The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2␣, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2␣ involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2␣ and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2␣ contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.
We have shown previously that palmitate treatment of C2C12 skeletal muscle myotubes causes inhibition of the protein kinase B (PKB) pathway and hence reduces insulin-stimulated glycogen synthesis through the elevation of intracellular ceramide levels. Ceramide is known to activate both atypical protein kinase C (aPKC) and protein phosphatase (PP) 2A, and each of these effectors has been reported to inhibit PKB. In the present study, palmitate pretreatment was found to elevate PP2A-like activity in myotubes and to prevent its inhibition by insulin. Incubation with the phosphatase inhibitor okadaic acid before insulin stimulation protected against the effect of the fatty acid on PKB phosphorylation. Palmitate was unable to inhibit PKB activity and glycogen synthesis in cells overexpressing the activated PKB mutant (T308D,S473D)-PKB␣, which is unaffected by phosphatase. In contrast, PKB activity and glycogen synthesis were still inhibited by palmitate in cells overexpressing a membrane-targeted and, hence, activated PKB mutant that retains sensitivity to phosphatase. Although aPKC activity was also increased in palmitate-treated cells, overexpression of wild-type or kinase-dead aPKC did not alter the inhibitory effects of the lipid on either stimulation of PKB or glycogen synthesis by insulin. We conclude that palmitate disrupts insulin signaling in C2C12 myotubes by promoting PP2A-like activity and, therefore, the dephosphorylation of PKB, which in turn reduces the stimulation of glycogen synthesis.
Thromboxane A2 (TXA2) is a biologically active metabolite of arachidonic acid formed by the action of the terminal synthase, thromboxane A2 synthase (TXA2S), on prostaglandin endoperoxide (PGH2). TXA2 is responsible for multiple biological processes through its cell surface receptor, the T-prostanoid (TP) receptor. Thromboxane A2 synthase and TP are the two necessary components for the functioning of this potent bioactive lipid. Thromboxane A2 is widely implicated in a range of cardiovascular diseases, owing to its acute and chronic effects in promoting platelet aggregation, vasoconstriction, and proliferation. In recent years, additional functional roles for both TXA2S and TP in cancer progression have been indicated. Increased cyclooxygenase (COX)-2 expression has been described in a variety of human cancers, which has focused attention on TXA2 as a downstream metabolite of the COX-2-derived PGH2. Several studies suggest potential involvement of TXA2S and TP in tumor progression, especially tumor cell proliferation, migration, and invasion that are key steps in cancer progression. In addition, the regulation of neovascularization by TP has been identified as a potent source of control during oncogenesis. There have been several recent reviews of TXA2S and TP but thus far none have discussed its role in cancer progression and metastasis in depth. This review will focus on some of the more recent findings and advances with a significant emphasis on understanding the functional role of TXA2S and TP in cancer progression and metastasis.
SummaryPhospholipase D (PLD) hydrolyzes the phosphodiester bond of the predominant membrane phospholipid, phosphatidylcholine producing phosphatidic acid and free choline. This activity can participate in signal transduction pathways and impact on vesicle trafficking for secretion and endocytosis, as well as receptor signalling. Phospholipids can regulate PLD activity directly, through specific intermolecular interactions, or indirectly, through their effect on the localization or activity of PLD's protein effectors. This short review highlights these various phospholipid inputs into the regulation of PLD activity and also reviews potential roles for PLD-generated phosphatidic acid, particularly a mechanism by which the phospholipid may participate in the process of vesicular trafficking.
