BBA - Molecular and Cell Biology of Lipids (v.1811, #6)

Hepoxilin A3 protects β-cells from apoptosis in contrast to its precursor, 12-hydroperoxyeicosatetraenoic acid by Maria-Patapia Zafiriou; Laura Cecilia Zelarayan; Claudia Noack; Anke Renger; Santosh Nigam; Athanassia Siafaka-Kapadai (361-369).
Pancreatic β-cells have a deficit of scavenging enzymes such as catalase (Cat) and glutathione peroxidase (GPx) and therefore are susceptible to oxidative stress and apoptosis. Our previous work showed that, in the absence of cytosolic GPx in insulinoma RINm5F cells, an intrinsic activity of 12 lipoxygenase (12(S)-LOX) converts 12S-hydroperoxyeicosatetraenoic acid (12(S)-HpETE) to the bioactive epoxide hepoxilin A3 (HXA3). The aim of the present study was to investigate the effect of HXA3 on apoptosis as compared to its precursor 12(S)-HpETE and shed light upon the underlying pathways. In contrast to 12(S)-HpETE, which induced apoptosis via the extrinsic pathway, we found HXA3 not only to prevent it but also to promote cell proliferation. In particular, HXA3 suppressed the pro-apoptotic BAX and upregulated the anti-apoptotic Bcl-2. Moreover, HXA3 induced the anti-apoptotic 12(S)-LOX by recruiting heat shock protein 90 (HSP90), another anti-apoptotic protein. Finally, a co-chaperone protein of HSP90, protein phosphatase 5 (PP5), was upregulated by HXA3, which counteracted oxidative stress-induced apoptosis by dephosphorylating and thus inactivating apoptosis signal-regulating kinase 1 (ASK1). Taken together, these findings suggest that HXA3 protects insulinoma cells from oxidative stress and, via multiple signaling pathways, prevents them from undergoing apoptosis.► 12(S)-HpETE induces apoptosis of RINm5F cells by activating the extrinsic pathway. ► HXA3 protects RINm5F cells against apoptosis and promotes cell survival. ► HXA3 protects RINm5F cells from apoptosis by promoting Bcl-2 and downregulating Bax. ► HXA3 induces 12(S)-LOX transcription via HSP90 upregulation. ► HXA3 induces PP5 that subsequently inactivates ASK1 and inhibits apoptosis.
Keywords: HXA3; 12(S)-HpETE; Apoptosis; Pancreatic beta cell; Oxidative stress;

Mild stretch activates cPLA2 in alveolar type II epithelial cells independently through the MEK/ERK and PI3K pathways by Eleftheria Letsiou; Ei. Kitsiouli; George Nakos; Marilena E. Lekka (370-376).
Alveolar epithelial type II cells (AT II) in which lung surfactant synthesis and secretion take place, are subjected to low magnitude stretch during normal breathing. The aim of the study was to explore the effect of mild stretch on phospholipase A2 (PLA2) activation, an enzyme known to be involved in surfactant secretion. In A549 cells (a model of AT II cells), we showed, using a fluorometric assay, that stretch triggers an increase of total PLA2 activity. Western blot experiments revealed that the cytosolic isoform cPLA2 is rapidly phosphorylated under stretch, in addition to a modest increase in cPLA2 mRNA levels. Treatment of A549 cells with selective inhibitors of the MEK/ERK pathway significantly attenuated the stretch-induced cPLA2 phosphorylation. A strong interaction of cPLA2 and pERK enzymes was demonstrated by immunoprecipitation. We also found that inhibition of PI3K pathway attenuated cPLA2 activation after stretch, without affecting pERK levels. Our results suggest that low magnitude stretch can induce cPLA2 phosphorylation through the MEK/ERK and PI3K–Akt pathways, independently.► Mechanical stretch induces rapid but transient phosphorylation of cPLA2 in A549 cells. ► ERK1/2 co-immunoprecipitate with cPLA2 in quiescent A549 cells. ► ERK1/2 and PI3 kinase independently regulate the cPLA2 phosphorylation under stretch.
Keywords: Mechanical stretch; A549 cells; Cytosolic PLA2; ERK1/2; PI3 kinase; Alveolar epithelial cells;

