Discovery of Human Intestinal MGAT Inhibitors Using High-Throughput Mass Spectrometry.

Monoacylglycerol acyltransferase (MGAT) activity catalyzes the synthesis of diacylglycerol (DAG) from fatty acyl-CoA and monoacylglycerol as substrates. It is important for the resynthesis of triacylglycerol (TAG) in the intestine. In the present study, we developed a MGAT enzymatic assay of human intestinal microsomes using a high-throughput mass spectrometry (MS)-based detection system. After screening with small-molecular-weight libraries for compounds exhibiting inhibitions against DAG and the consequent TAG syntheses, we identified multiple compounds that specifically inhibit intestinal MGAT activity. The inhibitory activities of these compounds were correlated to those determined using a recombinant human MGAT2 enzyme. An aryl-sulfonamide compound T1 showed potent inhibitory activity toward human intestinal MGAT and recombinant human MGAT2, with selectivity over MGAT3. This high-throughput MS-based assay provides a novel platform for the discovery of DAG or TAG synthesis inhibitors. The identified aryl-sulfonamide compound T1 is a promising starting compound for optimization studies of inhibitors with selectivity toward MGAT2.

Novel LC/MS/MS and High-Throughput Mass Spectrometric Assays for Monoacylglycerol Acyltransferase Inhibitors.

Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography-tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.

Pharmacological Inhibition of Monoacylglycerol O-Acyltransferase 2 Improves Hyperlipidemia, Obesity, and Diabetes by Change in Intestinal Fat Utilization.

Monoacylglycerol O-acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DG), a triacylglycerol precursor and potential peripheral target for novel anti-obesity therapeutics. High-throughput screening identified lead compounds with MGAT2 inhibitory activity. Through structural modification, a potent, selective, and orally bioavailable MGAT2 inhibitor, compound A (compA), was discovered. CompA dose-dependently inhibited postprandial increases in plasma triglyceride (TG) levels. Metabolic flux analysis revealed that compA inhibited triglyceride/diacylglycerol resynthesis in the small intestine and increased free fatty acid and acyl-carnitine with shorter acyl chains than originally labelled fatty acid. CompA decreased high-fat diet (HFD) intake in C57BL/6J mice. MGAT2-null mice showed a similar phenotype as compA-treated mice and compA did not suppress a food intake in MGAT2 KO mice, indicating that the anorectic effects were dependent on MGAT2 inhibition. Chronic administration of compA significantly prevented body weight gain and fat accumulation in mice fed HFD. MGAT2 inhibition by CompA under severe diabetes ameliorated hyperglycemia and fatty liver in HFD-streptozotocin (STZ)-treated mice. Homeostatic model assessments (HOMA-IR) revealed that compA treatment significantly improved insulin sensitivity. The proximal half of the small intestine displayed weight gain following compA treatment. A similar phenomenon has been observed in Roux-en-Y gastric bypass-treated animals and some studies have reported that this intestinal remodeling is essential to the anti-diabetic effects of bariatric surgery. These results clearly demonstrated that MGAT2 inhibition improved dyslipidemia, obesity, and diabetes, suggesting that compA is an effective therapeutic for obesity-related metabolic disorders.

Exercise-induced cardiac performance in autoimmune (type 1) diabetes is associated with a decrease in myocardial diacylglycerol.

