BBA - Molecular and Cell Biology of Lipids (v.1791, #8)
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Intracellular- and extracellular-derived Ca2+ influence phospholipase A2-mediated fatty acid release from brain phospholipids by Angelo O. Rosa; Stanley I. Rapoport (697-705).
Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca2+-dependent cytosolic phospholipase A2 (cPLA2) and Ca2+-independent phospholipase A2 (iPLA2), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca2+, even though iPLA2 has been thought to be Ca2+-independent. The source of Ca2+ for activation of cPLA2 is largely extracellular, whereas Ca2+ released from the endoplasmic reticulum can activate iPLA2 by a number of mechanisms. This review focuses on the role of Ca2+ in modulating cPLA2 and iPLA2 activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca2+ signal.
Keywords: Phospholipase A2; Calcium; Arachidonic acid; Docosahexaenoic acid; Endoplasmic reticulum; Signaling;
The cAMP-responsive element binding protein (CREB) regulates the expression of acid ceramidase (ASAH1) in H295R human adrenocortical cells by Natasha Lucki; Marion B. Sewer (706-713).
Acid ceramidase (encoded by ASAH1) is a lipid hydrolase that catalyzes the conversion of ceramide (cer) into sphingosine (SPH) and a free fatty acid. Adrenocortical steroidogenesis is regulated by the trophic peptide hormone adrenocorticotropin (ACTH), which induces the expression of steroidogenic genes in the human adrenal cortex primarily via a cAMP/protein kinase A (PKA)-dependent pathway. ACTH also stimulates sphingolipid metabolism in H295R adrenocortical cells leading to changes in steroidogenic gene expression. Based on our previous data identifying SPH as an antagonist for the nuclear receptor steroidogenic factor 1 (SF-1) and the role of ACTH-stimulated changes in sphingolipid metabolism on steroidogenic gene transcription, the aim of the current study was to determine the role of ACTH signaling in regulating the expression of the ASAH1 gene in H295R cells. We show that activation of the ACTH signaling pathway induces ASAH1 gene expression by stimulating the binding of the cAMP-responsive element binding protein (CREB) to multiple regions of the ASAH1 promoter. CREB binding promotes the recruitment of the coactivators CREB binding protein (CBP) and p300 to the CREB-responsive regions of the promoter. Consistent with transcriptional activation, we show that cAMP signaling increases the trimethylation of Lys 4 on histone H3 (H3K4) along the ASAH1 promoter. Finally, RNA interference (RNAi) experiments demonstrate that CREB is indispensable for cAMP-induced ASAH1 transcription. These data identify the ACTH/cAMP signaling pathway and CREB as transcriptional regulators of the ASAH1 gene in the human adrenal cortex.
Keywords: Acid ceramidase; cAMP; CREB; Ceramide; Sphingosine; Adrenocorticotropin;
Plasma pre β-HDL formation is decreased by atorvastatin treatment in type 2 diabetes mellitus: Role of phospholipid transfer protein by G.M. Dallinga-Thie; A. van Tol; R.P.F. Dullaart (714-718).
Atorvastatin lowers plasma phospholipid transfer protein (PLTP) activity, which stimulates pre-β-HDL generation in vitro. We determined the effect of atorvastatin on pre-β-HDL formation and its relation with PLTP activity in type 2 diabetes. Methods: Plasma pre-β-HDL formation as well as plasma apo A-I, LpA, LpAI:AII, cholesteryl ester transfer protein (CETP) and PLTP activity were measured before and after 30 weeks treatment in 40 patients randomized to atorvastatin 80 mg daily and 41 placebo receiving patients. Pre-β HDL formation was measured by crossed immunoelectrophoresis under conditions of lecithin:cholesterol acyltransferase (LCAT) inhibition. Results: Plasma pre-β-HDL formation, triglycerides, LDL cholesterol, PLTP activity, and CETP decreased after statin treatment (all P < 0.001 vs placebo), whereas HDL cholesterol increased (P < 0.005). Plasma apo A-I, LpAI and LpAI:AII remained unchanged compared to placebo. In all patients combined, the changes in pre-β-HDL formation were independently related to the decrease in plasma triglycerides (β = 0.31; P = 0.006) and PLTP activity (β = 0.23; P = 0.038), without a contribution of CETP. In the atorvastatin treated patients, the decrease in pre-β-HDL formation tended to be related to the decrease in PLTP activity (β = 0.30, P = 0.061) after controlling for decreases in triglycerides (β = 0.22, P = 0.22). Conclusion: High dose atorvastatin decreases the capacity of plasma to generate pre-β-HDL particles in type 2 diabetic patients, probably via lowering of plasma PLTP activity and triglycerides. This could contribute to an improvement in the atherogenic lipoprotein profile.
