Thin layer chromatography of coordinated Ag+-complexes of plant diacylglycerols

Coordination complexes of unsaturated rac-1,2-diacylglycerols (DAGs) with silver ions were separated by adsorption and reversed-phase TLC (Ag-TLC and Ag-rpTLC, respectively). During the Ag-TLC, the silver ion complexes were shown to be formed by the DAG coordination centers of various structures and only on the adsorbent surface. Separation of the complexes proceeds according to the adsorption mechanism, and there is an inverse exponential functional relationship between the DAG mobility and their double bond number. Meanwhile, during the Ag-rpTLC, the Ag(+)-complexes are formed only with double bonds, only in solution, and at a 1:1 ratio. The complexes are fractionated by partitioning between two liquid phases, and the relationship between the mobility and unsaturation of these complexes is directly proportional. Nevertheless, despite all the differences between the two TLC methods, the polarity of DAGs with a bent configuration of their acyl chains greatly exceeds that of DAGs with the same unsaturation but with the acyl-chain conformation close to extended: it is two to three times greater in Ag-TLC and 30-40% greater in Ag-rpTLC. In addition, the relationship between the mobility and unsaturation of DAG complexes exhibits quantitative rather than qualitative differences in both versions of argentation TLC. Thus, under all conditions of argentation liquid chromatography, the mobility of complexes and, therefore, their polarity are determined not only by their composition (unsaturation), but also by the spatial structure (conformation) of their molecules.     

Effect of diacylglycerols on the activity of cobra venom, bee venom, and pig pancreatic phospholipases A2.

The effects of a series of diacylglycerols (DAGs) with varying acyl chain lengths and degree of unsaturation on the activity of cobra venom, bee venom, and pig pancreatic phospholipases A2 (PL-A2S) were studied using two lipid substrates: dipalmitoylphosphatidylcholine (DPPC) or bovine liver phosphatidylcholine (BL-PC). The activities of the phospholipases critically depended on the chain length and degree of unsaturation of the added DAGs and on the chemical composition of the substrate. The effects of DAGs on cobra or bee venom PL-A2S were similar, but significantly different from the pig pancreatic PL-A2. The data, taken together with our previous NMR studies on physicochemical effects of these DAGs on lipid bilayer structure [De Boeck, H., & Zidovetzki, R. (1989) Biochemistry 28, 7439; (1992) Biochemistry 31, 623], allowed detailed correlation of the type of a bilayer perturbation induced by DAG with the activation or inhibition of the phospholipase on the same system. In general, the activation of the phospholipases correlated with the DAG-induced defects of the lipid bilayer structure. The results, however, argue against general designation of DAGs as "activators" or "inhibitors" of PL-A2S. Thus, for example, diolein activated phospholipases with the BL-PC lipid substrate, but inhibited them with the DPPC substrate. Dihexanoylglycerol and dioctanoylglycerol inhibited pig pancreatic PL-A2 with both lipid substrates and inhibited cobra or been venom PL-A2 with the DPPC substrate, but activated the latter two enzymes with the BL-PC substrate. Longer-chain DAGs (C greater than 12), which induce lateral phase separation of the bilayers into the regions of different fluidities, activated all PL-A2S with both lipid substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the Positional-Species Composition of Plant Reserve Triacylglycerols by Partial Chemical Deacylation

To determine the positional-species composition (PSC) of the sunflower oil triacylglycerols (TAGs), these TAGs were separated in a native state by preparative TLC on alumina and subjected to a partial chemical deacylation with the Grignard reagent under anhydrous conditions. The rac-1,2-diacylglycerols (DAGs) formed in this reaction were identified by comparing their TLC mobilities with the respective standard separated from the products of enzymatic hydrolysis of lecithin. Subsequently, the DAGs as their borate complexes were isolated from the reaction mixture by preparative TLC on silica gel. The quantitative fatty acid composition of both the DAGs and the initial TAGs was determined by capillary GLC. The PSC of TAGs calculated from the data obtained using a computer program was consistent with the data on sunflower TAG composition reported by other researchers using an independent method. Thus, the technique developed here makes it possible to obtain reliable data on the concentration of all TAG positional species in the plant tissue.     

