Mitochondria: the indispensable players in innate immunity and guardians of the inflammatory response.

Mitochondria, the dynamic organelles and power house of eukaryotic cells function as metabolic hubs of cells undergoing continuous cycles of fusion and fission. Recent findings have made it increasingly apparent that mitochondria essentially involved in energy production have evolved as principal intracellular signaling platforms regulating not only innate immunity but also inflammatory responses. Perturbations in mitochondrial dynamics, including fusion/fission, electron transport chain (ETC) architecture and cristae organization have now been actively correlated to modulate metabolic activity and immune function of innate and adaptive immune cells. Several newly identified mitochondrial proteins in mitochondrial outer membrane such as mitochondrial antiviral signaling protein (MAVS) and with mitochondrial DNA acting as danger-associated molecular pattern (DAMP) and mitochondrial ROS generated from mitochondrial sources have potentially established mitochondria as key signaling platforms in antiviral immunity in vertebrates and thereby orchestrating adaptive immune cell activations respectively. A thorough understanding of emerging and intervening role of mitochondria in toll-like receptor-mediated innate immune responses and NLRP3 inflammasome complex activation has gained lucidity in recent years that advocates the imposing functions of mitochondria in innate immunity. Fascinatingly, also how the signals stemming from the endoplasmic reticulum co-operate with the mitochondria to activate the NLRP3 inflammasome is now looked ahead as a stage to unravel as to how different mitochondrial and associated organelle stress responses co-operate to bring about inflammatory consequences. This has also opened avenues of research for revealing mitochondrial targets that could be exploited for development of novel therapeutics to treat various infectious, inflammatory, and autoimmune disorders. Thus, this review explores our current understanding of intricate interplay between mitochondria and other cellular processes like autophagy in controlling mitochondrial homeostasis and regulation of innate immunity and inflammatory responses.

IRF2BP2 Reduces Macrophage Inflammation and Susceptibility to Atherosclerosis.

RATIONALE: Inflammation impairs macrophage cholesterol clearance from vascular tissues and promotes atherosclerosis. Inflammatory macrophages suppress expression of the transcription cofactor interferon regulatory factor 2-binding protein 2 (IRF2BP2), and genetic variants near IRF2BP2 associate with ischemic heart disease progression in humans. OBJECTIVES: To test whether IRF2BP2 in macrophages affects atherosclerosis in mice and humans. METHODS AND RESULTS: We generated mice that delete IRF2BP2 in macrophages. IRF2BP2-deficient macrophages worsened atherosclerosis in irradiated low-density lipoprotein receptor null-recipient mice and in apolipoprotein E null mice. IRF2BP2-deficient macrophages were inflammatory and had impaired cholesterol efflux because of their inability to activate the cholesterol transporter ABCA1 in response to cholesterol loading. Their expression of the anti-inflammatory transcription factor Krüppel-like factor 2 was markedly reduced. Promoter studies revealed that IRF2BP2 is required for MEF2-dependent activation of Krüppel-like factor 2. Importantly, restoring Krüppel-like factor 2 in IRF2BP2-deficient macrophages attenuated M1 inflammatory and rescued M2 anti-inflammatory gene activation and improved the cholesterol efflux deficit by restoring ABCA1 activation in response to cholesterol loading. In a cohort of 1066 angiographic cases and 1011 controls, homozygous carriers of a deletion polymorphism (rs3045215) in the 3' untranslated region sequence of human IRF2BP2 mRNA had a higher risk of coronary artery disease (recessive model, odds ratio [95% confidence interval]=1.560 [1.179-2.065], P=1.73E-03) and had lower IRF2BP2 (and Krüppel-like factor 2) protein levels in peripheral blood mononuclear cells. The effect of this deletion polymorphism to suppress protein expression was confirmed in luciferase reporter studies. CONCLUSION: Ablation of IRF2BP2 in macrophages worsens atherosclerosis in mice, and a deletion variant that lowers IRF2BP2 expression predisposes to coronary artery disease in humans.

Chronic stress induces anxiety via an amygdalar intracellular cascade that impairs endocannabinoid signaling.