Keywords: Atorvastatin; High density lipoprotein; Triglyceride; Phospholipid transfer protein; Cholesteryl ester transfer protein; Pre-β HDL;
First evidence for the salt-dependent folding and activity of an esterase from the halophilic archaea Haloarcula marismortui by Marcelo Müller-Santos; Emanuel M. de Souza; Fabio de O. Pedrosa; David Alexander Mitchell; Sonia Longhi; Frédéric Carrière; Stéphane Canaan; Nadia Krieger (719-729).
A gene encoding an esterase from Haloarcula marismortui, a halophilic archaea from the Dead Sea, was cloned, expressed in Escherichia coli, and the recombinant protein (Hm EST) was biochemically characterized. The enzymatic activity of Hm EST was shown to exhibit salt dependence through salt-dependent folding. Hm EST exhibits a preference for short chain fatty acids and monoesters. It is inhibited by phenylmethylsulfonyl fluoride, diethyl-p-nitrophenyl phosphate, and 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one, confirming the conclusion from sequence alignments that Hm EST is a serine carboxylesterase belonging to the hormone-sensitive lipase family. The activity of Hm EST is optimum in the presence of 3 M KCl and no activity was detected in the absence of salts. Far–UV circular dichroism showed that Hm EST is totally unfolded in salt-free medium and secondary structure appears in the presence of 0.25–0.5 M KCl. After salt depletion, the protein was able to recover 60% of its initial activity when 2 M KCl was added. A 3D model of Hm EST was built and its surface properties were analyzed, pointing to an enrichment in acidic residues paralleled by a depletion in basic residues. This peculiar charge repartition at the protein surface supports a better stability of the protein in a high salt environment.
Keywords: Enzyme; Esterase; Haloarchaea; Haloadaptation; Hormone-sensitive lipase; Protein folding;
Downregulation of neutral ceramidase by gemcitabine: Implications for cell cycle regulation by Bill X. Wu; Youssef H. Zeidan; Yusuf A. Hannun (730-739).
Gemcitabine (GMZ) is a chemotherapeutic agent with well established effects on cell growth arrest and apoptosis. In this study, we investigated the potential roles of bioactive sphingolipids in mediating the growth suppressing effects of GMZ on a polyoma middle T transformed murine endothelial cell line. After 12-hour GMZ (0.6 μM) treatment, cell growth was arrested at the G0/G1 phase as detected by flow cytometric cell cycle analysis and MTT cell viability analysis, and this was accompanied by dephosphorylation of the retinoblastoma protein (Rb). Furthermore, GMZ treatment resulted in increased levels of specifically the very long chain ceramides as determined by mass spectrometry. Mechanistically, GMZ did not appear to affect the activities of many enzymes of ceramide metabolism; however, GMZ caused a selective reduction in the protein levels of neutral ceramidase (NCDase), as indicated by Western blot analysis, with a concomitant decrease in NCDase activity. The significance of NCDase loss on cell cycle regulation was investigated by specific knockdown of the enzyme using small interfering RNA (siRNA). Interestingly, NCDase siRNA transfection was sufficient to induce a cell cycle arrest at G0/G1 and an increase in total ceramide levels, with significant elevation in very long chain ceramides (C24:1 and C24:0). NCDase siRNA also induced Rb dephosphorylation. These data provide evidence for a novel mechanism of action for GMZ and highlight downregulation of NCDase as a critical step in GMZ-mediated ceramide elevation and cell cycle arrest.
Keywords: Neutral ceramidase; Ceramide; Cell cycle arrest; Gemcitabine; Sphingolipid;
Oleoylethanolamide, a natural ligand for PPAR-alpha, inhibits insulin receptor signalling in HTC rat hepatoma cells by María Martínez de Ubago; Inmaculada García-Oya; Antonio Pérez-Pérez; Alberto Canfrán-Duque; Rocio Quintana-Portillo; Fernando Rodríguez de Fonseca; Carmen González-Yanes; Víctor Sánchez-Margalet (740-745).