Determination of the molecular species composition of diacylglycerols in human adipose tissue by solid-phase extraction and gas chromatography on a polar phase

The free diacylglycerols (DAGs) in adipose tissue are involved in the metabolism of stored lipids and hence are related to the supply of fatty acids for other tissues. This paper describes a simple, fast, and reproducible method for the identification and quantification of different molecular species of DAGs in human adipose tissue. The method comprised solid-phase extraction on a diol-bonded phase column combined with capillary GC analysis of silylated DAG derivatives on a polar phase (65% phenylmethylsilicone). Separation of the DAGs was achieved based on chain length, isomeric structure (1,2- and 1,3-DAGs), and degree of unsaturation. The main DAGs were 1,2-OO, 1,2-OP, 1,2-LO and 1,2-LP. The composition was corroborated by analysis of the component fatty acids of the DAGs, 18:1(n-9), 16:0, and 18:2(n-6) being the three major fatty acids obtained.     

Effects of diacylglycerols on the structure of phosphatidylcholine bilayers: a 2H and 31P NMR study

The interaction of four diacylglycerols (DAGs) with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts was investigated by a combination of 31P and 2H NMR spectrometry. We found that saturated and unsaturated long-chain DAGs induce different types of perturbations into the bilayer structure. The saturated DAGs dipalmitin and distearin induce lateral phase separation of the lipids into (i) DAG-enriched gellike domains and (ii) relatively DAG-free regions in the liquid-crystalline phase. In the latter regions, the order parameters along the fatty acyl chains of DPPC are practically identical with the control. This phase separation effect was observed in both model systems studied, and its extent is dependent upon DAG concentration and temperature. Only bilayer phases were present upon addition of dipalmitin or distearin at all concentrations and temperatures studied. The unsaturated DAGs diolein and DAG derived from egg PC (egg-DAG) affect PC bilayers in the following two ways: (i) by increasing the order parameters of the side chains, as observed for both DPPC and BL-PC model systems; (ii) by inducing nonbilayer lipid phases, as observed for BL-PC, but not DPPC. At a concentration of 25 mol % of an unsaturated DAG in mixed PC bilayers, a peak corresponding to isotropic lipid conformation appeared and increased in intensity with increase in temperature, while at 32 mol % hexagonal and bilayer phases coexisted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diacylglycerols interact with the L2 lipidation site in TRPC3 to induce a sensitized channel state

Coordination of lipids within transient receptor potential canonical channels (TRPCs) is essential for their Ca²⁺ signaling function. Single particle cryo‐EM studies identified two lipid interaction sites, designated L1 and L2, which are proposed to accommodate diacylglycerols (DAGs). To explore the role of L1 and L2 in TRPC3 function, we combined structure‐guided mutagenesis and electrophysiological recording with molecular dynamics (MD) simulations. MD simulations indicate rapid DAG accumulation within both L1 and L2 upon its availability within the plasma membrane. Electrophysiological experiments using a photoswitchable DAG‐probe reveal potentiation of TRPC3 currents during repetitive activation by DAG. Importantly, initial DAG exposure generates a subsequently sensitized channel state that is associated with significantly faster activation kinetics. TRPC3 sensitization is specifically promoted by mutations within L2, with G652A exhibiting sensitization at very low levels of active DAG. We demonstrate the ability of TRPC3 to adopt a closed state conformation that features partial lipidation of L2 sites by DAG and enables fast activation of the channel by the phospholipase C‐DAG pathway.     

A stochastic approach to estimating earliest start times of nodes for scheduling DAGs on heterogeneous distributed computing systems

Previously, DAG scheduling schemes used the mean (average) of computation or communication time in dealing with temporal heterogeneity. However, it is not optimal to consider only the means of computation and communication times in DAG scheduling on a temporally (and spatially) heterogeneous distributed computing system. In this paper, it is proposed that the second order moments of computation and communication times, such as the standard deviations, be taken into account in addition to their means, in scheduling “stochastic” DAGs. An effective scheduling approach which accurately estimates the earliest start time of each node and derives a schedule leading to a shorter average parallel execution time has been developed. Through an extensive computer simulation, it has been shown that a significant improvement (reduction) in the average parallel execution times of stochastic DAGs can be achieved by the proposed approach.     