One of the fundamental biochemical defects underlying the complications of diabetic cardiovascular system is elevation of diacylglycerol (DAG) and its effects on protein kinase C (PKC) signaling. It has been noted that exercise training attenuates poor cardiac performance in Type 1 diabetes. However, the role of PKC signaling in exercise-induced alleviation of cardiac abnormalities in diabetes is not clear. We investigated the possibility that exercise training modulates PKC-ßII signaling to elicit its beneficial effects on the diabetic heart. bio-breeding diabetic resistant rats, a model reminiscent of Type 1 diabetes in humans, were randomly assigned to four groups: 1) nonexercised nondiabetic (NN); 2) nonexercised diabetic (ND); 3) exercised nondiabetic; and 4) exercised diabetic. Treadmill training was initiated upon the onset of diabetes. At the end of 8 wk, left ventricular (LV) hemodynamic assessment revealed compromised function in ND compared with the NN group. LV myocardial histology revealed increased collagen deposition in ND compared with the NN group, while electron microscopy showed a reduction in the viable mitochondrial fraction. Although the PKC-ßII levels and activity were unchanged in the diabetic heart, the DAG levels were increased. With exercise training, the deterioration of LV structure and function in diabetes was attenuated. Notably, improved cardiac performance in training was associated with a decrease in myocardial DAG levels in diabetes. Exercise-induced benefits on cardiac performance in diabetes may be mediated by prevention of an increase in myocardial DAG levels.

The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response.

Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.

Vitellogenesis in Oncopeltus fasciatus: PLC/IP(3), DAG/PK-C pathway triggered by CaM.

In Oncopeltus fasciatus, evidence shown here indicates it is calmodulin (CaM) that activates phospholipase-C (PLC), beginning a signalling pathway necessary for endocytic uptake of yolk precursor molecules. Epithelial cell-produced CaM, transported to oocytes via gap junctions, has been shown to be required for receptor-mediated endocytic uptake of vitellogenins (Vgs, the protein precursors of yolk). To determine if CaM was directly or indirectly stimulating the phospholipase-C (PLC) signalling cascade and thus controlling Vg endocytosis we used a series of molecules known to inactivate various elements of the pathway. W-7 prevents CaM from interacting with other molecules. Neomycin isolates PIP(2) from PLC. U-73122 directly inactivates PLC. 2-APB blocks IP(3) receptors which would otherwise cause release of Ca(2+). Verapamil and CdCl(2) block Ca(2+) release channels. Staurosporin and calphostin are inhibitors of PK-C. 1-Hexadecyl-2-acetyl glycerol (HAG) binds to diacylglycerol (DAG). Through the use of these antagonists we show here that: (1) the activation of phospholipase-C in this system requires CaM. (2) Stimulated phospholipase-C converts PIP(2) into IP(3) and DAG. (3) IP(3) causes increase in cytosolic Ca(2+). (4) DAG and Ca(2+) each stimulate phosphokinase-C, resulting in endocytosis of Vgs.

Protein kinase D-dependent trafficking of the large Herpes simplex virus type 1 capsids from the TGN to plasma membrane.

The biosynthetic pathway carries cargos from the endoplasmic reticulum (ER) to the trans Golgi network (TGN) via a typical passage through the Golgi. Interestingly, large particles such as procollagen, chylomicrons and some viruses all reach the TGN by atypical routes. Given this dichotomy, we anticipated that such cargos might rely on non-classical machineries downstream of the TGN. Using Herpes simplex virus type 1 (HSV-1) as a model and a synchronized infection protocol that focuses on TGN to plasma membrane transport, the present study revealed the surprising implication of the cellular serine-threonine protein kinase D in HSV-1 egress. These findings, confirmed by a variety of complementary means [pharmacological inhibitors, dominant negative mutant, RNA interference and electron microscopy (EM)], identify one of possibly several cellular factors that modulate the egress of viruses transiting at the TGN. Moreover, the involvement of this kinase, previously known to regulate the transport of small basolateral cargos, highlights the trafficking of both small and exceptionally large entities by a common machinery downstream of the TGN, in sharp contrast to earlier steps of transport. Conceptually, this indicates the TGN is not only a sorting station from which cargos can depart towards different destinations but also a meeting point where conventional and unconventional routes can meet along the biosynthetic pathway. Lastly, given the apical release of HSV-1 in neurons, it opens up the possibility that this kinase might regulate some apical sorting.

TRPC channels and diacylglycerol dependent calcium signaling in rat sensory neurons.