Oleoylethanolamide (OEA) is a lipid mediator belonging to the fatty acid ethanolamides family. It is produced by intestine and adipose tissue. It inhibits food intake and body weight gain, and has hypolipemiant action in vivo, as well as a lipolytic effect in vitro. OEA is a PPAR-alpha agonist, and recently it has been found that OEA is an endogenous ligand of an orphan receptor. Previously, we have shown that OEA inhibits insulin-stimulated glucose uptake in isolated adipocytes, and produces glucose intolerance in rats. In the present work, we have studied another insulin target cell, the hepatocyte using a rat hepatoma cell line (HTC), and we have studied the cross-talk of OEA signalling with metabolic and mitotic signal transduction of insulin receptor. OEA dose-dependently activates JNK and p38 MAPK, and inhibits insulin receptor phosphorylation. OEA inhibits insulin receptor activation, blunting insulin signalling in the downstream PI3K pathway, decreasing phosphorylation of PKB and its target GSK-3. OEA also inhibits insulin-dependent MAPK pathway, as assessed by immunoblot of phosphorylated MEK and MAPK. These effects were reversed by blocking JNK or p38 MAPK using pharmacological inhibitors (SP 600125, and SB 203580). Since OEA is an endogenous PPAR-alpha agonist, we investigated whether a pharmacologic agonist (WY 14643) may mimic the OEA effect on insulin receptor signalling. Activation of PPAR-alpha by the pharmacological agonist WY14643 in HTC hepatoma cells is sufficient to inhibit insulin signalling and this effect is also dependent on p38 MAPK but not JNK kinase. In summary, OEA inhibits insulin metabolic and mitogenic signalling by activation of JNK and p38 MAPK via PPAR-alpha.
Keywords: Oleylethanolamide; PPAR-alpha; Insulin receptor; Insulin resistance; Signal transduction;
Serine palmitoyltransferase subunit 1 is present in the endoplasmic reticulum, nucleus and focal adhesions, and functions in cell morphology by Jia Wei; Tokunbo Yerokun; Martina Leipelt; Chris A. Haynes; Harish Radhakrishna; Amin Momin; Samuel Kelly; Hyejung Park; Elaine Wang; Jill M. Carton; David J. Uhlinger; Alfred H. Merrill (746-756).
Serine palmitoyltransferase (SPT) has been localized to the endoplasmic reticulum (ER) by subcellular fractionation and enzymatic assays, and fluorescence microscopy of epitope-tagged SPT; however, our studies have suggested that SPT subunit 1 might be present also in focal adhesions and the nucleus. These additional locations have been confirmed by confocal microscopy using HEK293 and HeLa cells, and for focal adhesions by the demonstration that SPT1 co-immunoprecipitates with vinculin, a focal adhesion marker protein. The focal adhesion localization of SPT1 is associated with cell morphology, and possibly cell migration, because it is seen in most cells before they reach confluence but disappears when they become confluent, and is restored by a standard scratch-wound healing assay. Conversely, elimination of SPT1 using SPTLC1 siRNA causes cell rounding. Thus, in addition to its “traditional” localization in the ER for de novo sphingolipid biosynthesis, SPT1 is present in other cellular compartments, including focal adhesions where it is associated with cell morphology.
Keywords: Sphingolipid metabolizing enzyme; Subcellular localization; Focal adhesion;
In vivo assessment of hepatic triglycerides in murine non-alcoholic fatty liver disease using magnetic resonance spectroscopy by Ian R. Corbin; Emma E. Furth; Stephen Pickup; Evan S. Siegelman; Edward J. Delikatny (757-763).