The effects of acyl chain length and saturation of diacylglycerols and phosphatidylcholines on membrane monolayer curvature

The second messenger, diacylglycerol (DAG), introduces negative curvature in phospholipid monolayers and strongly induces the lamellar (L(alpha)) to reverse hexagonal (H(II)) phase transition. The chain lengths and degree of unsaturation of symmetric DAGs influence this effect. Within dioleoylphosphatidylcholine (DOPC) monolayers, the apparent spontaneous radius of curvature (R(0)) of the short, saturated dicaprylglycerol (C10-DCG) itself was determined to be -13.3 A, compared with an R(0) value of -10.1 A for the long, di-monounsaturated dioleoylglycerol (C18-DOG). Such increased length and unsaturation of the DAG acyl chains produces this small change. Di-saturated phosphatidylcholines (PCs) with equal length chains (from C10-C18) with 25 mol % DOG do not form the H(II) phase, even under the unstressed conditions of excess water and alkane. Di-unsaturated PCs with equal chain length (from C14-C18) with 25 mol % DOG do form the H(II) phase. Asymmetric chained PCs (position 1 saturated with varying lengths, position 2 differentially unsaturated with varying lengths) all form the H(II) phase in the presence of 25 mol % DOG. As a general rule for PCs, their unsaturation is critical for the induction of the H(II) phase by DOG. The degree of curvature stress induced by the second messenger DOG in membranes, and any protein that might be affected by it, would appear to depend on chain unsaturation of neighboring PCs.     

Des-acyl-ghrelin (DAG) normalizes hyperlactacidemia and improves survival in a lethal rat model of burn trauma

Critical illness, including burn injury, results in elevated plasma lactate levels. Dysregulation of PI3K/Akt signaling has been shown to play a predominant role in the inactivation of skeletal muscle PDC and, hence, in hyperlactacidemia in rat models of sepsis and endotoxemia. This observation, and our previous finding that DAG can reverse burn-induced skeletal muscle proteolysis through the activation of PI3K/Akt pathway, led us to hypothesize that DAG may also attenuate hyperlactacidemia in burn injury. Our investigations revealed that burn injury significantly elevated both skeletal muscle lactate production and plasma lactate levels. Moreover, this was accompanied in skeletal muscle by a 5–7 fold increase in mRNA expression of pyruvate dehydrogenase kinases (PDK) 2 and 4, and a ∼30% reduction in PDC activity. DAG treatment of burn rats completely normalized not only the mRNA expression of the PDKs and PDC activity, but also hyperlactacidemia within 24 h of burn injury. DAG also normalized epinephrine-induced lactate production by isolated skeletal muscles from normal rats. Moreover, DAG also improved survival in a lethal rat model of burn trauma. These findings with DAG may have clinical implications because chances of survival for critically ill patients are greatly improved if plasma lactate levels are normalized within 24 h of injury.     

Monounsaturated fatty acids are required for membrane translocation of protein kinase C-theta induced by lipid overload in skeletal muscle

Abstract

Protein kinase C (PKC) activation induced by diacylglycerols (DAGs) is one of the sequels of the dysregulation of intramuscular lipid metabolism and is thought to play an important role in the development of insulin resistance (IR). We tested the hypothesis that DAGs with different acyl chains have different biological effects and that DAG species enriched in monounsaturated fatty acids (MUFA) act as better activators of PKC. The experiments were performed in vitro on C2C12 myotubes treated with palmitate (16:0), stearate (18:0) or oleate (18:1) and in vivo on the skeletal muscles of rats fed high-fat (HF), high-tristearin (TS) or high-triolein (TO) diets. To define the importance of endogenously synthesized MUFA on DAG-induced PKCθ activation, we performed experiments on stearoyl-CoA desaturase 1 knockout mice (SCD1-/-) as well. The results show that the content of total DAGs and the levels of saturated DAG species are significantly increased in both insulin-resistant (16:0, HF and TO) and highly insulin-sensitive (18:0 and TS) groups. An increase in MUFA-containing DAGs levels was most constantly related to increase in PKCθ membrane translocation and IR. In the muscles of MUFA-deficient SCD1-/- mice, the DAG content and the induction of PKCθ translocation by the HF diet were significantly reduced. Collectively, our data from both the cell and animal experiments show that DAGs composed of 16:1 and/or 18:1, rather than the levels of total or saturated DAGs, are related to PKCθ membrane translocation. Moreover, our results show that the availability of dietary MUFA and/or the activity of endogenous desaturases play an important role in muscle DAG accumulation.     