Transient receptor potential (TRP) channels of the TRPV, TRPA, and TRPM subfamilies play important roles in somatosensation including nociception. While particularly the Thermo TRPs have been extensively investigated in sensory neurons, the relevance of the subclass of "canonical" TRPC channels in primary afferents is yet elusive. In the present study, we investigated the presence and contribution to Ca(2+) transients of TRPC channels in dorsal root ganglion neurons. We found that six of the seven known TRPC subtypes were expressed in lumbar DRG, with TRPC1, C3, and C6 being the most abundant. Microfluorimetric calcium measurements showed Ca(2+) influx induced by oleylacylglycerol (OAG), an activator of the TRPC3/C6/C7 subgroup. Furthermore, OAG induced rises in [Ca(2+)](i) were inhibited by SKF96365, an inhibitor of receptor and store operated calcium channel. OAG induced calcium transients were also inhibited by blockers of diacylglycerol (DAG) lipase, lipoxygenase or cyclooxygenase and, intriguingly, by inhibitors of the capsaicin receptor TRPV1. Notably, SKF96365 did not affect capsaicin-induced calcium transients. Taken together, our findings suggest that TRPC are functionally expressed in subpopulations of DRG neurons. These channels, along with TRPV1, contribute to calcium homeostasis in rat sensory neurons.

The Na+/Ca2+ exchange inhibitor KB-R7943 potently blocks TRPC channels.

Na(+)/Ca(2+) exchangers (NCXs) and members of the canonical transient receptor potential (TRPC) channels play an important role in Ca(2+) homeostasis in heart and brain. With respect to their overlapping expression and their role as physiological Ca(2+) influx pathways a functional discrimination of both mechanisms seems to be necessary. Here, the effect of the reverse-mode NCX inhibitor KB-R7943 was investigated on different TRPC channels heterologously expressed in HEK293 cells. In patch-clamp recordings KB-R7943 potently blocked currents through TRPC3 (IC(50)=0.46 microM), TRPC6 (IC(50)=0.71 microM), and TRPC5 (IC(50)=1.38 microM). 1-Oleoyl-2-acetyl-sn-glycerol-induced Ca(2+) entry was nearly completely suppressed by 10 microM KB-R7943 in TRPC6-transfected cells. Thus, KB-R7943 is able to block receptor-operated TRP channels at concentrations which are equal or below those required to inhibit reverse-mode NCX activity. These data further suggest that the protective effects of KB-R7943 in ischemic tissue may, at least partly, be due to inhibition of TRPC channels.

Adiponectin inhibits superoxide generation by human neutrophils.

Adiponectin (Ad), a member of the adipocytokine family, has been reported to possess antiinflammatory properties. We investigated the effects of full-length human Ad (hAd) on phorbol 12-myristate 13-acetate (PMA)-induced O2-* generation by human neutrophils. hAd, even at the lowest tested concentration of 0.001 microg/ml, after 30-min pretreatment of cells, significantly inhibited O2-* generation by neutrophils stimulated with PMA (100 nM). However, no relation between the dose of hAd and extent of inhibition of PMA-induced O2-* generation was observed with increasing the concentration of hAd up to 1 microg/ml. hAd also significantly inhibited neutrophil O2-* generation stimulated by N-formyl-methionyl-leucyl-phenylalanine (100 microM) and diacylglycerol (500 nM), as well as the PMA-induced neutrophil nitroblue tetrazolium reduction and H2O2 formation. Pretreatment of neutrophils with pronase-digested hAd failed to inhibit the PMA-induced O2-* generation. For the first time, this study revealed that Ad inhibited O2-* generation by neutrophils, possibly through regulation of NADPH oxidase.

Targeting of protein kinase C-epsilon during Fcgamma receptor-dependent phagocytosis requires the epsilonC1B domain and phospholipase C-gamma1.

