 8) and 14 +/-0.8 nmol/mg phospholipid (n = 7) respectively. The DAG content increased by approximately 50 nmol (100%) within 5 s of treatment with both alkalosis and ATP, returned to control levels by 1 min, and increased again at 5 min by approximately 20 nmol. The PI content decreased maximally by approximately 6 nmol (40%) at 5 s and returned to control levels by 30 s with both alkalosis and ATP, but was unchanged thereafter. Mass-balance analysis of net changes in DAG and PI pools suggests that additional sources, possibly PC, must also contribute to the DAG increase. ATP or alkalosis also increased the hydrolysis of PC. The labelling of phosphocholine was increased (approximately 60%) at as early as 5 s and remained elevated at subsequent time points, whereas labelling of choline was higher only with ATP at 50 s and later, suggesting activation of phospholipase C by both agonists, and of phospholipase D by only ATP. Our studies demonstrate that ATP and alkalosis stimulate rapid hydrolysis of inositol and choline phospholipids to increase the DAG mass in type II cells, and that phospholipase C-stimulated PC hydrolysis is the major pathway for DAG formation.

Inositol lipid signalling occurs in brush-border membranes during initiation of compensatory renal growth in the rat.

Using highly specific mass assays, concentrations of inositol lipids and 1,2-diacylglycerol (DAG) were determined in plasma membranes isolated from rat kidney cortex. Significantly higher concentrations of inositol lipids were determined in brush-border (BBM) than in basal-lateral (BLM) plasma membranes, although DAG concentrations were similar in both. After unilateral nephrectomy, a decrease in PtdIns(4,5)P2 and PtdIns4P, with a concomitant increase in DAG and translocation of protein kinase C (PKC), were observed in BBM but not in BLM isolated from the remaining kidney. On the other hand, stimulation of renal cortical slices with insulin-like growth factor II (IGF-II) or phenylephrine caused similar effects in BLM but not in BBM. Stimulation of phospholipase C activity with translocation of PKC only to BBM in one kidney was also induced by occlusion of blood flow through the contralateral kidney for 15 min. At 30 min after the occlusion was removed and reflow established, DAG concentration and the amount of PKC in BBM returned to control values. These results suggest that an early signal after unilateral nephrectomy is transmitted to cells through BBM and can be switched on and off by blood occlusion and reflow through the contralateral kidney, while hormonal signals caused by IGF-II and phenylephrine are transmitted to cells through BLM.

Diacylglycerol/protein kinase C signalling: a mechanism for insulin resistance?

It is proposed that an intracellular cycle exists to limit or terminate the insulin signal. The cycle involves increased synthesis of sn-1,2-diacylglycerol (DAG) in response to insulin. The DAG activates protein kinase C (PKC) which phosphorylates glycogen synthase either directly or through other protein kinases to render it inactive. Protein kinase C may also inhibit the insulin receptor by phosphorylation of receptor serine residues. Insulin resistance could then arise as a consequence of a persistent increase in DAG levels. Such an increase could occur in three different ways. Chronic hyperinsulinaemia could increase DAG levels by de-novo synthesis from phosphatidic acid, by hydrolysis of phosphatidylcholine, or by hydrolysis of glycosyl-phosphatidylinositol; DAG is also formed by hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2). This reaction, known as the 'PI response,' may be the connection between hypertension and insulin resistance. A third mechanism for an increase in DAG involves neural abnormalities. Thus, muscle denervation in the rat is characterized both by a profound insulin resistance and a large increase in DAG. It is possible that a similar increase occurs in humans and may explain the association between denervation, inactivity, and insulin resistance.

Generation of diacylglycerol and ceramide during homologous complement activation.