CTP:phosphocholine cytidylyltransferase α (CCTα) is a nuclear enzyme that catalyzes the rate-limiting step in the CDP–choline pathway for phosphatidylcholine (PC) synthesis. Lipid activation of CCTα results in its translocation to the nuclear envelope and expansion of an intranuclear membrane network termed the nucleoplasmic reticulum (NR) by a mechanism involving membrane deformation. Nuclear lamins are also required for stability and proliferation of the NR, but whether this unique structure, or the nuclear lamina in general, is required for PC synthesis is not known. To examine this relationship, the nuclear lamina was depleted by RNAi or disrupted by expression of the Hutchinson–Gilford progeria syndrome (HGPS) mutant lamin A (progerin), and the effect on CCTα and choline metabolism was analyzed. siRNA-mediated silencing of lamin A/C or lamin B1 in CHO cells to diminish the NR had no effect on PC synthesis, while double knockdown non-specifically inhibited the pathway. Confirming this minor role in PC synthesis, only 10% of transiently overexpressed choline/ethanolamine phosphotransferase was detected in the NR. In CHO cells, CCTα was nucleoplasmic and co-localized with GFP-progerin in nuclear folds and invaginations; however, HGPS fibroblasts displayed an abnormal distribution of CCTα in the cytoplasm and nuclear envelope that was accompanied by a 2-fold reduction in PC synthesis. In spite of its altered localization, choline-labeling experiments showed that CCT activity was unaffected, and inhibition of PC synthesis was traced to reduced activity of a hemicholinium-sensitive choline transporter. We conclude that CCTα and lamins specifically cooperate to form the NR, but the overall structure of the nuclear envelope has a minimal impact on CCT activity and PC synthesis.► CCTα catalyzes the rate-limiting step in phosphatidylcholine synthesis at the nuclear envelope. ► In conjunction with lamins, CCTa can also deform membranes into the nucleoplasmic reticulum. ► The nuclear lamina was disrupted by RNAi or mutant lamin expression. ► The catalytic activity of CCT and PC synthesis is not affected by nuclear membrane structure. ► The membrane-deforming and catalytic activities are independent nuclear functions of CCT.
Keywords: CCTα; Phosphatidylcholine; Nucleoplasmic reticulum; Lamins; Hutchinson–Gilford progeria syndrome; Choline transport;

The acylated peptide ghrelin (AG) and its endogenous non-acylated isoform (UAG) protect cardiomyocytes, pancreatic β-cells, and preadipocytes from apoptosis, and induce preadipocytes differentiation into adipocytes. These events are mediated by AG and UAG binding to a still unidentified receptor, which determines the activation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) ERK1/2. AG and UAG also possess antilipolytic activity in vitro, but the underlying mechanism remains unknown. Thus, the objective of the current study was to characterize the molecular events involved in AG/UAG receptor signaling cascade. We treated rat primary visceral adipocytes with isoproterenol (ISO) and forskolin (FSK) to stimulate lipolysis, simultaneously incubating them with or without AG or UAG. Both peptides blocked ISO- and FSK-induced lipolysis. By direct measurement of cAMP intracellular content, we demonstrated that AG/UAG effect was associated to a reduction of ISO-induced cAMP accumulation. Moreover, the cAMP analog 8Br-cAMP abolished AG/UAG effect. As AG and UAG were ineffective against lipolysis induced by db-cAMP, another poorly hydrolyzable cAMP analog, phosphodiesterase (PDE) involvement was hypothesized. Indeed, cilostamide, a specific PDE3B inhibitor, blocked AG/UAG effect on ISO-induced lipolysis. Furthermore, the PI3K inhibitor wortmannin and AKT inhibitor 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4piperidinyl)-2H-benzimidazol-2-one trifluoroacetate also blocked AG/UAG action, suggesting a role in PDE3B activation. In particular, PI3K isoenzyme gamma (PI3Kγ) selective inhibition through the compound AS605240 prevented AG/UAG effect on ISO-stimulated lipolysis, hampering AKT phosphorylation on Ser473. Taken together, these data demonstrate for the first time that AG/UAG attenuation of ISO-induced lipolysis involves PI3Kγ/AKT and PDE3B.► AG/UAG attenuate cAMP elevating agent induced lipolysis in rat primary adipocytes. ► AG/UAG impair isoproterenol-induced cAMP accumulation. ► PDE3B mediates AG and UAG anti-lipolytic effect. ► The GPCR-activatable PI3Kγ and AKT are required for AG/UAG effect.
Keywords: Ghrelin; Isoproterenol; Insulin; Lipolysis; Phosphodiesterase 3B; Phosphoinositide 3-kinase gamma;