In vivo 1H magnetic resonance spectroscopy (MRS) was used to examine the progression of fatty liver in two murine models of progressive hepatic steatosis: leptin-deficient obese (ob/ob) mice and mice maintained on a diet deficient in methionine and choline (MCDD). Ob/ob mice displayed high levels of intracellular hepatic triglycerides as early as 9 weeks after birth, as observed with MRS and histopathology. Single voxel spectra of ob/ob liver displayed strong resonances arising from saturated (1.3 ppm) and unsaturated (2.8 and 5.3 ppm) fatty acyl chains that could be resolved in the absence of water suppression. Hepatic inflammation, induced by lipopolysaccharide administration, led to a significant increase in unsaturated and polyunsaturated fatty acyl chain resonances (P < 0.05), indicating a change in the composition of hepatic triglycerides in lipid droplets. Mice maintained on the MCDD displayed histological evidence of hepatic steatosis as early as two weeks, progressing to macrovesicular steatohepatitis at 10 weeks. The histological changes were accompanied by significant increases in saturated and unsaturated fatty acyl chain resonances and a significant decrease in the lipid/(water + lipid) ratio (P < 0.05). These results indicate that in vivo 1H MRS may be a suitable method to monitor the progression of steatohepatitis.
Keywords: In vivo MR spectroscopy; Fatty liver; Non-alcoholic steatohepatitis; Non-alcoholic fatty liver disease; Ob/ob mice; Methionine–choline deficiency;
Differential regulation of the human versus the mouse apolipoprotein AV gene by PPARalpha by Xavier Prieur; Philippe Lesnik; Martine Moreau; Joan C. Rodríguez; Chantal Doucet; M. John Chapman; Thierry Huby (764-771).
Mice have been used widely to define the mechanism of action of fibric acid derivatives. The fibrates are pharmacological agonists of the peroxisome proliferator-activated receptor α (PPARα), whose activation in human subjects promotes potent reduction in plasma levels of triglycerides (TG) with concomitant increase in those of HDL-cholesterol. The impact of PPARα agonists on gene expression in humans and rodents is however distinct; such distinctions include differential regulation of key genes of lipid metabolism. We evaluated the question as to whether the human and murine genes encoding apolipoprotein apoAV, a regulator of plasma concentrations of TG-rich lipoproteins, might be differentially regulated in response to fibrates. Fenofibrate, a classic PPARα agonist, repressed expression of mouse Apoa5 in vivo in a mouse model transgenic for the human APOA5 gene; by contrast, expression of the human ortholog was up-regulated. Our findings are consistent with the presence of a functional PPAR-binding element in the promoter of the human APOA5 gene; this element is however degenerate and non-functional in the corresponding mouse Apoa5 sequence, as demonstrated by reporter assays and gel shift analyses. These data further highlights the distinct mechanisms which are implicated in the metabolism of TG-rich lipoproteins in mice as compared to man. They equally emphasize the importance of the choice of a mouse model for investigation of the impact of pharmaceutical modifiers on hypertriglyceridemia.
Keywords: Apolipoprotein A5; PPAR; Gene expression; Triglyceride;
Lipid aldehyde-mediated cross-linking of apolipoprotein B-100 inhibits secretion from HepG2 cells by Benjamin J. Stewart; James R. Roede; Jonathan A. Doorn; Dennis R. Petersen (772-780).
Hepatic oxidative stress and lipid peroxidation are common features of several prevalent disease states, including alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), a common component of the metabolic syndrome. These conditions are characterized in part by excessive accumulation of lipids within hepatocytes, which can lead to autocatalytic degradation of cellular lipids giving rise to electrophilic end products of lipid peroxidation. The pathobiology of reactive lipid aldehydes remains poorly understood. We therefore sought to investigate the effects of 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) on the transport and secretion of very low-density lipoprotein using HepG2 cells as a model hepatocyte system. Physiologically relevant concentrations of 4-HNE and 4-ONE rapidly disrupted cellular microtubules in a concentration-dependent manner. Interestingly, 4-ONE reduced apolipoprotein B-100 (ApoB) secretion while 4-HNE did not significantly impair secretion. Both 4-HNE and 4-ONE formed adducts with ApoB protein, but 4-HNE adducts were detectable as mono-adducts, while 4-ONE adducts were present as protein–protein cross-links. These results demonstrate that reactive aldehydes generated by lipid peroxidation can differ in their biological effects, and that these differences can be mechanistically explained by the structures of the protein adducts formed.
Keywords: 4-hydroxynonenal; 4-oxononenal; Apolipoprotein B-100; Microtubules; Lipid peroxidation;
Avian phospholipid transfer protein causes HDL conversion without affecting cholesterol efflux from macrophages by Jani Saarela; Jari Metso; Wolfgang J. Schneider; Matti Jauhiainen (781-789).