Diacylglycerol metabolism in mast cells. Analysis of lipid metabolic pathways using molecular species analysis of intermediates

These studies assess the metabolic source and fate of cellular 1,2-diacylglycerol (DAG), an intermediate that increases with physiologic stimulation, participates in the regulation of protein phosphorylation, and acts as a substrate for arachidonic acid release. The quantitation of the molecular species of DAG and one of its metabolic products, phosphatidic acid (PA), was assessed in the purified rat mast cell, a model system with marked quantitative constraints but with rapid and extensive secretion after receptor stimulation. Cellular DAG was extracted, partially purified, radioactively phosphorylated to form [32P]PA, and, after conversion to its dimethyl phosphoric acid ester, molecular species separations were undertaken using reversed phase HPLC and/or argentation TLC. Quantitation of 0.5 pmol of a single molecular species of cellular DAG was achieved, but HPLC was not alone sufficient to resolve all molecular species of interest. More importantly, comparison of mast cell DAG with [32P]PA generated in 32Pi-prelabeled cells revealed that the sub-classes that contained arachidonic acid species represent only 11% of the total DAG, while that of [32P]PA was 41% in resting cells. [32P]PA and, to a variable extent, DAG showed preferential increases in arachidonate-containing subclasses after stimulation (to 50.9 and 13.9%, respectively). These data suggest that a large portion of the increased mass of DAG seen during stimulation was probably not derived by phosphoinositide hydrolysis. This type of molecular species analysis of intermediates of important phospholipid metabolic pathways should help to establish the metabolic origin and fate of these and other compounds.     

The interaction of dianhydrogalactitol with DNA in cultured Yoshida sarcoma cells

Using alkaline sucrose gradient sedimentation centrifugation it was found that treatment of Yoshida sarcoma cells in culture for 1 h with increasing concentrations of dianhydrogalactitol (DAG) enhanced the sedimentation rate of DNA in a dose-dependent manner. There was no difference between the amount of protein which co-sedimented with DNA released from treated and untreated cells. When DNA was extracted from the cells using a p-amino-salicylate-phenol mixture, the protein content of DNA seemed not to be affected by DAG. The possibility that DAG could form interstrand cross-linking in cellular DNA was suggested from renaturation studies. The appearance of a fast sedimenting DNA in the alkaline sucrose gradient and the evidence for a cross-linked DNA detected by renaturation technique, only appeared later than 6 h after treatment. A similar delayed effect on the depression in the rate of DNA synthesis was also observed. These data suggest that the inhibition of DNA synthesis may be related to the delayed formation of DNA interstrand cross-linked.     

Diacylglycerols containing Omega 3 and Omega 6 fatty acids bind to RasGRP and modulate MAP kinase activation

We elucidated the effects of different diacylglycerols (DAGs), i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG), and 1-stearoyl-2-eicosapentaenoyl-sn-glycerol (SEG), on [3H]PDBu binding to RasGRP. The competition studies with these DAGs on [3H]PDBu binding to RasGRP revealed different Ki values for these DAG molecular species. Furthermore, we transfected human Jurkat T cells by a plasmid containing RasGRP and assessed the implication of endogenous DAGs on activation of MAP kinases ERK1/ERK2, induced by phorbol-12-myristate-13-acetate (PMA). In control cells, GF109203X, a protein kinase C inhibitor, inhibited ERK1/ERK2 activation. However, this agent curtailed but failed to completely diminish ERK1/ERK2 phosphorylation in RasGRP-overexpressing cells, though calphostin C, a DAG binding inhibitor, suppressed the phosphorylation of MAP kinases in these cells. In cells incubated with arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), PMA induced the production of endogenous DAGs containing these fatty acids, respectively: DAG-AA, DAG-DHA, and DAG-EPA. The inhibition of production of DAG-AA and DAG-DHA significantly inhibited MAP kinase activation in RasGRP overexpressing, but not in control, cells. Our study demonstrates that three DAG molecular species bind to RasGRP, but only DAG-AA and DAG-DHA participate in the modulation of RasGRP-mediated activation of MAP kinases in Jurkat T cells.     

Quantitative analysis of molecular species of diacylglycerol and phosphatidate formed upon muscarinic receptor activation of human SK-N-SH neuroblastoma cells.

