BBA - Molecular and Cell Biology of Lipids (v.1851, #12)

Docosahexaenoic acid inhibits proteolytic processing of sterol regulatory element-binding protein-1c (SREBP-1c) via activation of AMP-activated kinase by Xiong Deng; Qingming Dong; Dave Bridges; Rajendra Raghow; Edwards A. Park; Marshall B. Elam (1521-1529).
In hyperinsulinemic states including obesity and T2DM, overproduction of fatty acid and triglyceride contributes to steatosis of the liver, hyperlipidemia and hepatic insulin resistance. This effect is mediated in part by the transcriptional regulator sterol responsive element binding protein-1c (SREBP-1c), which stimulates the expression of genes involved in hepatic fatty acid and triglyceride synthesis. SREBP-1c is up regulated by insulin both via increased transcription of nascent full-length SREBP-1c and by enhanced proteolytic processing of the endoplasmic reticulum (ER)-bound precursor to yield the transcriptionally active n-terminal form, nSREBP-1c. Polyunsaturated fatty acids of marine origin (n-3 PUFA) prevent induction of SREBP-1c by insulin thereby reducing plasma and hepatic triglycerides. Despite widespread use of n-3 PUFA supplements to reduce triglycerides in clinical practice, the exact mechanisms underlying their hypotriglyceridemic effect remain elusive. Here we demonstrate that the n-3 PUFA docosahexaenoic acid (DHA; 22:5 n-3) reduces nSREBP-1c by inhibiting regulated intramembrane proteolysis (RIP) of the nascent SREBP-1c. We further show that this effect of DHA is mediated both via activation of AMP-activated protein kinase (AMPK) and by inhibition of mechanistic target of rapamycin complex 1 (mTORC1). The inhibitory effect of AMPK on SREBP-1c processing is linked to phosphorylation of serine 365 of SREBP-1c in the rat. We have defined a novel regulatory mechanism by which n-3 PUFA inhibit induction of SREBP-1c by insulin. These findings identify AMPK as an important negative regulator of hepatic lipid synthesis and as a potential therapeutic target for hyperlipidemia in obesity and T2DM.
Keywords: Insulin; AMP kinase; SREBP-1c; Lipogenesis; Docosahexanaenoic acid;

Lipogenesis mitigates dysregulated sarcoplasmic reticulum calcium uptake in muscular dystrophy by Christopher W. Paran; Kai Zou; Patrick J. Ferrara; Haowei Song; John Turk; Katsuhiko Funai (1530-1538).
Muscular dystrophy is accompanied by a reduction in activity of sarco/endoplasmic reticulum Ca2 +-ATPase (SERCA) that contributes to abnormal Ca2 + homeostasis in sarco/endoplasmic reticulum (SR/ER). Recent findings suggest that skeletal muscle fatty acid synthase (FAS) modulates SERCA activity and muscle function via its effects on SR membrane phospholipids. In this study, we examined muscle's lipid metabolism in mdx mice, a mouse model for Duchenne muscular dystrophy (DMD). De novo lipogenesis was ~ 50% reduced in mdx muscles compared to wildtype (WT) muscles. Gene expressions of lipogenic and other ER lipid-modifying enzymes were found to be differentially expressed between wildtype (WT) and mdx muscles. A comprehensive examination of muscles' SR phospholipidome revealed elevated phosphatidylcholine (PC) and PC/phosphatidylethanolamine (PE) ratio in mdx compared to WT mice. Studies in primary myocytes suggested that defects in key lipogenic enzymes including FAS, stearoyl-CoA desaturase-1 (SCD1), and Lipin1 are likely contributing to reduced SERCA activity in mdx mice. Triple transgenic expression of FAS, SCD1, and Lipin1 (3TG) in mdx myocytes partly rescued SERCA activity, which coincided with an increase in SR PE that normalized PC/PE ratio. These findings implicate a defect in lipogenesis to be a contributing factor for SERCA dysfunction in muscular dystrophy. Restoration of muscle's lipogenic pathway appears to mitigate SERCA function through its effects on SR membrane composition.
Keywords: Lipogenesis; Muscular dystrophy; Sarcoplasmic reticulum; Membrane phospholipid; Calcium;