Circulatory phospholipid transfer protein (PLTP) has two major functions: 1) transfer of phospholipids towards HDL particles; and 2) modulation of HDL size and composition via the HDL conversion process. In the laying hen (Gallus gallus), the massive oocyte-targeted lipid flow is achieved through the concerted actions of lipases, lipid transfer proteins, and relatives of the LDL receptor family. The aim of the study was to gain insights into the structure and functions of chicken PLTP. The results demonstrate that PLTP is highly conserved from chicken to mammals, as (i) chicken PLTP is associated with plasma HDL; (ii) it clearly possesses phospholipid transfer activity; (iii) it is inactivated at + 58 °C; and (iv) it mediates conversion of avian and human HDL into small preβ-mobile HDL and large fused α-mobile HDL particles. Our data show that HDL from different chicken models is similar in chemical and physical properties to that of man based on PLTP activity, cholesterol efflux, and HDL conversion assays. In contrast to mammals, PLTP-facilitated HDL remodeling did not enhance cholesterol efflux efficiency of chicken HDL particles.
Keywords: Phospholipid transfer protein; High-density lipoprotein; HDL remodeling; Atherosclerosis; Lipid; Animal model;
Reduction of HDL levels lowers plasma PLTP and affects its distribution among lipoproteins in mice by Hannelore Samyn; Matthijs Moerland; Teus van Gent; Rien van Haperen; Arie van Tol; Rini de Crom (790-796).
Phospholipid transfer protein (PLTP) is associated with HDL particles in plasma, where it transfers phospholipids between lipoproteins and remodels HDL particles. Tangier disease patients, with a mutated ABCA1 transporter, have extremely low plasma HDL concentration and reduced PLTP activity levels, a phenotype that is also observed in mice lacking ABCA1. We investigated whether low HDL levels and low PLTP activity are mechanistically related. Firstly, we studied PLTP expression and distribution among lipoproteins in mice lacking ABCA1 (ABCA1−/−). Parallel to the strong reduction in PLTP activity in plasma of ABCA1−/− mice, decreased PLTP protein levels were observed. Neither PLTP synthesis in liver or macrophages nor the ability of the macrophages to secrete PLTP were impaired in ABCA1−/− mice. However, the PLTP activity level in the medium of cultured macrophages was determined by HDL levels in the medium. PLTP was associated with HDL particles in wild type mice, whereas in ABCA1−/− mice, PLTP was associated with VLDL and LDL particles. Secondly, we treated different mouse models with varying plasma HDL and PLTP levels (wild type, ABCA1−/−, apoE−/− and PLTPtg mice, overexpressing human PLTP) with a synthetic LXR ligand, and investigated the relationship between LXR-mediated PLTP induction and HDL levels in plasma. Plasma PLTP activity in wild type mice was induced 5.6-fold after LXR activation, whereas in ABCA1−/−, apoE−/− and PLTPtg mice, all having reduced HDL levels, induction of PLTP activity was 2.4- , 3.2- and 2.0-fold, respectively. The less pronounced PLTP induction in these mice compared to wild type mice was not caused by a decreased PLTP gene expression in the liver or macrophages. Our findings indicate that the extent of LXR-mediated PLTP induction depends on plasma HDL levels. In conclusion, we demonstrate that ABCA1 deficiency in mice affects plasma PLTP level and distribution through an indirect effect on HDL metabolism. In addition, we show that the extent of LXR-mediated PLTP induction is HDL-dependent. These findings indicate that plasma HDL level is an important regulator of plasma PLTP and might play a role in the stabilization of PLTP in plasma.
Keywords: Lipoprotein metabolism; Phospholipid transfer protein; High density lipoprotein; ATP-binding cassette transporter A1; Liver X receptor;
Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1 by Tohko Hirano; Mikiko Kishi; Hiroyuki Sugimoto; Ryo Taguchi; Hideru Obinata; Noriyasu Ohshima; Kazuaki Tatei; Takashi Izumi (797-805).
Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca2+ for its maximum activity. The K M values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 μM, and the V max values were 27.3 and 1.62 μmol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.