Quantitative changes in the total mass and the molecular species of 1,2-diacyl-sn-glycerol (DAG) and phosphatidic acid (PA) formed upon muscarinic receptor activation were studied in cultured human SK-N-SH neuroblastoma cells. DAG was isolated from the total lipid extracts of carbachol (CCh)-stimulated and unstimulated cells and after benzoylation, was subjected to reverse phase high performance liquid chromatography to separate the component species. The molecular species of DAG were identified by analyzing the fatty acid composition of each separated fraction by gas chromatography, and their total and individual masses were quantified from the known amount of an internal standard, 1,2-distearoyl-sn-glycerol, added during the extraction of the lipid. Relatively high basal levels of DAG (1.5 nmol/mg protein) are present in these cells, and addition of CCh elicited a 50-60% increase in the total amounts of DAG within 5 min. The increase was biphasic: an initial major peak at 5 min was followed by a sustained increase that persisted for at least 30 min. An increase in DAG was elicited by both full and partial muscarinic agonists and was blocked by atropine. The presence of extracellular Ca2+ was necessary for muscarinic receptor-activated formation of DAG. To determine the source of the DAG, the molecular species of the major phospholipids present in SK-N-SH cells were also analyzed. The phospholipids were first enzymatically hydrolyzed to DAGs which were then analyzed as described above. A number of unusual fatty acids, the major one being 20:3 (n-9), were present in these lipids especially in the phosphoinositides and also in the DAG formed after CCh stimulation. Within 5 s of CCh stimulation there were transient increases in the DAG species representative of phosphoinositides. By 5 min the newly formed molecular species of DAG resembled a mixture of phosphoinositides and phosphatidylcholine (PC). Quantitative comparison of the molecular species compositions of phosphoinositides, PC, and newly formed DAGs indicated that at time periods up to 10 min, approximately 30% of the DAG originated from the phosphoinositides and the rest from PC. At longer intervals (greater than 20 min), most (85%) of DAGs originated from PC. Activation of muscarinic receptors in SK-N-SH cells also elicited an increase in PA (200% in 5 min). A quantitative molecular species analysis, using 1,2-distearoyl-sn-glycerol-3-P as internal standard, was performed by enzymatic (alkaline phosphatase) hydrolysis of PA to DAG and subsequent analysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of Modulators of Protein Kinase C Activity on Ca2+ Transport in Retinal Rod Microsomes

The effect of modulators of protein kinase C (PKC) activity on Ca²⁺ translocation in retinal rod microsomes was studied. It is shown that PKC activators (phorbol 12-myristate-13-acetate (PMA) and diacylglycerol (DAG)) and inhibitors (chelerythrine chloride, polymyxin B, and phloretin) stimulate and inhibit ATP-dependent Ca²⁺ uptake in retinal rod microsomes, respectively. This effect is apparently due to an influence of PKC on Ca-ATPase contained in these vesicular structures. It was found that PKC inhibitors (chelerythrine chloride, polymyxin B, and phloretin) and activators (PMA and DAG) potentiate Ca²⁺ release from Ca²⁺ -loaded retinal rod microsomes. Specific and nonspecific mechanisms of Ca-release stimulation by the modulators of PKC activity are discussed.     

The sustained second phase of hormone-stimulated diacylglycerol accumulation does not activate protein kinase C in GH3 cells

Numerous hormones activate cells through receptor-regulated hydrolysis of phosphoinositides resulting in elevated cellular diacylglycerol (DAG), an activator of protein kinase C (PKC). Our previous studies showed that thyrotropin-releasing hormone (TRH) treatment of GH3 cells stimulated a rapid (less than 10 s) but transient (less than 60 s) association of cytosolic PKC with the membrane. In this study, we investigated the roles of hormone-stimulated Ca2+ and DAG levels in initiating and terminating the membrane association of PKC. The initial effects of TRH were not mimicked by elevating CA2+ levels, however, inhibiting TRH-stimulated Ca2+ increases blocked hormone-stimulated PKC translocation. Hence, the TRH stimulation of both Ca2+ and DAG levels were essential for the initial PKC translocation. The termination of PKC membrane association could not be attributed to proteolysis of PKC nor to limiting Ca2+ levels. Treatment of cells with phorbol diesters potentiated and prolonged the effects of TRH on PKC translocation, suggesting that DAG levels limited the membrane association of PKC. Since TRH stimulated a sustained increase in DAG levels, DAG composition was analyzed. There was a marked shift in DAG from tetraenoic (at 15 s) to more saturated DAGs at longer times. In addition, increases in plasma membrane DAG in response to TRH were transient rather than sustained. We propose that the TRH stimulation of PKC translocation is short-lived due to the metabolism of plasma membrane DAGs which are effective in promoting PKC activation. In contrast, DAGs which accumulate in intracellular membranes during the sustained phase of TRH treatment appear to be ineffective as activators of PKC.     

