BBA - Molecular and Cell Biology of Lipids (v.1841, #11)

Ceramide modulates pre-mRNA splicing to restore the expression of wild-type tumor suppressor p53 in deletion-mutant cancer cells by Gauri A. Patwardhan; Salman B. Hosain; David X. Liu; Sachin K. Khiste; Yunfeng Zhao; Jacek Bielawski; S. Michal Jazwinski; Yong-Yu Liu (1571-1580).
Mutants of tumor suppressor p53 not only lose the activity in genome stabilizing and in tumor suppression, but also exhibit oncogenic function in cancer cells. Most efforts in restoring p53 biological activity focus on either altering mutant-protein conformation or introducing an exogenous p53 gene into cells to eliminate p53-mutant cancer cells. Being different from these, we report that ceramide can restore the expression of wild-type p53 and induce p53-dependent apoptosis in deletion-mutant cancer cells. We show that endogenous long-carbon chain ceramide species (C16- to C24-ceramides) and exogenous C6-ceramide, rather than other sphingolipids, restore wild-type mRNA (intact exon-5), phosphorylated protein (Ser15 in exon-5) of p53, and p53-responsive proteins, including p21 and Bax, in ovarian cancer cells, which predominantly express a deleted exon-5 of p53 mutant before treatments. Consequently, the restored p53 sensitizes these p53-mutant cancer cells to DNA damage-induced growth arrest and apoptosis. Furthermore, we elucidate that ceramide activates protein phosphatase-1, and then the dephosphorylated serine/arginine-rich splicing-factor 1 (SRSF1) is translocated to the nucleus, thus promoting pre-mRNA splicing preferentially to wild-type p53 expression. These findings disclose an unrecognized mechanism that pre-mRNA splicing dysfunction can result in p53 deletion-mutants. Ceramide through SRSF1 restores wild-type p53 expression versus deletion-mutant and leads cancer cells to apoptosis. This suggests that heterozygous deletion-mutants of p53 can be restored in posttranscriptional level by using epigenetic approaches.Display Omitted
Keywords: Ceramide; Pre-mRNA splicing; p53 mutant; Restoration; SRSF1; Cancer;

Granule-mediated release of sphingosine-1-phosphate by activated platelets by Deepa Jonnalagadda; Manjula Sunkara; Andrew J. Morris; Sidney W. Whiteheart (1581-1589).
Sphingosine-1-phosphate (S1P) is an intracellularly generated bioactive lipid essential for development, vascular integrity, and immunity. These functions are mediated by S1P-selective cell surface G-protein coupled receptors. S1P signaling therefore requires extracellular release of this lipid. Several cell types release S1P and evidence for both plasma membrane transporter-mediated and vesicle-dependent secretion has been presented. Platelets are an important source of S1P and can release it in response to agonists generated at sites of vascular injury. S1P release from agonist-stimulated platelets was measured in the presence of a carrier molecule (albumin) using HPLC-MS/MS. The kinetics and agonist-dependence of S1P release were similar to that of other granule cargo e.g. platelet factor IV (PF4). Agonist-stimulated S1P release was defective in platelets from Unc13dJinx (Munc13-4 null) mice demonstrating a critical role for regulated membrane fusion in this process. Consistent with this observation, platelets efficiently converted fluorescent NBD-sphingosine to its phosphorylated derivative which accumulated in granules. Fractionation of platelet organelles revealed the presence of S1P in both the plasma membrane and in α-granules. Resting platelets contained a second pool of constitutively releasable S1P that was more rapidly labeled by exogenously added sphingosine. Our studies indicate that platelets contain two pools of S1P that are released extracellularly: a readily-exchangeable, metabolically active pool of S1P, perhaps in the plasma membrane, and a granular pool that requires platelet activation and regulated exocytosis for release.
Keywords: Platelet; Sphingosine; Secretion; Munc13-4; Platelet activation;

MicroRNAs are key regulators of brown adipogenesis by Joseph Yi Zhou; Lixin Li (1590-1595).
The recent discovery of microRNA, thousands of short, non-coding strands of RNA that regulate gene expressions on the transcriptional level throughout the body, raises the possibility of their roles as therapeutic targets in the treatment of a diverse range of diseases including diabetes, cancer, cardiovascular disease, and obesity. Specifically, their potential as therapeutic targets in the treatment of obesity has been highlighted. Brown adipose tissue containing a large number of mitochondria and expressing Ucp-1 is metabolically active through dissipating energy as heat in cold temperatures. Brown adipose, which was previously thought to be present only in neonatal and infants, has been recently unexpectedly identified in various anatomical regions of the adult human body. Furthermore, brown adipocytes have been shown to originate from skeletal and cardiovascular myoblast progenitor cells. Several identified microRNAs participate in the regulation of brown adipocyte differentiation through pathways involving the Prdm16 and C/ebp-β program. These miRNAs are potential therapeutic targets in the induction of brown adipocyte lineage differentiation from myoblast and white adipose, through which the Ucp-1 expression is regulated to increase calorie expenditure and reduce body weight in obese individuals. This review focuses on the current understanding of miRNAs on the regulation of brown adipogenesis.
Keywords: Brown adipocytes; White adipose tissue; MicroRNA; Beige fat; Obesity; Prdm16;