Keywords: Acylprotein thioesterase; Lysophospholipase; Palmitoylation; Substrate selectivity; Subcellular localization;
Mitochondrial long chain fatty acid β-oxidation in man and mouse by Malika Chegary; Heleen te Brinke; Jos P.N. Ruiter; Frits A. Wijburg; Maria S.K. Stoll; Paul E. Minkler; Michel van Weeghel; Horst Schulz; Charles L. Hoppel; Ronald J.A. Wanders; Sander M. Houten (806-815).
Several mouse models for mitochondrial fatty acid β-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD−/−), very long chain acyl-CoA dehydrogenase knockout (VLCAD−/−) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.
Keywords: Cellular metabolism; Inborn errors of metabolism; Fatty acid oxidation; Mouse model; Long chain acyl-CoA dehydrogenase; Very long chain acyl-CoA dehydrogenase;
Expression regulation and function of Pref-1 during adipogenesis of human mesenchymal stem cells (MSCs) by Kaipeng Jing; Jun-Young Heo; Kyoung-Sub Song; Kang-Sik Seo; Ji-Hoon Park; Jong-Seok Kim; Yeon-Joo Jung; Deog-Yeon Jo; Gi-Ryang Kweon; Wan-Hee Yoon; Byung-Doo Hwang; Kyu Lim; Jong-Il Park (816-826).
Preadipocyte Factor 1 (Pref-1), also known as Delta-like Protein 1 (DLK-1) is an epidermal growth factor-like domain-containing trans-membrane protein that is involved in adipogenesis and cell fate decision. Its function in adipogenesis is reported inconsistently based on different cellular model systems. Here, by using human mesenchymal stem cells (MSCs), we show that Pref-1 is modulated by both dexamethasone and 3-isobutyl-1methylxanthine (IBMX), two components of the adipogenic induction mixture during the adipogenesis in vitro. IBMX induces the expression of Pref-1 in a time- and dose-dependent manner through cyclic AMP and cyclic GMP independent pathway and attenuates adipocyte differentiation by down-regulating PPARγ (peroxisome proliferator activated receptor gamma) expression. Dexamethasone, on the other hand, is capable of subduing the inhibitory effect of IBMX-induced Pref-1 and initiating the adipogenesis by up-regulating PPARγ expression. Moreover, the treatment of IBMX or dexamethasone alone fails to develop MSCs into mature adipocytes, however, treating cells with both IBMX and dexamethasone leads to a complete adipocyte differentiation as evaluated by lipid-droplet formation. Taken together, our study demonstrates that IBMX accelerates accumulation of lipid in MSCs only under the circumstance that the negative effect of Pref-1 induced by IBMX on the adipogenesis is overcome by dexamethasone.
Keywords: Lipid accumulation; Adipocyte differentiation; Pref-1; IBMX; Dexamethasone;
Structural basis for pH-dependent alterations of reaction specificity of vertebrate lipoxygenase isoforms by M. Walther; J. Roffeis; C. Jansen; M. Anton; I. Ivanov; H. Kuhn (827-835).
Lipoxygenases have been classified according to their specificity of fatty acid oxygenation and for several plant enzymes pH-dependent alterations in the product patterns have been reported. Assuming that the biological role of mammalian lipoxygenases is based on the formation of specific reaction products, pH-dependent alterations would impact enzymes' functionality. In this study we systematically investigated the pH-dependence of vertebrate lipoxygenases and observed a remarkable stability of the product pattern in the near physiological range for the wild-type enzyme species. Site-directed mutagenesis of selected amino acids and alterations in the substrate concentrations induced a more pronounced pH-dependence of the reaction specificity. For instance, for the V603H mutant of the human 15-lipoxygenase-2 8-lipoxygenation was dominant at acidic pH (65%) whereas 15-H(p)ETE was the major oxygenation product at pH 8. Similarly, the product pattern of the wild-type mouse 8-lipoxygenase was hardly altered in the near physiological pH range but H604F exchange induced strong pH-dependent alterations in the positional specificity. Taken together, our data suggest that the reaction specificities of wild-type vertebrate lipoxygenase isoforms are largely resistant towards pH alterations. However, we found that changes in the assay conditions (low substrate concentration) and introduction/removal of a critical histidine at the active site impact the pH-dependence of reaction specificity for some lipoxygenase isoforms.
Keywords: Eicosanoids; Lipoxygenase; Fatty acid metabolism; Molecular enzymology; Catalytic mechanism;