从薄层层析到天然产物工业制备层析的理论与实践

基于分离理论的共性,通过薄层层析优化难分离物质对的最佳分离条件并直接放大到植物天然产物的工业制备分离。本文对实际应用中的溶剂组成、溶剂强度、溶剂用量及溶剂再利用等问题作了详细论述。最佳溶剂选择性可通过不同溶剂系统在薄层板上所给难分离物质对的最大Rf比值确定;在正相硅胶层析中,主要极性溶剂决定了物质的洗脱顺序;洗脱剂中极少量强极性溶剂(O．5％)或pH值的改变可显著改善拖尾现象;根据Rf值和容量因子(K)及洗脱体积三者之间的关系,Rf在0．1～0．3为用于柱层析的洗脱溶剂强度的最佳范围。     

Streptomyces chromofuscus phospholipase D interaction with lipidic activators at the air-water interface

The phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme that interacts with membranes to catalyse phosphatidylcholine (PC) transformation. In this work, we focused on the interaction between PLDSc and two lipid activators: a neutral lipid, diacylglycerol (DAG), and an anionic one, phosphatidic acid (PA). DAG is a naturally occurring alcohol, so it is a potent nucleophile for the transphosphatidylation reaction catalysed by PLD. Concerning PA, it is a widely described activator of PLDSc-catalysed hydrolysis of PC.The monolayer technique allowed us to define PLDSc interaction with DAG and PA. In the case of DAG, the results suggest an insertion of PLDSc within the acyl chains of the lipid with an exclusion pressure of approximately 45 mN/m. PLDSc-DAG interaction seemed to occur preferentially with the lipid in the liquid-expanded (LE) phase.PLDSc interaction with PA was found to be more effective at high surface pressures. The overall results obtained with PA show a preferential interaction of the protein with condensed PA domains. No exclusion pressure could be found for PLDSc-PA interaction indicating only superficial interaction with the polar head of this lipid. Brewster angle microscopy (BAM) images were acquired in order to confirm these results and to visualise the patterns induced by PLDSc adsorption.     

Arachidonate activation of protein kinase C may be involved in the stimulation of protein synthesis by insulin in L6 myoblasts

Insulin stimulated protein synthesis in L6 myoblasts but did not increase the labelling of DAG or the release of phosphocholine from phosphatidylcholine. The DAG lipase inhibitor, RHC 80267, more than doubled the amount of label appearing in DAG but did not stimulate protein synthesis. Even in the presence of the DAG lipase inhibitor insulin failed to have any effect on DAG labelling, and conversely RHC 80267 did not modify the insulin-induced increase in protein synthesis. These results suggest that endogenous DAG production is not involved in the stimulation of protein synthesis by insulin. However, exogenous diacylglycerols (1-oleoyl-2-acetyl glycerol and 1-stearoyl-2-arachidonoyl glycerol) both stimulated protein synthesis in L6 myoblasts. The efficacy of the former (arachidonatefree) DAG suggested that their action was by activation of protein kinase C rather than by arachidonate release and prostaglandin formation. Ibuprofen, an inhibitor of cyclo-oxygenase failed to block the effects of insulin whereas a second cyclo-oxygenase inhibitor, indomethacin had only a partial inhibitory effect. The protein kinase C (PKC) inhibitor, RO-31-8220, totally blocked the effect of insulin. Since indomethacin is also recognised to inhibit phospholipase A₂, the data suggests that insulin acts on protein synthesis in myoblasts by arachidonate activation of PKC.     

Interactions of saturated diacylglycerols with phosphatidylcholine bilayers: A 2H NMR study.

The interactions of a series of saturated diacylglycerols (DAGs) with fatty acid side chain lengths of 6-14 carbons with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts were studied by 2H NMR spectrometry. We found that the perturbation induced by the DAGs into the bilayer structure strongly depends on the length of the DAG fatty acid side chain. Shorter chain 1,2-sn-dihexanoylglycerol and, to a larger degree, 1,2-sn-dioctanoylglycerol (diC8) induce transverse perturbation of the bilayer structure: the order parameters of the phospholipid side chains are increased by the intercalating DAG molecules in the region adjacent to the phospholipid headgroups and decreased toward the terminal methyls, corresponding to the bilayer interior. The longer chain DAGs (C greater than or equal to 12) studied in this and previous [De Boeck & Zidovetzki (1989) Biochemistry 28, 7439] work induce lateral phase separation of the lipids into DAG-enriched gellike domains and relatively DAG-free regions in the liquid-crystalline phase. Each of the DAGs studied induces a decrease in the area per phospholipid molecule, and a corresponding increase in the lateral surface pressure of the bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)