Collapse of endocannabinoid (eCB) signaling in the amygdala contributes to stress-induced anxiety, but the mechanisms of this effect remain unclear. eCB production is tied to the function of the glutamate receptor mGluR5, itself dependent on tyrosine phosphorylation. Herein, we identify a novel pathway linking eCB regulation of anxiety through phosphorylation of mGluR5. Mice lacking LMO4, an endogenous inhibitor of the tyrosine phosphatase PTP1B, display reduced mGluR5 phosphorylation, eCB signaling, and profound anxiety that is reversed by genetic or pharmacological suppression of amygdalar PTP1B. Chronically stressed mice exhibited elevated plasma corticosterone, decreased LMO4 palmitoylation, elevated PTP1B activity, reduced amygdalar eCB levels, and anxiety behaviors that were restored by PTP1B inhibition or by glucocorticoid receptor antagonism. Consistently, corticosterone decreased palmitoylation of LMO4 and its inhibition of PTP1B in neuronal cells. Collectively, these data reveal a stress-responsive corticosterone-LMO4-PTP1B-mGluR5 cascade that impairs amygdalar eCB signaling and contributes to the development of anxiety.

Cross talk between Leishmania donovani CpG DNA and Toll-like receptor 9: an immunoinformatics approach.

The precise and potential contribution of Toll-like receptors (TLRs) signaling pathways in fighting parasitic infections of Leishmania spp., an intracellular protozoan parasite, has gained significant attention during the last decades. Although it is well established that TLR9 recognizes CpG motifs in microbial genomes, the specificity of the CpG DNA pattern of Leishmania parasite interacting with endosomal TLR9 is still unknown. Hence in our study to identify the CpG DNA pattern of Leishmania donovani acting as ligand for TLR9, consecutive homology searches were performed using known CpG ODN 2216 as initial template until a consistent CpG pattern in L. donovani was found. A reliable model of TLR9 ectodomains (ECDs) as well as CpG DNA patterns was predicted to develop the 3D structural complexes of TLR9 ECD-CpG DNA utilizing molecular modeling and docking approaches. The results revealed the preferential specificity of L. donovani CpG DNA to TLR9 compared to control ODN and other CpG patterns. The interface between TLR9 and L. donovani CpG DNA was also found to be geometrically complementary with the LRR11 region of TLR9, acting as the critical region for ligand recognition. The L. donovani CpG pattern identified can be employed to derive a platform for development of an innate immunomodulatory agent for deadly disease.

Functional properties of Claramine: a novel PTP1B inhibitor and insulin-mimetic compound.

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling, interfering with its control of glucose homeostasis and metabolism. PTP1B activity is elevated in obesity and type 2 diabetes and is a major cause of insulin resistance. Trodusquemine (MSI-1436) is a "first-in-class" highly selective inhibitor of PTP1B that can cross the blood-brain barrier to suppress feeding and promote insulin sensitivity and glycemic control. Trodusquemine is a naturally occurring cholestane that can be purified from the liver of the dogfish shark, Squalus acanthias, but it can also be manufactured synthetically by a fairly laborious process that requires several weeks. Here, we tested a novel easily and rapidly (2 days) synthesized polyaminosteroid derivative (Claramine) containing a spermino group similar to Trodusquemine for its ability to inhibit PTP1B. Like Trodusquemine, Claramine displayed selective inhibition of PTP1B but not its closest related phosphatase TC-PTP. In cultured neuronal cells, Claramine and Trodusquemine both activated key components of insulin signaling, with increased phosphorylation of insulin receptor-ß (IRß), Akt and GSK3ß. Intraperitoneal administration of Claramine or Trodusquemine effectively restored glycemic control in diabetic mice as determined by glucose and insulin tolerance tests. A single intraperitoneal dose of Claramine, like an equivalent dose of Trodusquemine, suppressed feeding and caused weight loss without increasing energy expenditure. In summary, Claramine is an alternative more easily manufactured compound for the treatment of type II diabetes.

LMO4 is required to maintain hypothalamic insulin signaling.

