BBA - Molecular and Cell Biology of Lipids (v.1851, #2)

Fructose supplementation impairs rat liver autophagy through mTORC activation without inducing endoplasmic reticulum stress by Miguel Baena; Gemma Sangüesa; Natalia Hutter; Rosa M. Sánchez; Núria Roglans; Juan C. Laguna; Marta Alegret (107-116).
Supplementation with 10% liquid fructose to female rats for 2 weeks caused hepatic steatosis through increased lipogenesis and reduced peroxisome proliferator activated receptor (PPAR) α activity and fatty acid catabolism, together with increased expression of the spliced form of X-binding protein-1 (Rebollo et al., 2014). In the present study, we show that some of these effects are preserved after sub-chronic (8 weeks) fructose supplementation, specifically increased hepatic expression of lipid synthesis-related genes (stearoyl-CoA desaturase, × 6.7-fold; acetyl-CoA carboxylase, × 1.6-fold; glycerol-3-phosphate acyltransferase, × 1.65-fold), and reduced fatty acid β-oxidation (× 0.77-fold), resulting in increased liver triglyceride content (× 1.69-fold) and hepatic steatosis. However, hepatic expression of PPARα and its target genes was not modified and, further, livers of 8-week fructose-supplemented rats showed no sign of unfolded protein response activation, except for an increase in p-IRE1 levels. Hepatic mTOR phosphorylation was enhanced (× 1.74-fold), causing an increase in the phosphorylation of UNC-51-like kinase 1 (ULK-1) (× 2.8-fold), leading to a decrease in the ratio of LC3B-II/LC3B-I protein expression (× 0.39-fold) and an increase in the amount of the autophagic substrate p62, indicative of decreased autophagy activity. A harmful cycle may be established in the liver of 8-week fructose-supplemented rats where lipid accumulation may cause defective autophagy, and reduced autophagy may result in decreased free fatty acid formation from triglyceride depots, thus reducing the substrates for β-oxidation and further increasing hepatic steatosis. In summary, the length of supplementation is a key factor in the metabolic disturbances induced by fructose: in short-term studies, PPARα inhibition and ER stress induction are critical events, whereas after sub-chronic supplementation, mTOR activation and autophagy inhibition are crucial.
Keywords: Fructose; Hepatic steatosis; Autophagy; mTORC;

Homeostasis of phospholipids — The level of phosphatidylethanolamine tightly adapts to changes in ethanolamine plasmalogens by Fabian Dorninger; Alexander Brodde; Nancy E. Braverman; Ann B. Moser; Wilhelm W. Just; Sonja Forss-Petter; Britta Brügger; Johannes Berger (117-128).
Ethanolamine plasmalogens constitute a group of ether glycerophospholipids that, due to their unique biophysical and biochemical properties, are essential components of mammalian cellular membranes. Their importance is emphasized by the consequences of defects in plasmalogen biosynthesis, which in humans cause the fatal disease rhizomelic chondrodysplasia punctata (RCDP). In the present lipidomic study, we used fibroblasts derived from RCDP patients, as well as brain tissue from plasmalogen-deficient mice, to examine the compensatory mechanisms of lipid homeostasis in response to plasmalogen deficiency. Our results show that phosphatidylethanolamine (PE), a diacyl glycerophospholipid, which like ethanolamine plasmalogens carries the head group ethanolamine, is the main player in the adaptation to plasmalogen insufficiency. PE levels were tightly adjusted to the amount of ethanolamine plasmalogens so that their combined levels were kept constant. Similarly, the total amount of polyunsaturated fatty acids (PUFAs) in ethanolamine phospholipids was maintained upon plasmalogen deficiency. However, we found an increased incorporation of arachidonic acid at the expense of docosahexaenoic acid in the PE fraction of plasmalogen-deficient tissues. These data show that under conditions of reduced plasmalogen levels, the amount of total ethanolamine phospholipids is precisely maintained by a rise in PE. At the same time, a shift in the ratio between ω-6 and ω-3 PUFAs occurs, which might have unfavorable, long-term biological consequences. Therefore, our findings are not only of interest for RCDP but may have more widespread implications also for other disease conditions, as for example Alzheimer's disease, that have been associated with a decline in plasmalogens.
Keywords: Plasmalogen; Compensation; Docosahexaenoic acid; Arachidonic acid; Alzheimer's disease; Peroxisome;

Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region by Jannet Kamoun; Mathieu Schué; Wala Messaoud; Justine Baignol; Vanessa Point; Eduardo Mateos-Diaz; Pascal Mansuelle; Youssef Gargouri; Goetz Parsiegla; Jean-François Cavalier; Frédéric Carrière; Ahmed Aloulou (129-140).
Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.Display Omitted
Keywords: Enzyme; Lipolysis; Pichia pastoris; Regioselectivity; Signal peptide; Yeast;

The mannoprotein TIR3 (CAGL0C03872g) is required for sterol uptake in Candida glabrata by Tatsuya Inukai; Minoru Nagi; Akihiro Morita; Koichi Tanabe; Toshihiro Aoyama; Yoshitsugu Miyazaki; Martin Bard; Hironobu Nakayama (141-151).
Sterol uptake in the pathogenic fungus, Candida glabrata, occurs via the sterol transporter, CgAus1p. Azole inhibition of sterol biosynthesis can under certain circumstances be reversed by adding exogenously sterol. Here we demonstrate that the CgTIR3 (CAGL0C03872g) gene product is also required for sterol uptake, since Cgtir3Δ strains fail to take up sterol both aerobically and under hypoxic conditions. Western analysis using an HA-tagged TIR3 strain showed that CgTir3p localizes to the cell wall, and its expression is induced by serum. Semi-quantitative reverse transcriptase-PCR also showed that two transcription regulatory genes, CgUPC2A and CgUPC2B, control CgTIR3 as well as CgAUS1 gene expression. Interestingly, complementation studies using Cgtir3Δ showed that ScDAN1, a mannoprotein required for sterol uptake in Saccharomyces cerevisiae, could not complement the C. glabrata TIR3 function. Furthermore, sterol analyses, in which both the CgAUS1 and CgTIR3 genes were constitutively expressed, resulted in aerobic sterol uptake although the amount of uptake was considerably less than that of cells cultured aerobically with serum. These results suggest that additional factors other than CgAUS1 and CgTIR3 are required for sterol uptake in C. glabrata.
Keywords: Sterol uptake; Mannoprotein; DAN/TIR; Candida glabrata; UPC2;

Decreased lipogenesis in white adipose tissue contributes to the resistance to high fat diet-induced obesity in phosphatidylethanolamine N-methyltransferase-deficient mice by Xia Gao; Jelske N. van der Veen; Martin Hermansson; Marta Ordoñez; Antonio Gomez-Muñoz; Dennis E. Vance; René L. Jacobs (152-162).
Mice lacking phosphatidylethanolamine N-methyltransferase (PEMT, Pemt / mice) are resistant to high-fat diet (HFD)-induced obesity (DIO) but develop non-alcoholic steatohepatitis. PEMT expression is strongly induced during differentiation of 3T3-L1 adipocytes. Hence, we hypothesized that white adipose tissue (WAT) might be a key player in the protection against DIO in Pemt −/− mice. We fed Pemt −/− and Pemt +/+ mice the HFD for 2 weeks, after which we examined adipocyte differentiation, adipogenesis and lipolysis in WAT. Pemt −/− mice gained less body weight, had reduced WAT mass and had smaller adipocytes than Pemt +/+ mice. The protein levels of adipose differentiation markers FABP4, PPARγ and C/EBPβ were not altered by genotype, but acetyl-CoA carboxylase expression and activation was reduced in the Pemt −/− mice. The in vivo conversion of [14C]acetate to [14C]TG in WAT was also lower in Pemt −/− mice. The release of glycerol from WAT explants was comparable between Pemt+/+ and Pemt −/− mice under basal condition and in the presence of isoproterenol, indicating unaffected lipolytic capacity. Furthermore, the amounts of leptin, cytokines and chemokines in WAT were not altered by genotype in mice fed the HFD for 2 weeks. However, after 10 weeks of HFD, WAT from Pemt −/− mice had dramatically lower leptin, inflammatory cytokines (IL-1 and TNF-α) and chemokines (MCP-1 and RANTES), and significantly higher anti-inflammatory cytokine IL-10 than Pemt+/+ mice. Together, our data show that PEMT deficiency did not affect the capability for differentiation and lipolysis in WAT. Decreased lipogenesis in WAT may contribute to the resistance to DIO in Pemt −/− mice.
Keywords: Obesity; White adipose tissue; Phosphatidylethanolamine N-methyltransferase; Fatty acids;