We have previously shown that glycogen synthesis is reduced in lipid-treated C(2)C(12) skeletal muscle myotubes and that this is independent of changes in glucose uptake. Here, we tested whether mitochondrial metabolism of these lipids is necessary for this inhibition and whether the activation of specific protein kinase C (PKC) isoforms is involved. C(2)C(12) myotubes were pretreated with fatty acids and subsequently stimulated with insulin for the determination of glycogen synthesis. The carnitine palmitoyltransferase-1 inhibitor etomoxir, an inhibitor of beta-oxidation of acyl-CoA, did not protect against the inhibition of glycogen synthesis caused by the unsaturated fatty acid oleate. In addition, although oleate caused translocation, indicating activation, of individual PKC isoforms, inhibition of PKC by pharmacological agents or adenovirus-mediated overexpression of dominant negative PKC-alpha, -epsilon, or -theta mutants was unable to prevent the inhibitory effects of oleate on glycogen synthesis. We conclude that neither mitochondrial lipid metabolism nor activation of PKC-alpha, -epsilon, or -theta plays a role in the direct inhibition of glycogen synthesis by unsaturated fatty acids.
Aims/hypothesis Insulin resistance in skeletal muscle is strongly associated with lipid oversupply, but the intracellular metabolites and underlying mechanisms are unclear. We therefore sought to identify the lipid intermediates through which the common unsaturated fatty acid linoleate causes defects in IRS-1 signalling in L6 myotubes and mouse skeletal muscle. Materials and methods Cells were pre-treated with 1 mmol/l linoleate for 24 h. Subsequent insulin-stimulated IRS-1 tyrosine phosphorylation and its association with the p85 subunit of phosphatidylinositol 3-kinase were determined by immunoblotting. Intracellular lipid species and protein kinase C activation were modulated by overexpression of diacylglycerol kinase ɛ, which preferentially converts unsaturated diacylglycerol into phosphatidic acid, or by inhibition of lysophosphatidic acid acyl transferase with lisofylline, which reduces phosphatidic acid synthesis. Phosphatidic acid species in linoleate-treated cells or muscle from insulin-resistant mice fed a safflower oil-based high-fat diet that was rich in linoleate were analysed by mass spectrometry. Results Linoleate pretreatment reduced IRS-1 tyrosine phosphorylation and p85 association. Overexpression of diacylglycerol kinase ɛ reversed the activation of protein kinase C isoforms by linoleate, but paradoxically further diminished IRS-1 tyrosine phosphorylation. Conversely, lisofylline treatment restored IRS-1 phosphorylation. Mass spectrometry indicated that the dilinoleoyl-phosphatidic acid content increased from undetectable levels to almost 20% of total phosphatidic acid in L6 cells and to 8% of total in the muscle of mice fed a high-fat diet. Micelles containing dilinoleoyl-phosphatidic acid specifically inhibited IRS-1 tyrosine phosphorylation and glycogen synthesis in L6 cells. Conclusions/interpretation These data indicate that linoleatederived phosphatidic acid is a novel lipid species that contributes independently of protein kinase C to IRS-1 signalling defects in muscle cells in response to lipid oversupply.
Over the last two decades, there has been an increasing awareness of the role of eicosanoids in the development and progression of several types of cancer, including breast, prostate, lung, and colorectal cancers. Several processes involved in cancer development, such as cell growth, migration, and angiogenesis, are regulated by the arachidonic acid derivative thromboxane A2 (TXA2). Higher levels of circulating TXA2 are observed in patients with multiple cancers, and this is accompanied by overexpression of TXA2 synthase (TBXAS1, TXA2S) and/or TXA2 receptors (TBXA2R, TP). Overexpression of TXA2S or TP in tumor cells is generally associated with poor prognosis, reduced survival, and metastatic disease. However, the role of TXA2 signaling in the stroma during oncogenesis has been underappreciated. TXA2 signaling regulates the tumor microenvironment by modulating angiogenic potential, tumor ECM stiffness, and host immune response. Moreover, the by-products of TXA2S are highly mutagenic and oncogenic, adding to the overall phenotype where TXA2 synthesis promotes tumor formation at various levels. The stability of synthetic enzymes and receptors in this pathway in most cancers (with few mutations reported) suggests that TXA2 signaling is a viable target for adjunct therapy in various tumors to reduce immune evasion, primary tumor growth, and metastasis.
Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.