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The tumour suppressor PTEN is a negative regulator of the PI3K/AKT signalling pathway. Liver‐specific deletion of Pten in mice results in the hyper‐activation PI3K/AKT signalling accompanied by enhanced genome duplication (polyploidization), marked lipid accumulation (steatosis) and formation of hepatocellular carcinomas. However, it is unknown whether polyploidization in this model has an impact on the development of steatosis and the progression towards liver cancer. Here, we used a liver‐specific conditional knockout approach to delete Pten in combination with deletion of E2f7/8, known key inducers of polyploidization. As expected, Pten deletion caused severe steatosis and liver tumours accompanied by enhanced polyploidization. Additional deletion of E2f7/8 inhibited polyploidization, alleviated Pten‐induced steatosis without affecting lipid species composition and accelerated liver tumour progression. Global transcriptomic analysis showed that inhibition of polyploidization in Pten‐deficient livers resulted in reduced expression of genes involved in energy metabolism, including PPAR‐gamma signalling. However, we find no evidence that deregulated genes in Pten‐deficient livers are direct transcriptional targets of E2F7/8, supporting that reduction in steatosis and progression towards liver cancer are likely consequences of inhibiting polyploidization. Lastly, flow cytometry and image analysis on isolated primary wildtype mouse hepatocytes provided further support that polyploid cells can accumulate more lipid droplets than diploid hepatocytes. Collectively, we show that polyploidization promotes steatosis and function as an important barrier against liver tumour progression in Pten‐deficient livers. [ABSTRACT FROM AUTHOR]

Background: Hepatic lipidosis is increasing in incidence in the Western world, with cats being particularly sensitive. When cats stop eating and start utilizing their fat reserves, free fatty acids (FFAs) increase in blood, causing an accumulation of triacylglycerol (TAG) in the liver. Objective: Identifying potential new drugs that can be used to treat hepatic lipidosis in cats using a feline hepatic organoid system. Animals: Liver organoids obtained from 6 cats. Methods: Eight different drugs were tested, and the 2 most promising were further studied using a quantitative TAG assay, lipid droplet staining, and qPCR. Results: Both T863 (a diacylglycerol O‐acyltransferase 1 [DGAT1] inhibitor) and 5‐aminoimidazole‐4‐carboxamide 1‐β‐D‐ribofuranoside (AICAR; an adenosine monophosphate kinase activator) decreased TAG accumulation by 55% (P <.0001) and 46% (P =.0003), respectively. Gene expression of perilipin 2 (PLIN2) increased upon the addition of FFAs to the medium and decreased upon treatment with AICAR but not significantly after treatment with T863. Conclusions and Clinical Importance: Two potential drugs useful in the treatment of hepatic lipidosis in cats were identified. The drug T863 inhibits DGAT1, indicating that DGAT1 is the primary enzyme responsible for TAG synthesis from external fatty acids in cat organoids. The drug AICAR may act as a lipid‐lowering compound via decreasing PLIN2 mRNA. Liver organoids can be used as an in vitro tool for drug testing in a species‐specific system and provide the basis for further clinical testing of drugs to treat steatosis. [ABSTRACT FROM AUTHOR]

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation. [ABSTRACT FROM AUTHOR]

The anticancer drug hexadecylphosphocholine (HePC), an alkyl-lysophospholipid analog (ALP), has been shown to induce apoptosis and inhibit the synthesis of phosphatidylcholine (PC) in a number of cell lines. We investigated whether inhibition of PC synthesis plays a major causative role in the induction of apoptosis by HePC. We therefore directly compared the apoptosis caused by HePC in CHO cells to the apoptotic process in CHO-MT58 cells, which contain a genetic defect in PC synthesis. HePC-provoked apoptosis was found to differ substantially from the apoptosis observed in MT58 cells, since it was (i) not accompanied by a large decrease in the amount of PC and diacylglycerol (DAG), (ii) not preceded by induction of the pro-apoptotic protein GADD153/CHOP, and (iii) not dependent on the synthesis of new proteins. Furthermore, lysoPC as well as lysophosphatidylethanolamine (lysoPE) could antagonize the apoptosis induced by HePC, whereas only lysoPC was able to rescue MT58 cells. HePC also induced a rapid externalisation of phosphatidylserine (PS). These observations suggest that inhibition of PC synthesis is not the primary pathway in HePC-induced apoptosis. [Copyright &y& Elsevier]