Cardiac lipoprotein lipase (LPL) is a pivotal enzyme controlling heart metabolism by providing the majority of fatty acids required by this organ. From activation in cardiomyocytes to secretion to the vascular lumen, cardiac LPL is regulated by multiple pathways, which are altered during diabetes. Hence, dimerization/activation of LPL is modified following diabetes, a process controlled by lipase maturation factor 1. The role of AMP-activated protein kinase, protein kinase D, and heparan sulfate proteoglycans, intrinsic factors that regulate the intracellular transport of LPL is also shifted, and is discussed. More recent studies have identified several exogenous factors released from endothelial cells (EC) and adipose tissue that are required for proper functioning of LPL. In response to hyperglycemia, both active and latent heparanase are released from EC to facilitate LPL secretion. Diabetes also increased the expression of glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) in EC, which mediates the transport of LPL across EC. Angiopoietin-like protein 4 secreted from the adipose tissue has the potential to reduce coronary LPL activity. Knowledge of these intrinsic and extrinsic factors could be used develop therapeutic targets to normalize LPL function, and maintain cardiac energy homeostasis after diabetes.
Keywords: Lipoprotein lipase; Diabetes; Heart metabolism; Post-translational regulation; Cardiomyocytes; Endothelial cells;

Opposing regulation of megakaryopoiesis by LPA receptors 2 and 3 in K562 human erythroleukemia cells by Ya-Hsuan Ho; Chao-Ling Yao; Kuan-Hung Lin; Fen-Han Hou; Wei-min Chen; Chi-Ling Chiang; Yu-Nung Lin; Meng-Wei Li; Shi-Hung Lin; Ya-Jan Yang; Chu-Cheng Lin; Jenher Lu; Gabor Tigyi; Hsinyu Lee (172-183).
Erythrocytes and megakaryocytes (MK) are derived from a common progenitor that undergoes lineage specification. Lysophosphatidic acid (LPA), a lipid growth factor was previously shown to be a regulator for erythropoietic process through activating LPA receptor 3 (LPA3). However, whether LPA affects megakaryopoiesis remains unclear. In this study, we used K562 leukemia cell line as a model to investigate the roles of LPA in MK differentiation. We demonstrated that K562 cells express both LPA2 and LPA3, and the expression levels of LPA2 are higher than LPA3. Treatment with phorbol 12-myristate 13-acetate, a commonly used inducer of megakaryopoiesis, reciprocally regulates the expressions of LPA2 and LPA3. By pharmacological blockers and knockdown experiments, we showed that activation of LPA2 suppresses whereas, LPA3 promotes megakaryocytic differentiation in K562. The LPA2-mediated inhibition is dependent on β-catenin translocation, whereas reactive oxygen species (ROS) generation is a downstream signal for activation of LPA3. Furthermore, the hematopoietic transcriptional factors GATA-1 and FLI-1, appear to be involved in these regulatory mechanisms. Taken together, our results suggested that LPA2 and LPA3 may function as a molecular switch and play opposing roles during megakaryopoiesis of K562 cells.
Keywords: Megakaryopoiesis; Hematopoiesis; Lysophosphatidic acid; Phorbol ester; LPA receptor; CD61;