Formation of sublytic terminal complement complexes (TCC) on nucleated cells produces transient increase in [Ca2+]i and activates protein kinase C. The present study is to evaluate whether TCC can generate endogenous signal messengers other than Ca2+ that regulate cell activities by measuring mass-levels of sn-1,2-diacylglycerol (DAG) and ceramide. As targets, lymphoblastoid human B cell lines JY25 and its mutant JY5 were used. JY5, cells deficient in glycosylphosphatidylinositol-anchored proteins with higher lytic susceptibility to human complement, are four times more efficient in forming C5b-9. When cells sensitized with limited anti-class II IgG were exposed to human serum to generate sublytic TCC, a sustained increase in DAG and ceramide was observed with a maximum 3.6-fold DAG increase over basal level in JY25 and 2.8-fold in JY5, and 6.3-fold ceramide increase in JY25 and 2.8-fold in JY5. The effect of TCC was evaluated with C7-deficient human serum (C7D) +/- C7 and also with C5b6, C7, C8, and C9 proteins. The DAG and ceramide increase by C7D + C7 over C7D control were 1.6- and 1.8-fold, respectively, in JY25, and 2.3-, and two-fold in JY5. TCC activation also induced an increased hydrolysis of sphyingomyelin and phosphatidylcholine. In addition, DAG increase by TCC was primarily achieved by C5b-7 and preincubation of cells with pertussis toxininhibited DAG increase, suggesting an involvement of a pertussis toxin-sensitive GTP-binding protein. As important signal transduction molecules, DAG and ceramide generated in response to TCC assembly, could participate in cell activation during inflammation and repair.

The hepatitis B virus transactivator HBx causes elevation of diacylglycerol and activation of protein kinase C

Chronic infection with hepatitis B virus (HBV) is accompanied by an increasing risk of developing hepatocellular carcinoma. There are indications that the HBx protein of HBV is involved in the process of tumour formation. HBx also transactivates several transcription factor binding sites. Recently, we reported that HBx can use a tumour promotor pathway for transactivation. In particular, we found that transactivation of the binding motif for transcription factor AP-1 (JUN/FOS) by HBx is dependent on functional protein kinase C (PKC), as indicated by abolition of the transcriptional stimulation following downregulation or inhibition of the enzyme. Moreover, HBx activates PKC, probably via increasing the endogenous PKC activator sn-1,2-diacylglycerol (DAG). Here we extend these data and report on the time course of PKC activation. We found that activation of PKC by HBx is transient and differs from activation of PKC by the ras oncogene product or phorbol ester in that it does not lead to rapid downregulation of the enzyme subsequent to the activation. Moreover, we provide evidence that an increase in cellular DAG is observable not only as an early event in response to HBx but also in cell lines transformed after transfection with HBV DNA and stably expressing HBx. Besides its important role in the regulation of cellular genes, PKC is also the intracellular receptor for tumour-promoting agents and an activator of proto-oncogenes, suggesting that our observations might provide an explanation for the oncogenic properties of HBx.

Calcium-activated phosphatidylcholine-specific phospholipase C and D in MDCK epithelial cells.

Calcium ionophore exposure generates diglycerides (DAG) from phosphatidylcholine (PC) hydrolysis in Madin-Darby canine kidney (MDCK) epithelial cells. This study compares calcium ionophore-activated PC hydrolysis with the previously described phorbol ester-stimulated PC hydrolysis pathway using MDCK cells labeled with [14C]-linoleic acid. Lipid species were measured using thin-layer chromatography. DAG resulted in part from PC hydrolysis because DAG increased in cells labeled with [palmitoyl-2-14C]phosphatidylcholine. Neither protein kinase C (PKC) inhibitors nor PKC depletion affected the ionomycin (IONO)-induced increase in DAG. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the increased DAG after IONO but not after phorbol 12,13-dibutyrate (PDBu) exposure. The EGTA effect was reversed by adding excess calcium but was not reversed by adding excess Mg2+. IONO exposure also increased phosphatidic acid (PA) production. The PA was produced by phospholipase D (PLD) because phosphatidylethanol was produced when IONO was added to the cells in the presence of ethanol. Although increasing concentrations of ethanol resulted in progressively less PA, it had no effect on increased DAG after IONO exposure at any time point tested. These data are consistent with both increased phospholipase C (PLC) and increased PLD activity following ionomycin. In contrast to IONO exposure, ethanol completely prevented the increase in DAG after PDBu exposure, consistent with DAG produced by PLD activation. These results demonstrate that calcium activates both PC-specific PLC and PLD in MDCK cells and that the calcium-activated pathway is independent of the previously described PKC activation pathways.