Protein kinase C-epsilon (PKC-epsilon) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-epsilon mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding epsilonC1 and epsilonC1B domains, or the epsilonC1B point mutant epsilonC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that epsilonC259G, epsilonC1, and epsilonC1B accumulation at phagosomes was significantly less than that of intact PKC-epsilon. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-epsilon translocation. Thus, DAG binding to epsilonC1B is necessary for PKC-epsilon translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-gamma1, and PI-PLC-gamma2 in PKC-epsilon accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-epsilon localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-epsilon accumulation. Although expression of PI-PLC-gamma2 is higher than that of PI-PLC-gamma1, PI-PLC-gamma1 but not PI-PLC-gamma2 consistently concentrated at phagosomes. Macrophages from PI-PLC-gamma2-/- mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-epsilon at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-gamma1 as the enzyme that supports PKC-epsilon localization and phagocytosis. That PI-PLC-gamma1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-gamma1 provides DAG that binds to epsilonC1B, facilitating PKC-epsilon localization to phagosomes for efficient IgG-mediated phagocytosis.

An essential role for phospholipase D in the activation of protein kinase C and degranulation in mast cells.

Activation of phospholipase D (PLD) and protein kinase C (PKC) as well as calcium mobilization are essential signals for degranulation of mast cells. However, the exact role of PLD in degranulation remains undefined. In this study we have tested the hypothesis that the PLD product, phosphatidic acid, and diacylglycerides generated therefrom might promote activation of PKC. Studies were conducted in two rodent mast cell lines that were stimulated with Ag via FcepsilonRI and a pharmacologic agent, thapsigargin. Diversion of production of phosphatidic acid to phosphatidylbutanol (the transphosphatidylation reaction) by addition of l-butanol suppressed both the translocation of diacylglyceride-dependent isoforms of PKC to the membrane and degranulation. Tertiary-butanol, which is not a substrate for the transphosphatidylation, had a minimal effect on PKC translocation and degranulation, and 1-butanol itself had no effect on PKC translocation when PKC was stimulated directly with phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. Also, in cells transfected with small inhibitory RNAs directed against PLD1 and PLD2, activation of PLD, generation of diacylglycerides, translocation of PKC, and degranulation were all suppressed. Phorbol ester, which did not stimulate degranulation by itself, restored degranulation when used in combination with thapsigargin whether PLD function was disrupted with 1-butanol or the small inhibitory RNAs. However, degranulation was not restored when cells were costimulated with Ag and phorbol ester. These results suggested that the production of phosphatidic acid by PLD facilitates activation of PKC and, in turn, degranulation, although additional PLD-dependent processes appear to be critical for Ag-mediated degranulation.

Intramolecular occlusion of the diacylglycerol-binding site in the C1 domain of munc13-1.

Protein kinase C (PKC) isozymes and other receptors of diacylglycerol (DAG) bind to this widespread second messenger through their C(1) domains. These alternative DAG receptors include munc13-1, a large neuronal protein that is crucial for DAG-dependent augmentation of neurotransmitter release. Whereas the structures of several PKC C(1) domains have been determined and have been shown to require little conformational changes for ligand binding, it is unclear whether the C(1) domains from other DAG receptors contain specific structural features with key functional significance. To gain insight into this question, we have determined the three-dimensional structure in solution of the munc13-1 C(1) domain using NMR spectroscopy. The overall structure includes two beta-sheets, a short C-terminal alpha-helix, and two Zn(2+)-binding sites, resembling the structures of PKC C(1) domains. However, the munc13-1 C(1) domain exhibits striking structural differences with the PKC C(1) domains in the ligand-binding site. These differences result in occlusion of the binding site of the munc13-1 C(1) domain by a conserved tryptophan side chain that in PKCs adopts a completely different orientation. As a consequence, the munc13-1 C(1) domain requires a considerable conformational change for ligand binding. This structural distinction is expected to decrease the DAG affinity of munc13-1 compared to that of PKCs, and is likely to be critical for munc13-1 function. On the basis of these results, we propose that augmentation of neurotransmitter release may be activated at higher DAG levels than PKCs as a potential mechanism for uncoupling augmentation of release from the multitude of other signaling processes mediated by DAG.

Role of diacylglycerol induced by hypoxia in the regulation of HIF-1alpha activity.