Neutrophil effector responses are suppressed by secretory phospholipase A2 modified HDL by Sanja Curcic; Michael Holzer; Robert Frei; Lisa Pasterk; Rudolf Schicho; Akos Heinemann; Gunther Marsche (184-193).
Secretory phospholipase A2 (sPLA2) generates bioactive lysophospholipids implicated in acute and chronic inflammation, but the pathophysiologic role of sPLA2 is poorly understood. Given that high-density lipoprotein (HDL) is the major substrate for sPLA2 in plasma, we investigated the effects of sPLA2-mediated modification of HDL (sPLA2-HDL) on neutrophil function, an essential arm of the innate immune response and atherosclerosis.Treatment of neutrophils with sPLA2-HDL rapidly prevented agonist-induced neutrophil activation, including shape change, neutrophil extracellular trap formation, CD11b activation, adhesion under flow and migration of neutrophils. The cholesterol-mobilizing activity of sPLA2-HDL was markedly increased when compared to native HDL, promoting a significant reduction of cholesterol-rich signaling microdomains integral to cellular signaling pathways. Moreover, sPLA2-HDL effectively suppressed agonist-induced rise in intracellular Ca2 + levels. Native HDL showed no significant effects and removing lysophospholipids from sPLA2-HDL abolished all anti-inflammatory activities.Overall, our studies suggest that the increased cholesterol-mobilizing activity of sPLA2-HDL and suppression of rise in intracellular Ca2 + levels are likely mechanism that counteracts agonist-induced activation of neutrophils. These counterintuitive findings imply that neutrophil trafficking and effector responses are altered by sPLA2-HDL during inflammatory conditions.
Keywords: HDL; Secretory phospholipase A2; Lysophospholipid; Neutrophil;

CTGF/CCN2 exerts profibrotic action in myoblasts via the up-regulation of sphingosine kinase-1/S1P3 signaling axis: Implications in the action mechanism of TGFβ by Gennaro Bruno; Francesca Cencetti; Irene Pertici; Lukasz Japtok; Caterina Bernacchioni; Chiara Donati; Paola Bruni (194-202).
The matricellular protein connective tissue growth factor (CTGF/CCN2) is recognized as key player in the onset of fibrosis in various tissues, including skeletal muscle. In many circumstances, CTGF has been shown to be induced by transforming growth factor beta (TGFβ) and accounting, at least in part, for its biological action. In this study it was verified that in cultured myoblasts CTGF/CCN2 causes their transdifferentiation into myofibroblasts by up-regulating the expression of fibrosis marker proteins α-smooth muscle actin and transgelin. Interestingly, it was also found that the profibrotic effect exerted by CTGF/CCN2 was mediated by the sphingosine kinase (SK)-1/S1P3 signaling axis specifically induced by the treatment with the profibrotic cue. Following CTGF/CCN2-induced up-regulation, S1P3 became the S1P receptor subtype expressed at the highest degree, at least at mRNA level, and was thus capable of readdressing the sphingosine 1-phosphate signaling towards fibrosis rather than myogenic differentiation. Another interesting finding is that CTGF/CCN2 silencing prevented the TGFβ-dependent up-regulation of SK1/S1P3 signaling axis and strongly reduced the profibrotic effect exerted by TGFβ, pointing at a crucial role of endogenous CTGF/CCN2 generated following TGFβ challenge in the transmission of at least part of its profibrotic effect. These results provide new insights into the molecular mechanism by which CTGF/CCN2 drives its biological action and strengthen the concept that SK1/S1P3 axis plays a critical role in the onset of fibrotic cell phenotype.
Keywords: Sphingosine kinase; S1P3 receptor; Connective tissue growth factor; Myoblasts; Transforming growth factor beta;