Phosphatidylcholine (PC) constitutes a major portion of cellular phospholipids and displays unique molecular species in different cell types and tissues. Inhibition of the CDP–choline pathway in most mammalian cells or overexpression of the hepatic phosphatidylethanolamine methylation pathway in hepatocytes leads to perturbation of PC homeostasis, growth arrest or even cell death. Although many agents that perturb PC homeostasis and induce cell death have been identified, the signaling pathways that mediate this cell death have not been well defined. This review summarizes recent progress in understanding the relationship between PC homeostasis and cell death. [Copyright &y& Elsevier]

Investigates the inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides. Crosstalk between glycerolophospholipid and sphingolipid metabolism; Dose and time-dependent inhibition of the CDP-pathways.

Regulation of CTP:choline-phosphate cytidylyltransferase activity was studied in regenerating rat liver. The formation of phosphatidylcholine from [14C]choline in hepatocytes isolated from regenerating liver at 22h after surgery was increased 1.9-fold when compared with hepatocytes from sham-operated animals. This effect was accompanied by a 1.4-fold increase in cytosolic cytidylyltransferase activity as well as by a 1.5-fold increase in the amount of immunoreactive cytidylyltransferase protein, and a 1.7-fold increase in [35S]methionine incorporation into cytidylyltransferase protein. Northern blot analysis of cytidylyltransferase mRNA showed two signals at 1.5 and 5.0 kb. Partial hepatectomy caused a significant 2–3-fold increase in the 1.5-kb and 5.0-kb messengers at 12 h after surgery. During the next 10 h alter partial hepatectomy cytidylyltransferase mRNA levels slightly decreased, although they were still elevated in comparison with sham-operated rats 20– 22 h after surgery. In contrast to the elevated cytidylyltransferase mRNA levels, the amount of acetyl-CoA carboxylase mRNA did not increase between 12 and 22 h alter surgery, which is in line with the unchanged activity of this enzyme. In conclusion, our data demonstrate that in regenerating liver phosphatidylcholine biosynthesis and cytidylyltransferase activity are regulated at a pretranslational level. [ABSTRACT FROM AUTHOR]

The role of potential target enzymes in the protein-kinase-C-independent eicosanoid response triggered by certain bacteria in murine peritoneal macrophages [Svensson, U., Holst, E. & Sundler, R. (1991) Eur. J. Biochem. 202, 699-705] has been investigated. The eicosanoid response was found to be due to an increase in the mobilization of arachidonate rather than to inhibition of arachidonate esterification or activation of the cyclooxygenase pathway and to be accompanied by a persistent increase in the activity of the arachidonate-mobilizing phospholipase A2 (PLA2-85). Also, down-regulation of protein-kinase C by prolonged treatment with 4 β-phorbol 12-myristate 13-acetate did not reduce the bacterial activation of PLA2-85. The increase in activity of PLA2-85, like the increase in eicosanoid formation, showed a lag period of approximately 10 min. Furthermore, exposure of 32P-labeled macrophages to either bacteria (Gardnerella vaginalis) or the protein-phosphatase inhibitor okadaic acid caused an increase in the phosphorylation of PLA2-85. Okadaic acid (0.5 µM), which itself caused arachidonate mobilization and activation of PLA2-85 after a lag period of approximately 45 min, greatly promoted the response to bacteria even at earlier time points. This study provides strong evidence that the eicosanoid response to bacteria in macrophages occurs via a protein-kinase-C-independent activation of PLA2-85 and that this activation is due to an increase in the phosphorylation of the enzyme. [ABSTRACT FROM AUTHOR]