The LDL receptor (LDLR) is an endocytic receptor that plays a major role in the clearance of atherogenic lipoproteins from the circulation. During the endocytic process, the LDLR first binds lipoprotein at the cell surface and then traffics to endosomes, where the receptor releases bound lipoprotein. Release is acid-dependent and correlates with the formation of an intramolecular contact within the receptor. Human mutations at residues that form the contact are associated with familial hypercholesterolemia (FH) and the goal of the present study was to determine the role of contact residues on LDLR function. We show that mutations at nine contact residues reduce the ability of the LDLR to support lipoprotein uptake. Unexpectedly, only four of the mutations (W515A, W541A, H562Y and H586Y) impaired acid-dependent lipoprotein release. The remaining mutations decreased the lipoprotein-binding capacity of the LDLR through either reduction in the number of surface receptors (H190Y, K560W, H562Y and K582W) or reduction in the fraction of surface receptors that were competent to bind lipoprotein (W144A and W193A). We also examined three residues, distal to the contact, which were predicted to be necessary for the LDLR to adopt the acidic conformation. Of the three mutations we tested (G293S, F362A and G375S), one mutation (F362A) reduced lipoprotein uptake. Together, these data suggest that the intramolecular interface plays multiple roles in LDLR function.► Mutations at residues that form an intramolecular contact in the LDLR cripple lipoprotein uptake. ► K560W, H562Y, K582W reduce the number of surface receptors. ► W144A, W193A reduce the ability of surface receptors to bind lipoprotein. ► F362A, W515A, W541A, H562Y, H586Y impair lipoprotein release at endosomal pH. ► Modeling suggests that the contact deforms a lipoprotein-binding surface.
Keywords: LDL; VLDL; LDL receptor; LDLR;

The metabolic cascade leading to eicosanoid precursors – desaturases, elongases, and phospholipases A2 – is altered in Zucker fatty rats by Cécile Fèvre; Sandrine Bellenger; Anne-Sophie Pierre; Mélaine Minville; Jérôme Bellenger; Joseph Gresti; Mickaël Rialland; Michel Narce; Christian Tessier (409-417).
Metabolic syndrome characterized by insulin resistance and obesity is accompanied by severe lipid metabolism perturbations and chronic low-grade inflammation. However, many unresolved questions remained regarding the regulation that underlie dyslipidemia, particularly the regulation of the metabolic cascade (synthesis and release) leading to eicosanoid precursors release. This study was undertaken to investigate the regulation of desaturases/elongases and phospholipases A2 during the establishment of metabolic syndrome. Our results showed that delta-6 desaturase as well as elongase-6 expressions were upregulated in 3-month-old Zucker fatty rats as compared to lean littermates, independently of SREBP-1c activation. We also demonstrated for the first time an increase of liver group VII phospholipase A2 gene expression in the obese animals together with a strong specific inhibition of type IVA and VIA phospholipases A2. These results suggest that the regulation of unsaturated fatty acids biosynthesis and signalling cascade could contribute to the development of liver lipid dysregulation related to metabolic syndrome and may be considered as new potential targets in such pathological conditions.► Liver lipid metabolism was studied in Zucker rats, model of metabolic syndrome. ► We observed a marked increase of liver saturated and mono-unsaturated fatty acids. ► SCD-1 and D6D were increased independently of SREBP-1c in fatty rats. ► Elongase-6 gene expression was specifically upregulated. ► Expression of type IVA and VIA PLA2 was specifically decreased in obese animals.
Keywords: Metabolic syndrome; Desaturase; Elongase; Phospholipase A2;