Role of white adipose lipolysis in the development of NASH induced by methionine- and choline-deficient diet by Naoki Tanaka; Shogo Takahashi; Zhong-Ze Fang; Tsutomu Matsubara; Kristopher W. Krausz; Aijuan Qu; Frank J. Gonzalez (1596-1607).
Methionine- and choline-deficient diet (MCD) is a model for nonalcoholic steatohepatitis (NASH) in rodents. However, the mechanism of NASH development by dietary methionine/choline deficiency remains undetermined. To elucidate the early metabolic changes associated with MCD-NASH, serum metabolomic analysis was performed using mice treated with MCD and control diet for 3 days and 1  week, revealing significant increases in oleic and linoleic acids after MCD treatment. These increases were correlated with reduced body weight and white adipose tissue (WAT) mass, increased phosphorylation of hormone-sensitive lipase, and up-regulation of genes encoding carboxylesterase 3 and β2-adrenergic receptor in WAT, indicating accelerated lipolysis in adipocytes. The changes in serum fatty acids and WAT by MCD treatment were reversed by methionine supplementation, and similar alterations were detected in mice fed a methionine-deficient diet (MD), thus demonstrating that dietary methionine deficiency enhances lipolysis in WAT. MD treatment decreased glucose and increased fibroblast growth factor 21 in serum, thus exhibiting a similar metabolic phenotype as the fasting response. Comparison between MCD and choline-deficient diet (CD) treatments suggested that the addition of MD-induced metabolic alterations, such as WAT lipolysis, to CD-induced hepatic steatosis promotes liver injury. Collectively, these results demonstrate an important role for dietary methionine deficiency and WAT lipolysis in the development of MCD-NASH.
Keywords: Fasting response; Linoleic acid; Lipolysis; Metabolomics; Oleic acid; Choline deficiency;

Dephosphorylation of CCAAT/enhancer-binding protein β by protein phosphatase 2A containing B56δ is required at the early time of adipogenesis by Miyoung Park; Yeon A Choi; Hee Gu Lee; Keun Il Kim; Jong-Seok Lim; Myeong-Sok Lee; Ki-Sook Oh; Young Yang (1608-1618).
It is known that protein phosphatase 2A (PP2A) expression is increased in high-fat diet (HFD)-induced obese mice, but the role of PP2A in adipogenesis as well as obesity remains to be addressed. In this study, the role of PP2A in adipogenesis was explored. Preadipocytes were treated with okadaic acid (OA) during adipogenesis and the degree of adipogenesis was determined. The OA treatment blocked adipogenesis at the early time of adipogenesis, but not at the late time. In the early time of adipogenesis, CCAAT/enhancer-binding protein β (C/EBPβ) activation is preceded by the expression of key adipogenic transcription factors including PPARγ and C/EBPα, which function at the late time of adipogenesis, and then C/EBPβ is degraded. However, the inhibition of PP2A by OA treatment sustained phosphorylation of C/EBPβ and delayed its degradation. In turn, PPARγ and C/EBPα activation was altered. Among the various regulatory B56 subunits consisting of PP2A holoenzyme, B56δ was directly bound to C/EBPβ and was responsible for the dephosphorylation of C/EBPβ by PP2A.Taken together, these findings suggest that the phosphorylation of C/EBPβ after hormonal induction has to be inactivated by PP2A containing B56δ at the early time of adipogenesis to allow the completion of adipogenesis.
Keywords: Adipogenesis; Protein phosphatase 2A (PP2A); C/EBPβ; Extracellular signal regulated kinase (ERK); B56δ;