• A straightforward enzymatic method was developed to synthesize specific lyso-phospholipids. • 20:1 lyso-PS is a true agonist of the TLR2/6 hetero-dimer. • Both chainlength and the headgroup of 20:1 lyso-PS determine this interaction. Toll-like receptor 2 (TLR2) is an important pattern recognition receptor on the surface of host immune cells that binds a variety of ligands that are released by microorganisms as well as by damaged or dying host cells. According to the current concept, TLR2/1 and TLR2/6 heterodimers are activated by tri- or di-acylated ligands, respectively. However, also mono-acyl phospholipid containing lipid fractions derived from parasites, were reported to be able to activate TLR2. In order to provide conclusive evidence for the TLR2 activating capacity of mono-acyl phospholipids derived from pathogens, we developed a biosynthetic method to enzymatically convert commercially available phospholipids into several mono-acyl-phospholipid variants that were examined for their TLR2 activating capacity. These investigations demonstrated that 1-(11Z-eicosenoyl)-glycero-3-phosphoserine 20:1 (20:1 lyso-PS) is a true agonist of the TLR2/6 heterodimer and that its polar headgroup as well as the length of the acyl chain are crucial for TLR2 activation. In silico modelling further confirmed 20:1 mono-acyl PS as a ligand for TLR2/6 heterodimer, as this predicted that multiple hydrogen bonds are formed between the polar headgroup of 20:1 mono-acyl PS and amino acid residues of both TLR2 and TLR6. Future studies can now be performed to further assess the functions of 20:1 lyso-PS as an immunological mediator, because this enzymatic method enables its preparation in larger quantities than is possible by isolation from the parasite that naturally produces this compound, Schistosoma mansoni , the source of the original discovery (Van der Kleij et al., 2002). [ABSTRACT FROM AUTHOR]

Background & Aims The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X receptor [FXR]) regulates bile acid synthesis, transport, and catabolism. FXR also regulates postprandial lipid and glucose metabolism. We performed quantitative proteomic analyses of liver tissues from mice to evaluate these functions and investigate whether FXR regulates amino acid metabolism. Methods To study the role of FXR in mouse liver, we used mice with a disruption of Nr1h4 (FXR-knockout mice) and compared them with floxed control mice. Mice were gavaged with the FXR agonist obeticholic acid or vehicle for 11 days. Proteome analyses, as well as targeted metabolomics and chromatin immunoprecipitation, were performed on the livers of these mice. Primary rat hepatocytes were used to validate the role of FXR in amino acid catabolism by gene expression and metabolomics studies. Finally, control mice and mice with liver-specific disruption of Nr1h4 (liver FXR-knockout mice) were re-fed with a high-protein diet after 6 hours fasting and gavaged a 15 NH 4 Cl tracer. Gene expression and the metabolome were studied in the livers and plasma from these mice. Results In livers of control mice and primary rat hepatocytes, activation of FXR with obeticholic acid increased expression of proteins that regulate amino acid degradation, ureagenesis, and glutamine synthesis. We found FXR to bind to regulatory sites of genes encoding these proteins in control livers. Liver tissues from FXR-knockout mice had reduced expression of urea cycle proteins, and accumulated precursors of ureagenesis, compared with control mice. In liver FXR-knockout mice on a high-protein diet, the plasma concentration of newly formed urea was significantly decreased compared with controls. In addition, liver FXR-knockout mice had reduced hepatic expression of enzymes that regulate ammonium detoxification compared with controls. In contrast, obeticholic acid increased expression of genes encoding enzymes involved in ureagenesis compared with vehicle in C57Bl/6 mice. Conclusions In livers of mice, FXR regulates amino acid catabolism and detoxification of ammonium via ureagenesis and glutamine synthesis. Failure of the urea cycle and hyperammonemia are common in patients with acute and chronic liver diseases; compounds that activate FXR might promote ammonium clearance in these patients. [ABSTRACT FROM AUTHOR]