Hypoxia-inducible factor 1 (HIF-1) is a critical transcription factor for the adaptation to lowered oxygen environments. We have previously reported that hypoxia induced phosphatidic acid (PA) accumulation through diacylglycerol kinase (DGK) activity and provided evidence that this PA production regulated HIF-1 expression. Here we report that hypoxia also produces a marked intracellular accumulation of diacylglycerol (DAG) in different cell types. The previously proposed inhibitor of phosphatidylcholine phospholipase C (PC-PLC)/sphingomyelin synthase (SMS) activities, D609, specifically abrogates both hypoxia-dependent DAG accumulation and hypoxia-induced HIF-1 expression. We show that DAG-dependent protein kinase C (PKC) isoforms do not play an essential role in the regulation of HIF-1 expression. D609 inhibits PA accumulation triggered by hypoxia, suggesting that DAG could act as substrate for its conversion into PA by DGK upon these conditions. Therefore, this work provides novel evidence for the existence of DAG/PA-dependent intracellular mechanisms involved in the regulation of HIF-1 expression.

AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists.

M-type (KCNQ2/3) potassium channels are suppressed by activation of G(q/11)-coupled receptors, thereby increasing neuronal excitability. We show here that rat KCNQ2 can bind directly to the multivalent A-kinase-anchoring protein AKAP150. Peptides that block AKAP150 binding to the KCNQ2 channel complex antagonize the muscarinic inhibition of the currents. A mutant form of AKAP150, AKAP(DeltaA), which is unable to bind protein kinase C (PKC), also attenuates the agonist-induced current suppression. Analysis of recombinant KCNQ2 channels suggests that targeting of PKC through association with AKAP150 is important for the inhibition. Phosphorylation of KCNQ2 channels was increased by muscarinic stimulation; this was prevented either by coexpression with AKAP(DeltaA) or pretreatment with PKC inhibitors that compete with diacylglycerol. These inhibitors also reduced muscarinic inhibition of M-current. Our data indicate that AKAP150-bound PKC participates in receptor-induced inhibition of the M-current.

Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin.

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.

Nuclear diacylglycerol produced by phosphoinositide-specific phospholipase C is responsible for nuclear translocation of protein kinase C-alpha.

It is well established that an independent inositide cycle is present within the nucleus, where it is involved in the control of cell proliferation and differentiation. Previous results have shown that when Swiss 3T3 cells are treated with insulin-like growth factor-I (IGF-I) a rapid and sustained increase in mass of diacylglycerol (DAG) occurs within the nuclei, accompanied by a decrease in the levels of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. However, it is unclear whether or not other lipids could contribute to this prolonged rise in DAG levels. We now report that the IGF-I-dependent increase in nuclear DAG production can be inhibited by the specific phosphatidylinositol phospholipase C inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or by neomycin sulfate but not by the purported phosphatidylcholine-phospholipase C specific inhibitor D609 or by inhibitors of phospholipase D-mediated DAG generation. Treatment of cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or neomycin sulfate inhibited translocation of protein kinase C-alpha to the nucleus. Moreover, exposure of cells to 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, but not to D609, dramatically reduced the number of cells entering S-phase upon stimulation with IGF-I. These results suggest that the only phospholipase responsible for generation of nuclear DAG after IGF-I stimulation of 3T3 cells is PI-PLC. When this activity is inhibited, neither DAG rise is seen nor PKC-alpha translocation to the nucleus occurs. Furthermore, this PI-PLC activity appears to be essential for the G0/G1 to S-phase transition.

Urokinase-dependent angiogenesis in vitro and diacylglycerol production are blocked by antisense oligonucleotides against the urokinase receptor.