Gestational diabetes mellitus modulates neonatal high-density lipoprotein composition and its functional heterogeneity by Ivana Sreckovic; Ruth Birner-Gruenberger; Carolin Besenboeck; Milica Miljkovic; Tatjana Stojakovic; Hubert Scharnagl; Gunther Marsche; Uwe Lang; Jelena Kotur-Stevuljevic; Zorana Jelic-Ivanovic; Gernot Desoye; Christian Wadsack (1619-1627).
Gestational diabetes mellitus (GDM) is related to neonatal macrosomia and an increased risk of vascular events. We hypothesized that GDM exerts qualitative effects on neonatal high-density lipoprotein (HDL). HDL was isolated from control (n = 11) and GDM maternal/neonatal donors (n = 9) and subjected to shotgun proteomics. Differences in HDL mobility were assessed by FPLC and native gel-electrophoresis. Paraoxonase (PON1) activity, cholesterol ester-transfer protein (CETP) mass and activity, phospholipid, triglyceride and cholesterol concentrations were quantified with commercial kits. Total anti-oxidative capacity and cholesterol efflux capability of HDLs were measured. Four proteins involved in lipid metabolism, inflammation and innate immunity were differentially expressed between controls and GDM neonates. ApoM (decreased, p  < 0.05) and SAA1 (increased, p  < 0.05) showed the same differences on both, maternal and neonatal GDM HDL. Lower PON1 protein expression was corroborated by lower activity (p  < 0.05) which in turn was associated with attenuated anti-oxidant capacity of GDM HDL. Protein changes were accompanied by increased levels of triglycerides and decreased levels of cholesterol esters, respectively. The observed differences in GDM HDL lipid moiety may be related to CETP mass and activity alterations. The rate of cholesterol efflux from term trophoblasts to maternal and from placental endothelial cells to neonatal GDM HDL was impaired (p  < 0.05). In conclusion, GDM causes changes in HDL composition and is intimately associated with impaired cholesterol efflux capability as well as diminished anti-oxidative particle properties. Remodeling of neonatal GDM HDL in utero supports the hypothesis that maternal conditions in pregnancy impact neonatal lipoprotein metabolism.
Keywords: Neonatal HDL; GDM; Anti-oxidative property;

Molecular characterization of the human COQ5 C-methyltransferase in coenzyme Q10 biosynthesis by Theresa P.T. Nguyen; Alberto Casarin; Maria Andrea Desbats; Mara Doimo; Eva Trevisson; Carlos Santos-Ocaña; Placido Navas; Catherine F. Clarke; Leonardo Salviati (1628-1638).
Coq5 catalyzes the only C-methylation involved in the biosynthesis of coenzyme Q (Q or ubiquinone) in humans and yeast Saccharomyces cerevisiae. As one of eleven polypeptides required for Q production in yeast, Coq5 has also been shown to assemble with the multi-subunit complex termed the CoQ-synthome. In humans, mutations in several COQ genes cause primary Q deficiency, and a decrease in Q biosynthesis is associated with mitochondrial, cardiovascular, kidney and neurodegenerative diseases. In this study, we characterize the human COQ5 polypeptide and examine its complementation of yeast coq5 point and null mutants. We show that human COQ5 RNA is expressed in all tissues and that the COQ5 polypeptide is associated with the mitochondrial inner membrane on the matrix side. Previous work in yeast has shown that point mutations within or adjacent to conserved COQ5 methyltransferase motifs result in a loss of Coq5 function but not Coq5 steady state levels. Here, we show that stabilization of the CoQ-synthome within coq5 point mutants or by over-expression of COQ8 in coq5 null mutants permits the human COQ5 homolog to partially restore coq5 mutant growth on respiratory media and Q6 content. Immunoblotting against the human COQ5 polypeptide in isolated yeast mitochondria shows that the human Coq5 polypeptide migrates in two-dimensional blue-native/SDS-PAGE at the same high molecular mass as other yeast Coq proteins. The results presented suggest that human and Escherichia coli Coq5 homologs expressed in yeast retain C-methyltransferase activity but are capable of rescuing the coq5 yeast mutants only when the CoQ-synthome is assembled.Display Omitted
Keywords: Human COQ gene; Mitochondrial metabolism; Protein complex; Q-biosynthetic intermediate; Saccharomyces cerevisiae; Ubiquinone;

Creatine reduces hepatic TG accumulation in hepatocytes by stimulating fatty acid oxidation by Robin P. da Silva; Karen B. Kelly; Kelly-Ann Leonard; René L. Jacobs (1639-1646).
Non-alcoholic fatty liver disease encompasses a wide spectrum of liver damage including steatosis, non-alcoholic steatohepatitis, fibrosis and cirrhosis. We have previously reported that creatine supplementation prevents hepatic steatosis and lipid peroxidation in rats fed a high-fat diet. In this study, we employed oleate-treated McArdle RH-7777 rat hepatoma cells to investigate the role of creatine in regulating hepatic lipid metabolism. Creatine, but not structural analogs, reduced cellular TG accumulation in a dose-dependent manner. Incubating cells with the pan-lipase inhibitor diethyl p-nitrophenylphosphate (E600) did not diminish the effect of creatine, demonstrating that the TG reduction brought about by creatine does not depend on lipolysis. Radiolabeled tracer experiments indicate that creatine increases fatty acid oxidation and TG secretion. In line with increased fatty acid oxidation, mRNA analysis revealed that creatine-treated cells had increased expression of PPARα and several of its transcriptional targets. Taken together, this study provides direct evidence that creatine reduces lipid accumulation in hepatocytes by the stimulation of fatty acid oxidation and TG secretion.
Keywords: Creatine; Non-alcoholic fatty liver disease; Fatty acid oxidation; Hepatocytes; PPARα;