Visibility of lipid resonances in HR-MAS spectra of brain biopsies subject to spinning rate variation by C. Precht; G. Diserens; A. Oevermann; M. Vermathen; J. Lang; C. Boesch; P. Vermathen (1539-1544).
Lipid resonances from mobile lipids can be observed by 1H NMR spectroscopy in multiple tissues and have also been associated with malignancy. In order to use lipid resonances as a marker for disease, a reference standard from a healthy tissue has to be established taking the influence of variable factors like the spinning rate into account. The purpose of our study was to investigate the effect of spinning rate variation on the HR-MAS pattern of lipid resonances in non-neoplastic brain biopsies from different regions and visualize polar and non-polar lipids by fluorescence microscopy using Nile Red staining. 1H HR-MAS NMR spectroscopy demonstrated higher lipid peak intensities in normal sheep brain pure white matter biopsies compared to mixed white and gray matter biopsies and pure gray matter biopsies. High spinning rates increased the visibility particularly of the methyl resonances at 1.3 and the methylene resonance at 0.89 ppm in white matter biopsies stronger compared to thalamus and brainstem biopsies, and gray matter biopsies. The absence of lipid droplets and presence of a large number of myelin sheaths observed in white matter by Nile Red fluorescence microscopy suggest that the observed lipid resonances originate from the macromolecular pool of lipid protons of the myelin sheath's plasma membranes. When using lipid contents as a marker for disease, the variable behavior of lipid resonances in different neuroanatomical regions of the brain and at variable spinning rates should be considered. The findings may open up interesting possibilities for investigating lipids in myelin sheaths.
Keywords: NMR spectroscopy; Gray matter; White matter; Lipid; Nile Red;

Synthesis, metabolism and systemic transport of a fluorinated mimic of the endogenous jasmonate precursor OPC-8:0 by Guillermo H. Jimenez-Aleman; Sandra S. Scholz; Monika Heyer; Michael Reichelt; Axel Mithöfer; Wilhelm Boland (1545-1553).
Jasmonates (JAs) are fatty acid derivatives that mediate many developmental processes and stress responses in plants. Synthetic jasmonate derivatives (commonly isotopically labeled), which mimic the action of the endogenous compounds are often employed as internal standards or probes to study metabolic processes. However, stable-isotope labeling of jasmonates does not allow the study of spatial and temporal distribution of these compounds in real time by positron emission tomography (PET). In this study, we explore whether a fluorinated jasmonate could mimic the action of the endogenous compound and therefore, be later employed as a tracer to study metabolic processes by PET. We describe the synthesis and the metabolism of (Z)-7-fluoro-8-(3-oxo-2-(pent-2-en-1-yl)cyclopentyl)octanoic acid (7F-OPC-8:0), a fluorinated analog of the JA precursor OPC-8:0. Like endogenous jasmonates, 7F-OPC-8:0 induces the transcription of marker jasmonate responsive genes (JRG) and the accumulation of jasmonates after its application to Arabidopsis thaliana plants. By using UHPLC–MS/MS, we could show that 7F-OPC-8:0 is metabolized in vivo similarly to the endogenous OPC-8:0. Furthermore, the fluorinated analog was successfully employed as a probe to show its translocation to undamaged systemic leaves when it was applied to wounded leaves. This result suggests that OPC-8:0 – and maybe other oxylipins – may contribute to the mobile signal which triggers systemic defense responses in plants. We highlight the potential of fluorinated oxylipins to study the mode of action of lipid-derived molecules in planta, either by conventional analytical methods or fluorine-based detection techniques.Fluorinated oxylipins as probes to study metabolism and signal transport in plantsDisplay Omitted
Keywords: Arabidopsis thaliana; Fatty acid metabolism; Jasmonate; JA-biosynthesis; UHPLC–MS; Systemic response; Signal translocation;