Insulin action at the hypothalamus controls glucose homeostasis by suppressing hepatic glucose production and promoting glucose uptake by muscle. However, the mechanisms that control central insulin signaling have not been fully elucidated. Previously, we showed that LMO4 is highly expressed in hypothalamic nuclei that regulate glucose homeostasis. Here, we determined how loss of LMO4 in the hypothalamus would affect central insulin signaling and glucose homeostasis. In transgenic mice that have LMO4 in ablated in glutamatergic neurons, we found that insulin signaling is impaired in the hypothalamus as well as in peripheral tissues (liver and skeletal muscle). Impaired glucose homeostasis was associated with a markedly elevation in hypothalamic protein tyrosine phosphatase 1B (PTP1B) activity. PTP1B is a key phosphatase that terminates insulin signaling by dephosphorylating its receptor and downstream signaling molecules. Importantly, we found that administration of a selective PTP1B inhibitor Trodusquemine to the hypothalamus restored central insulin signaling and improved the response of peripheral tissues to insulin in these LMO4-deficient mice. Thus, our study reveals an essential requirement for LMO4 to modulate central insulin signaling.

LMO4 is essential for paraventricular hypothalamic neuronal activity and calcium channel expression to prevent hyperphagia.

The dramatic increase in the prevalence of obesity reflects a lack of progress in combating one of the most serious health problems of this century. Recent studies have improved our understanding of the appetitive network by focusing on the paraventricular hypothalamus (PVH), a key region responsible for the homeostatic balance of food intake. Here we show that mice with PVH-specific ablation of LIM domain only 4 (Lmo4) become rapidly obese when fed regular chow due to hyperphagia rather than to reduced energy expenditure. Brain slice recording of LMO4-deficient PVH neurons showed reduced basal cellular excitability together with reduced voltage-activated Ca(2+) currents. Real-time PCR quantification revealed that LMO4 regulates the expression of Ca(2+) channels (Cacna1h, Cacna1e) that underlie neuronal excitability. By increasing neuronal activity using designer receptors exclusively activated by designer drugs technology, we could suppress food intake of PVH-specific LMO4-deficient mice. Together, these results demonstrate that reduced neural activity in LMO4-deficient PVH neurons accounts for hyperphagia. Thus, maintaining PVH activity is important to prevent hyperphagia-induced obesity.

The LIM domain only 4 protein is a metabolic responsive inhibitor of protein tyrosine phosphatase 1B that controls hypothalamic leptin signaling.

Protein tyrosine phosphatase 1B (PTP1B) counteracts leptin signaling and is a therapeutic target for obesity and diabetes. Here we found that LIM domain only 4 (LMO4) inhibits PTP1B activity by increasing the oxidized inactive form of PTP1B. Mice with neuronal ablation of LMO4 have elevated PTP1B activity and impaired hypothalamic leptin signaling, and a PTP1B inhibitor normalized PTP1B activity and restored leptin control of circulating insulin levels. LMO4 is palmitoylated at its C-terminal cysteine, and deletion of this residue prevented palmitoylation and retention of LMO4 at the endoplasmic reticulum and abolished its inhibitory effect on PTP1B. Importantly, LMO4 palmitoylation is sensitive to metabolic stress; mice challenged with a brief high-fat diet or acute intracerebroventricular infusion of saturated fatty acid had less palmitoylated LMO4, less oxidized PTP1B, and increased PTP1B activity in the hypothalamus. Thus, unleashed PTP1B activity attributable to loss of LMO4 palmitoylation may account for rapid loss of central leptin signaling after acute exposure to saturated fat.

Insulino-mimetic and anti-diabetic effects of zinc.

While it has long been known that zinc (Zn) is crucial for the proper growth and maintenance of normal biological functions, Zn has also been shown to exert insulin-mimetic and anti-diabetic effects. These insulin-like properties have been demonstrated in isolated cells, tissues, and different animal models of type 1 and type 2 diabetes. Zn treatment has been found to improve carbohydrate and lipid metabolism in rodent models of diabetes. In isolated cells, it enhances glucose transport, glycogen and lipid synthesis, and inhibits gluconeogenesis and lipolysis. The molecular mechanism responsible for the insulin-like effects of Zn compounds involves the activation of several key components of the insulin signaling pathways, which include the extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3-K)/protein kinase B/Akt (PKB/Akt) pathways. However, the precise molecular mechanisms by which Zn triggers the activation of these pathways remain to be clarified. In this review, we provide a brief history of zinc, and an overview of its insulin-mimetic and anti-diabetic effects, as well as the potential mechanisms by which zinc exerts these effects.