The plasminogen activator system is known to play a crucial role in the angiogenesis process by modulating the adhesive properties of endothelial cells to the extracellular matrix and cell-cell interaction. In the present study, we demonstrated that the urokinase-type plasminogen activator (u-PA) induced neovascular growth in the avascular rabbit cornea and dose-dependently promoted growth, chemotaxis, and matrix invasion of cultured endothelial cells. Interaction between u-PA and its receptor appears to be mandatory for the angiogenic effect of u-PA because monoclonal antibodies anti-u-PA and anti-u-PA receptor (u-PAR) blocked the proangiogenic effects of u-PA at the endothelial cell level. We then assessed the signaling pathway activated in endothelial cells by u-PA. u-PAR activation by u-PA produced de novo synthesis of diacylglycerol (DAG) from glucose by a cytochalasin B-inhibitable mechanism, indicating the involvement of a specific glucose transporter (GLUT). Endothelial cells expressed GLUT2, whose activation was tyrosine kinase-dependent and protein kinase C (PKC)-independent. The increase of glucose uptake led to DAG production, which resulted in PKC activation/translocation. Impairment of u-PAR availability by monoclonal antibodies and by antisense oligonucleotides (aODN) against u-PAR mRNA inhibited glucose uptake, DAG neosynthesis, and PKC activation, resulting in the blockade of endothelial cell proliferation, chemotaxis, and chemoinvasion. These data suggest that u-PAR activation consequent to the binding of u-PA can be regarded as an "angiogenic switch" and disclose the possibility that an anti-u-PAR aODN strategy may efficiently target endothelial cell function to control angiogenesis in vivo.

Adenosine A1 receptor stimulation antagonizes the negative inotropic effects of the PKC activator dioctanoylglycerol.

It has been suggested that adenosine cardioprotection occurs via adenosine A1 receptor-mediated activation of protein kinase C (PKC). However, adenosine has well-known vasodilatory effects in the myocardium, whereas PKC is a vasoconstrictor. This study examined whether adenosine A1 receptor activation alters the effects of the PKC activator. 1,2-dioctanoyl-s,n-glycerol (DOG) in isolated perfused rat hearts (left-ventricular developed pressure) and rat ventricular myocytes ([Ca2+]i and cell shortening). Exposure to DOG decreased left-ventricular developed pressure by 30%, an effect that was completely reversible. Pretreatment of isolated hearts with either the PKC inhibitor chelerythrine or the adenosine A1 agonist 2-chloro-N6-cyclo-cyclo-isolated pentlyadenosine (CCPA) attenuated the negative inotropic effects of DOG. In the isolated myocytes, DOG decreased [Ca2+]i and cell shortening by 25 and 28%, respectively, effects that were attenuated by both chelerythrine and CCPA. The CCPA attenuation of the DOG-induced decrease in [Ca2+]i and cell shortening was blocked by pretreating the myocytes with the adenosine A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). These results indicate that in rat ventricular myocardium, adenosine A1 receptor activation attenuates the apparent PKC-dependent negative inotropic effects of DOG via preservation of [Ca2+]i levels.

Retinoids inhibit protein kinase C-dependent transduction of 1,2-diglyceride signals in human colonic tumor cells.

1,2-Diglycerides with long-chain fatty acid residues related to nutritional fat (LCDGs) specifically affect growth and urokinase secretion in human colonic tumor cells, but not in normal mucosa. This allows them to advance and enhance carcinogenesis in the colon and rectum. SW480 colon carcinoma cells are LCDG sensitive in the same way as primary colonic tumor cells and have therefore been used as a model system to study the mechanism of LCDG action and to search for inhibitors of tumor development in the colon. Using this model system, we have shown that the effects of LCDGs are transmitted by protein kinase C and abolished by downregulation of the enzyme. Retinol, retinoic acid, and beta-carotene in nanomolar concentrations inhibit LCDG-induced growth and urokinase secretion and block stimulation of protein kinase C. Although retinol and retinoic acid at higher concentrations also display stimulatory activity, beta-carotene does not. At 100 nM, a concentration that can easily be reached in the plasma of humans, beta-carotene reduces LCDG-induced urokinase secretion about 50%. Inasmuch as beta-carotene does not have side effects due to intrinsic activities and storage effects, beta-carotene and foods rich in carotenes could be useful in the prevention of colorectal cancer.