Histological analyses by matrix-assisted laser desorption/ionization-imaging mass spectrometry reveal differential localization of sphingomyelin molecular species regulated by particular ceramide synthase in mouse brains by Masayuki Sugimoto; Yoichi Shimizu; Takeshi Yoshioka; Masato Wakabayashi; Yukari Tanaka; Kenichi Higashino; Yoshito Numata; Shota Sakai; Akio Kihara; Yasuyuki Igarashi; Yuji Kuge (1554-1565).
Sphingomyelin (SM) is synthesized by SM synthase (SMS) from ceramide (Cer). SM regulates signaling pathways and maintains organ structure. SM comprises a sphingoid base and differing lengths of acyl-chains, but the importance of its various forms and regulatory synthases is not known. It has been reported that Cer synthase (CerS) has restricted substrate specificity, whereas SMS has no specificity for different lengths of acyl-chains. We hypothesized that the distribution of each SM molecular species was regulated by expression of the CerS family. Thus, we compared the distribution of SM species and CerS mRNA expression using molecular imaging. Spatial distribution of each SM molecular species was investigated using ultra-high-resolution imaging mass spectrometry (IMS). IMS revealed that distribution of SM molecular species varied according to the lengths of acyl-chains found in each brain section. Furthermore, a combination study using in situ hybridization and IMS revealed the spatial expression of CerS1 to be associated with the localization of SM (d18:1/18:0) in cell body-rich gray matter, and CerS2 to be associated with SM (d18:1/24:1) in myelin-rich white matter. Our study is the first comparison of spatial distribution between SM molecular species and CerS isoforms, and revealed their distinct association in the brain. These observations were demonstrated by suppression of CerS2 using siRNA in HepG2 cells; that is, siRNA for CerS2 specifically decreased C22 very long-chain fatty acid (VLCFA)- and C24 VLCFA-containing SMs. Thus, histological analyses of SM species by IMS could be a useful approach to consider their molecular function and regulative mechanism.
Keywords: Imaging mass spectrometry; Fourier transform ion cyclotron resonance; Sphingomyelin; Ceramide synthase; Very long-chain fatty acid;

Acylglycerophosphate acyltransferase 4 (AGPAT4) is a mitochondrial lysophosphatidic acid acyltransferase that regulates brain phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol levels by Ryan M. Bradley; Phillip M. Marvyn; Juan J. Aristizabal Henao; Emily B. Mardian; Steve George; Marc G. Aucoin; Ken D. Stark; Robin E. Duncan (1566-1576).
The acylglycerophosphate acyltransferase/lysophosphatidic acid acyltransferase (AGPAT/LPAAT) family is a group of homologous acyl-CoA-dependent lysophospholipid acyltransferases. We performed studies to better understand the subcellular localization, activity, and in vivo function of AGPAT4/LPAATδ, which we found is expressed in multiple mouse brain regions. Endogenous brain AGPAT4 and AGPAT4 overexpressed in HEK293 or Sf9 insect cells localizes to mitochondria and is resident on the outer mitochondrial membrane. Further fractionation showed that AGPAT4 is present specifically in the mitochondria and not in the mitochondria-associated endoplasmic reticulum membrane (i.e. MAM). Lysates from Sf9 cells infected with baculoviral Agpat4 were tested with eight lysophospholipid species but showed an increased activity only with lysophosphatidic acid as an acyl acceptor. Analysis of Sf9 phospholipid species, however, indicated a significant 72% increase in phosphatidylinositol (PI) content. We examined the content of major phospholipid species in brains of Agpat4 / mice and found also a > 50% decrease in total levels of PI relative to wildtype mice, as well as significant decreases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), but no significant differences in phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid (PA). A compensatory upregulation of Agpats 1, 2, 3, 5, and 9 may help to explain the lack of difference in PA. Our findings indicate that AGPAT4 is a mitochondrial AGPAT/LPAAT that specifically supports synthesis of brain PI, PC, and PE. This understanding may help to explain apparent redundancies in the AGPAT/LPAAT family.
Keywords: Acylglycerophosphate acyltransferase 4 (AGPAT4)/lysophosphatidic acid acyltransferase delta (LPAAT4); Brain metabolism; Gene knockout; Kennedy pathway; Land's pathway; Mitochondria; Phospholipid metabolism; Phosphatidic acid; Phosphatidylinositol;