LIM domain only 4 (LMO4) regulates calcium-induced calcium release and synaptic plasticity in the hippocampus.

The LIM domain only 4 (LMO4) transcription cofactor activates gene expression in neurons and regulates key aspects of network formation, but the mechanisms are poorly understood. Here, we show that LMO4 positively regulates ryanodine receptor type 2 (RyR2) expression, thereby suggesting that LMO4 regulates calcium-induced calcium release (CICR) in central neurons. We found that CICR modulation of the afterhyperpolarization in CA3 neurons from mice carrying a forebrain-specific deletion of LMO4 (LMO4 KO) was severely compromised but could be restored by single-cell overexpression of LMO4. In line with these findings, two-photon calcium imaging experiments showed that the potentiation of RyR-mediated calcium release from internal stores by caffeine was absent in LMO4 KO neurons. The overall facilitatory effect of CICR on glutamate release induced during trains of action potentials was likewise defective in LMO4 KO, confirming that CICR machinery is severely compromised in these neurons. Moreover, the magnitude of CA3-CA1 long-term potentiation was reduced in LMO4 KO mice, a defect that appears to be secondary to an overall reduced glutamate release probability. These cellular phenotypes in LMO4 KO mice were accompanied with deficits in hippocampus-dependent spatial learning as determined by the Morris water maze test. Thus, our results establish LMO4 as a key regulator of CICR in central neurons, providing a mechanism for LMO4 to modulate a wide range of neuronal functions and behavior.

Cell-type-specific roles of IGF-1R and EGFR in mediating Zn2+-induced ERK1/2 and PKB phosphorylation.

Zn(2+) exerts insulin-mimetic and antidiabetic effects in rodent models of insulin resistance, and activates extracellular-signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key components of the insulin signaling pathway. Zn(2+)-induced signaling has been shown to be associated with an increase in the tyrosine phosphorylation of insulin receptor (IR), as well as of insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) in several cell types. However, the specific contribution of these receptor protein tyrosine kinases (R-PTKs) in mediating Zn(2+)-induced responses in a cell-specific fashion remains to be established. Therefore, using a series of pharmacological inhibitors and genetically engineered cells, we have investigated the roles of various R-PTKs in Zn(2+)-induced ERK1/2 and PKB phosphorylation. Pretreatment of Chinese hamster ovary (CHO) cells overexpressing a human IR (CHO-HIR cells) with AG1024, an inhibitor for IR protein tyrosine kinase (PTK) and IGF-1R-PTK, blocked Zn(2+)-induced ERK1/2 and PKB phosphorylation, but AG1478, an inhibitor for EGFR, was without effect in CHO cells. On the other hand, both of these inhibitors were able to attenuate Zn(2+)-induced phosphorylation of ERK1/2 and PKB in A10 vascular smooth muscle cells. In addition, in CHO cells overexpressing tyrosine kinase deficient IR, Zn(2+) was still able to induce the phosphorylation of these two signaling molecules, whereas the insulin effect was significantly attenuated. Furthermore, both Zn(2+) and insulin-like growth factor 1 failed to stimulate ERK1/2 and PKB phosphorylation in IGF-1R knockout cells. Also, Zn(2+)-induced responses in CHO-HIR cells were not associated with an increase in the tyrosine phosphorylation of the IR beta-subunit and insulin receptor substrate 1 in CHO-HIR cells. Taken together, these data suggest that distinct R-PTKs mediate Zn(2+)-evoked ERK1/2 and PKB phosphorylation in a cell-specific manner.

An induction in hepatic HDL secretion associated with reduced ATPase expression.