n-3 fatty acids effectively improve the reference memory-related learning ability associated with increased brain docosahexaenoic acid-derived docosanoids in aged rats by Michio Hashimoto; Masanori Katakura; Yoko Tanabe; Abdullah Al Mamun; Takayuki Inoue; Shahdat Hossain; Makoto Arita; Osamu Shido (203-209).
We investigated whether a highly purified eicosapentaenoic acid (EPA) and a concentrated n-3 fatty acid formulation (prescription TAK-085) containing EPA and docosahexaenoic acid (DHA) ethyl ester could improve the learning ability of aged rats and whether this specific outcome  had any relation with the brain levels of EPA-derived eicosanoids and DHA-derived docosanoids. The rats were tested for reference memory errors (RMEs) and working memory errors (WMEs) in an eight-arm radial maze. Fatty acid compositions were analyzed by GC, whereas brain eicosanoid/docosanoids were measured by LC–ESI-MS–MS-based analysis. The levels of lipid peroxides (LPOs) were measured by thiobarbituric acid reactive substances. The administration of TAK-085 at 300 mg·kg− 1  day− 1 for 17 weeks reduced the number of RMEs in aged rats compared with that in the control rats. Both TAK-085 and EPA administration increased plasma EPA and DHA levels in aged rats, with concurrent increases in DHA and decreases in arachidonic acid in the corticohippocampal brain tissues. TAK-085 administration significantly increased the formation of EPA-derived 5-HETE and DHA-derived 7-, 10-, and 17-HDoHE, PD1, RvD1, and RvD2. ARA-derived PGE2, PGD2, and PGF significantly decreased in TAK-085-treated rats. DHA-derived mediators demonstrated a significantly negative correlation with the number of RMEs, whereas EPA-derived mediators did not exhibit any relationship. Furthermore, compared with the control rats, the levels of LPO in the plasma, cerebral cortex, and hippocampus were significantly reduced in TAK-085-treated rats. The findings of the present study suggest that long-term EPA + DHA administration may be a possible preventative strategy against age-related cognitive decline.Display Omitted
Keywords: Brain; PUFA-derived mediators; Memory; Aging; EPA and DHA;

Overexpression of sphingosine kinase 1 in liver reduces triglyceride content in mice fed a low but not high-fat diet by Greg M. Kowalski; Joachim Kloehn; Micah L. Burch; Ahrathy Selathurai; Steven Hamley; Stéphanie A.M. Bayol; Séverine Lamon; Matthew J. Watt; Robert S. Lee-Young; Malcolm J. McConville; Clinton R. Bruce (210-219).
Hepatic insulin resistance is a major risk factor for the development of type 2 diabetes and is associated with the accumulation of lipids, including diacylglycerol (DAG), triacylglycerols (TAG) and ceramide. There is evidence that enzymes involved in ceramide or sphingolipid metabolism may have a role in regulating concentrations of glycerolipids such as DAG and TAG. Here we have investigated the role of sphingosine kinase (SphK) in regulating hepatic lipid levels. We show that mice on a high-fat high-sucrose diet (HFHS) displayed glucose intolerance, elevated liver TAG and DAG, and a reduction in total hepatic SphK activity. Reduced SphK activity correlated with downregulation of SphK1, but not SphK2 expression, and was not associated with altered ceramide levels. The role of SphK1 was further investigated by overexpressing this isoform in the liver of mice in vivo. On a low-fat diet (LFD) mice overexpressing liver SphK1, displayed reduced hepatic TAG synthesis and total TAG levels, but with no change to DAG or ceramide. These mice also exhibited no change in gluconeogenesis, glycogenolysis or glucose tolerance. Similarly, overexpression of SphK1 had no effect on the pattern of endogenous glucose production determined during a glucose tolerance test. Under HFHS conditions, normalization of liver SphK activity to levels observed in LFD controls did not alter hepatic TAG concentrations. Furthermore, DAG, ceramide and glucose tolerance were also unaffected. In conclusion, our data suggest that SphK1 plays an important role in regulating TAG metabolism under LFD conditions.
Keywords: Liver; Sphingosine kinase 1; Ceramide; Triacylglycerol; Diacylglycerol; Glucose tolerance;

Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells by Maidina Tuohetahuntila; Bart Spee; Hedwig S. Kruitwagen; Richard Wubbolts; Jos F. Brouwers; Chris H. van de Lest; Martijn R. Molenaar; Martin Houweling; J. Bernd Helms; Arie B. Vaandrager (220-230).
Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. We previously observed that the levels of triacylglycerol (TAG) species containing long polyunsaturated fatty acids (PUFAs) are increased in in vitro activated HSCs. Here we investigated the cause and consequences of the rise in PUFA-TAGs by profiling enzymes involved in PUFA incorporation. We report that acyl CoA synthetase (ACSL) type 4, which has a preference for PUFAs, is the only upregulated ACSL family member in activated HSCs. Inhibition of the activity of ACSL4 by siRNA-mediated knockdown or addition of rosiglitazone specifically inhibited the incorporation of deuterated arachidonic acid (AA-d8) into TAG in HSCs. In agreement with this, ACSL4 was found to be partially localized around lipid droplets (LDs) in HSCs. Inhibition of ACSL4 also prevented the large increase in PUFA-TAGs in HSCs upon activation and to a lesser extent the increase of arachidonate-containing phosphatidylcholine species. Inhibition of ACSL4 by rosiglitazone was associated with an inhibition of HSC activation and prostaglandin secretion. Our combined data show that upregulation of ACSL4 is responsible for the increase in PUFA-TAG species during activation of HSCs, which may serve to protect cells against a shortage of PUFAs required for eicosanoid secretion.Display Omitted
Keywords: Prostaglandin; Eicosanoid; Phosphatidylcholine; Lipidomics; Heavy isotope labeling; Arachidonic acid;

Enzymatic characterization of ELOVL1, a key enzyme in very long-chain fatty acid synthesis by Martin J.A. Schackmann; Rob Ofman; Inge M.E. Dijkstra; Ronald J.A. Wanders; Stephan Kemp (231-237).
X-linked adrenoleukodystrophy (X-ALD) is a neurometabolic disease that is caused by mutations in the ABCD1 gene. ABCD1 protein deficiency impairs peroxisomal very long-chain fatty acid (VLCFA) degradation resulting in increased cytosolic VLCFA-CoA levels, which are further elongated by the VLCFA-specific elongase, ELOVL1. In adulthood, X-ALD most commonly manifests as a gradually progressive myelopathy (adrenomyeloneuropathy; AMN) without any curative or disease modifying treatments. We recently showed that bezafibrate reduces VLCFA accumulation in X-ALD fibroblasts by inhibiting ELOVL1. Although, in a clinical trial, bezafibrate was unable to lower VLCFA levels in plasma or lymphocytes in X-ALD patients, inhibition of ELOVL1 remains an attractive therapeutic option.In this study, we investigated the kinetic characteristics of ELOVL1 using X-ALD fibroblasts and microsomal fractions from ELOVL1 over-expressing HEK293 cell lines and analyzed the inhibition kinetics of a series of fibrates. Our data show that the CoA esters of bezafibrate and gemfibrozil reduce chain elongation by specifically inhibiting ELOVL1. These fibrates can therefore serve as lead compounds for the development of more potent and more specific inhibitors for ELOVL1.
Keywords: Adrenoleukodystrophy; Peroxisome; Substrate-reduction; Fatty acids; Fibrates;