Destabilization of the torsioned conformation of a ligand side chain inverts the LXRβ activity by Lautaro D. Álvarez; M. Virginia Dansey; Diego Y. Grinman; Daniela Navalesi; Gisela A. Samaja; M. Celeste del Fueyo; Niek Bastiaensen; René Houtman; Darío A. Estrin; Adriana S. Veleiro; Adali Pecci; Gerardo Burton (1577-1586).
Liver X receptors (LXRs) are transcription factors activated by cholesterol metabolites containing an oxidized side chain. Due to their ability to regulate lipid metabolism and cholesterol transport, they have become attractive pharmacological targets. LXRs are closely related to DAF-12, a nuclear receptor involved in nematode lifespan and regulated by the binding of C-27 steroidal acids. Based on our recent finding that the lack of the C-25 methyl group does not abolish their DAF-12 activity, we evaluated the effect of removing it from the (25R)-cholestenoic acid, a LXR agonist.The binding mode and the molecular basis of action of 27-nor-5-cholestenoic acid were evaluated using molecular dynamics simulations. The biological activity was investigated using reporter gene expression assays and determining the expression levels of endogenous target genes. The in vitro MARCoNI assay was used to analyze the interaction with cofactors.27-Nor-5-cholestenoic acid behaves as an inverse agonist. This correlates with the capacity of the complex to better bind corepressors rather than coactivators. The C-25 methyl moiety would be necessary for the maintenance of a torsioned conformation of the steroid side chain that stabilizes an active LXRβ state.We found that a 27-nor analog is able to act as a LXR ligand. Interestingly, this minimal structural change on the steroid triggered a drastic change in the LXR response.Results contribute to improve our understanding on the molecular basis of LXRβ mechanisms of action and provide a new scaffold in the quest for selective LXR modulators.Display Omitted
Keywords: Liver X receptors; Inverse agonism; Molecular dynamics; Cholestenoic acid;

Lipoprotein profiles in human heterozygote carriers of a functional mutation P297S in scavenger receptor class B1 by Stefan A. Ljunggren; Johannes H.M. Levels; Kees Hovingh; Adriaan G. Holleboom; Menno Vergeer; Letta Argyri; Christina Gkolfinopoulou; Angeliki Chroni; Jeroen A. Sierts; John J. Kastelein; Jan Albert Kuivenhoven; Mats Lindahl; Helen Karlsson (1587-1595).
The scavenger receptor class B type 1 (SR-B1) is an important HDL receptor involved in cholesterol uptake and efflux, but its physiological role in human lipoprotein metabolism is not fully understood. Heterozygous carriers of the SR-B1P297S mutation are characterized by increased HDL cholesterol levels, impaired cholesterol efflux from macrophages and attenuated adrenal function. Here, the composition and function of lipoproteins were studied in SR-B1P297S heterozygotes.Lipoproteins from six SR-B1P297S carriers and six family controls were investigated. HDL and LDL/VLDL were isolated by ultracentrifugation and proteins were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. HDL antioxidant properties, paraoxonase 1 activities, apoA-I methionine oxidations and HDL cholesterol efflux capacity were assessed.Multivariate modeling separated carriers from controls based on lipoprotein composition. Protein analyses showed a significant enrichment of apoE in LDL/VLDL and of apoL-1 in HDL from heterozygotes compared to controls. The relative distribution of plasma apoE was increased in LDL and in lipid-free form. There were no significant differences in paraoxonase 1 activities, HDL antioxidant properties or HDL cholesterol efflux capacity but heterozygotes showed a significant increase of oxidized methionines in apoA-I.The SR-B1P297S mutation affects both HDL and LDL/VLDL protein compositions. The increase of apoE in carriers suggests a compensatory mechanism for attenuated SR-B1 mediated cholesterol uptake by HDL. Increased methionine oxidation may affect HDL function by reducing apoA-I binding to its targets. The results illustrate the complexity of lipoprotein metabolism that has to be taken into account in future therapeutic strategies aiming at targeting SR-B1.
Keywords: ApoE; ApoL-1; HDL; LDL/VLDL; P297S; SR-B1;