Linoleic acid-phospholipids stimulate high-density lipoprotein (HDL) net secretion from liver cells by blocking the endocytic recycling of apoA-I. Experiments were undertaken to determine whether apoA-I accumulation in the cell media is associated with membrane ATPase expression. Treatment of HepG2 cells with dilinoeoylphosphatidylcholine (DLPC) increased apoA-I secretion fourfold. DLPC also significantly reduced cell surface F1-ATPase expression and reduced cellular ATP binding cassette (ABC)A1 and ABCG1 protein levels by approximately 50%. In addition, treatment of HepG2 cells with the ABC transporter inhibitor, glyburide, stimulated the apoA-I secretory effects of both DLPC and clofibrate. Pretreatment of HepG2 cells with compounds that increased ABC transport protein levels (TO901317, N-Acetyl-L-leucyl-L-leucyl-L-norleucinal, and resveratrol) blocked the DLPC-induced stimulation in apoA-I net secretion. Furthermore, whereas HepG2 cells normally secrete nascent prebeta-HDL, DLPC treatment promoted secretion of alpha-migrating HDL particles. These data show that an linoleic acid-phospholipid induced stimulation in hepatic HDL secretion is related to the expression and function of membrane ATP metabolizing proteins.

CaMKII knockdown attenuates H2O2-induced phosphorylation of ERK1/2, PKB/Akt, and IGF-1R in vascular smooth muscle cells.

We have shown earlier a requirement for Ca(2+) and calmodulin (CaM) in the H(2)O(2)-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H(2)O(2)-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKII alpha. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H(2)O(2)-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H(2)O(2), calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281-309) of CaMKII and with siRNA of CaMKII alpha attenuated the H(2)O(2)-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H(2)O(2)-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKII alpha siRNA abolished the H(2)O(2)-induced IGF-1R phosphorylation. H(2)O(2) treatment also induced Thr(286) phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H(2)O(2) on ERK1/2, PKB, and IGF-1R phosphorylation.

Hepatic high-density lipoprotein secretion regulates the mobilization of cell-surface hepatic lipase.

HDL acts much like heparin to liberate hepatic lipase (HL) from cell surface proteoglycans and stimulate triglyceride clearance. Experiments were undertaken to evaluate the effects of factors that stimulate the secretion of HDL from the liver on the release of HL. Treatment of HepG2 cells with linoleic acid phospholipids (LAPL) (12 muM) promotes a similar increase in the accumulation of both HDL and HL in the cell media. LAPL also induce both apoA-I and HL release from primary human hepatocytes. Dilinoleoylphosphatidylcholine has a greater effect on both apoA-I secretion and HL release than palmitoyllinoleoylphosphatidylcholine. HL released from HepG2 cells is inactive and associated with a large HDL complex containing both apoA-I and apoA-II. Inclusion of the PPARalpha inhibitor, MK-886, or MAPK inhibitor, U0126, completely blocks the LAPL-induced apoA-I and HL accumulation in the media. LAPL-treated cell lysates, however, showed no change in HL protein expression nor HL mRNA. LAPL-induced HL release appears to be a consequence of the displacement ability of newly secreted HDL. Overexpression of pre-pro-apoA-I in HepG2 cells increased HL release, while siRNA inhibition of the apoA-I gene reduced HL in the media. The data show that factors that stimulate HDL secretion in hepatocytes act to also increase the release of HL. This may partly explain why HDL therapeutics often impact plasma triglyceride levels.

Phosphatidylinositol acts through mitogen-activated protein kinase to stimulate hepatic apolipoprotein A-I secretion.

Phosphatidylinositol (PI) has been shown to stimulate reverse cholesterol transport in animal models and to increase plasma apolipoprotein (apo) A-I levels and high-density lipoprotein cholesterol in human subjects. The objective of this study was to determine the molecular mechanism through which PI stimulates apo A-I secretion in hepatic cells. PI (12 mumol/L) significantly stimulates apo A-I secretion from HepG2 cells over 24 hours. The stimulation in apo A-I secretion is completely blocked by phospholipase C inhibitors (D609 and U73122) and the Ras inhibitor sulindac sulfide. Apolipoprotein A-I secretion is augmented with a protein kinase C agonist (dioctanoyl glycerol) and inhibited by a protein kinase C inhibitor (dioleoyl ethylene glycol). The PI-induced apo A-I secretion is unaffected by PI-3-kinase inhibitors but is sensitive to mitogen-activated protein kinase (MAPK) inhibitors. Whereas the p38MAPK inhibitor SB203580 has no effect on PI-induced apo A-I secretion, the MAPK kinase 1/2 inhibitor U0126 and the c-Jun-N-terminal kinase/stress-activated protein kinase inhibitor SP600125 block PI-induced apo A-I secretion. PI also increased extracellular-regulated protein kinase 1 and 2 phosphorylation in HepG2 cells in a time-dependent manner. PI does not appear to stimulate apo A-I gene transcription, as cellular apo A-I messenger RNA levels remained unchanged over the 24-hour incubation. However, PI significantly decreases apo A-I binding and degradation in HepG2 cells. Collectively, the data suggest that PI acts through MAPK pathways to increase plasma apo A-I levels by protecting it from reuptake and degradation.

Lipoprotein charge and vascular lipid metabolism.

Lipoproteins play a central role in transporting hydrophobic molecules through the bloodstream and between specific tissues. Lipoprotein molecules have a distinctive electrical charge and changes in electrostatic properties directly affect the metabolism of the lipoprotein. Lipoprotein charge controls interfacial interactions and determines the ability of the lipoprotein to interact with intravascular enzymes and cell surface proteins. Uniquely charged constituents of the lipoprotein thereby control the metabolism of lipoproteins by creating a regulatory system wherein the electrostatic properties of plasma lipoproteins determine the fate of intravascular lipids.

Phospholipids as cardiovascular therapeutics.

A uniquely formulated soy phospholipid is being developed as a potential therapeutic for the treatment and prevention of heart disease. Three phase I and one phase I/II clinical trials have been completed with soy phosphatidylinositol (PI). The compound was shown to be safe in all trials and at doses over 5 g. Clinical studies have also shown early-stage efficacy to suggest that PI is able to raise plasma HDL-cholesterol and apolipoprotein A-I levels, and reduce triglyceride levels in humans. PI directly impacts plasma HDL levels through a MAPK stimulation of HDL production by the liver. Research has shown that the linoleic acid content of soy PI is critical to a peroxisome proliferator-activated receptor alpha dependent stimulation of HDL secretion. Soy-derived phospholipids uniquely affect cellular signaling and transcriptional processes. These lipids are safe and efficacious in humans and may therefore offer a novel therapeutic opportunity to treat cardiovascular disease.

Linoleic acid-enriched phospholipids act through peroxisome proliferator-activated receptors alpha to stimulate hepatic apolipoprotein A-I secretion.

A uniquely formulated soy phospholipid, phosphatidylinositol (PI), is under development as a therapeutic agent for increasing plasma high-density lipoprotein (HDL) levels. Soy PI has been shown to increase plasma HDL and apolipoprotein A-I (apoA-I) levels in phase I human trials. Low micromolar concentrations of PI increase the secretion of apoA-I in model human hepatoma cell lines, through activation of G-protein and mitogen-activated protein (MAP) kinase pathways. Experiments were undertaken to determine the importance of the PI head group and acyl chain composition on hepatic apoA-I secretion. Phospholipids with choline and inositol head groups and one or more linoleic acid (LA) acyl chains were shown to stimulate apoA-I secretion by HepG2 cells and primary human hepatocytes. Phospholipids containing two LA groups (dilinoleoylphosphatidylcholine, DLPC) were twice as active as those with only one LA group and promoted a 4-fold stimulation in apoA-I secretion. Inhibition of cytosolic phospholipase A2 with pyrrolidine 1 (10 microM) resulted in complete attenuation of PI- and DLPC-induced apoA-I secretion. Pretreatment with the peroxisome proliferator-activated receptor alpha (PPARalpha) inhibitor MK886 (10 microM) also completely blocked PI- and DLPC-induced apoA-I secretion. Hepatic PPARalpha expression was significantly increased by both PI and DLPC. However, in contrast to that seen with the fibrate drugs, PI caused minimal inhibition of catalytic activities of cytochrome P450 and UGT1A1 enzymes. These data suggest that LA-enriched phospholipids stimulate hepatic apoA-I secretion through a MAP kinase stimulation of PPARalpha. LA-enriched phospholipids have a greater apoA-I secretory activity than the fibrate drugs and a reduced likelihood to interfere with concomitant drug therapies.