Hepatic stellate cells (HSCs) are liver-resident cells best known for their role in vitamin A storage under physiological conditions. Upon liver injury, HSCs activate into myofibroblast-like cells, a key process in the onset of liver fibrosis. Lipids play an important role during HSC activation. Here, we provide a comprehensive characterization of the lipidomes of primary rat HSCs during 17 days of activation in vitro. For lipidomic data interpretation, we expanded our previously described Lipid Ontology (LION) and associated web application (LION/Web) with the LION-PCA heatmap module, which generates heatmaps of the most typical LIONsignatures in lipidomic datasets. Furthermore, we used LION to perform pathway analysis to determine the significant metabolic conversions in lipid pathways. Together, we identify two distinct stages of HSC activation. In the first stage, we observe a decrease of saturated phosphatidylcholine, sphingomyelin, and phosphatidic acid and an increase in phosphatidylserine and polyunsaturated bis(monoacylglycero)phosphate (BMP), a lipid class typically localized at endosomes and lysosomes. In the second activation stage, BMPs, hexosylceramides, and ether-linked phosphatidylcholines are elevated, resembling a lysosomal lipid storage disease profile. The presence of isomeric structures of BMP in HSCs was confirmed ex vivo in MS-imaging datasets of steatosed liver sections. Finally, treatment with pharmaceuticals targeting the lysosomal integrity led to cell death in primary HSCs but not in HeLa cells. In summary, our combined data suggest that lysosomes play a critical role during a two-stage activation process of HSCs. [ABSTRACT FROM AUTHOR]

The gene for the proapoptotic transcription factor CCAAT/enhancer-binding protein (C/EBP)-homologous protein/growth arrest and DNA damage-inducible protein 153 (CHOP/GADD153) is induced by various cellular stresses. Previously, we described that inhibition of phosphatidylcholine (PC) synthesis in MT58 cells, which contain a temperature-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), results in apoptosis preceded by the induction of CHOP. Here we report that prevention of CHOP induction, by expression of antisense CHOP, delays the PC depletion-induced apoptotic process. By mutational analysis of the conserved region in the promoter of the CHOP gene, we provide evidence that the C/EBP-ATF composite site, but not the ER stress-responsive element or the activator protein-1 site, is required for the increased expression of CHOP during PC depletion. Inhibition of PC synthesis in MT58 cells also led to an increase in phosphorylation of the stress-related transcription factor ATF2 and the stress kinase JNK after 8 and 16 h, respectively. In contrast, no phosphorylation of p38 MAPK was observed in MT58 cultured at the nonpermissive temperature. Treatment of MT58 cells with the JNK inhibitor SP600125 could rescue the cells from apoptosis but did not inhibit the phosphorylation of ATF2 or the induction of CHOP. Taken together, our results suggest that increased expression of CHOP during PC depletion depends on a C/EBP-ATF element in its promoter and might be mediated by binding of ATF2 to this element. [ABSTRACT FROM AUTHOR]

Activation of hepatic stellate cells (HSCs) is a critical step in the development of liver fibrosis. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerols (TAGs), cholesteryl esters, and retinyl esters (REs). We previously provided evidence for the presence of two distinct LD pools, a preexisting and a dynamic LD pool. Here we investigate the mechanisms of neutral lipid metabolism in the preexisting LD pool. To investigate the involvement of lysosomal degradation of neutral lipids, we studied the effect of lalistat, a specific lysosomal acid lipase (LAL/Lipa) inhibitor on LD degradation in HSCs during activation in vitro. The LAL inhibitor increased the levels of TAG, cholesteryl ester, and RE in both rat and mouse HSCs. Lalistat was less potent in inhibiting the degradation of newly synthesized TAG species as compared with a more general lipase inhibitor orlistat. Lalistat also induced the presence of RE-containing LDs in an acidic compartment. However, targeted deletion of the Lipa gene in mice decreased the liver levels of RE, most likely as the result of a gradual disappearance of HSCs in livers of Lipa-/- mice. Lalistat partially inhibited the induction of activation marker α-smooth muscle actin (α-SMA) in rat and mouse HSCs. Our data suggest that LAL/Lipa is involved in the degradation of a specific preexisting pool of LDs and that inhibition of this pathway attenuates HSC activation. [ABSTRACT FROM AUTHOR]

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC–MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT −/− HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT −/− HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ± 9.0% small (≤ 700 nm) lipid droplets in LRAT −/− HSCs and 25.6 ± 1.4% large (1400–2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (≤ 700 nm) lipid droplets strongly increased both in wild type and in LRAT −/− HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In mammalian cells, phosphatidylethanolamine (PtdEtn) is mainly synthesized via the CDP-ethanolamine (Kennedy) pathway and by decarboxylation of phosphatidylserine (PtdSer). However, the extent to which these two pathways contribute to overall PtdEtn synthesis both quantitatively and qualitatively is still not clear. To assess their contributions, PtdEtn species synthesized by the two routes were labeled with pathway-specific stable isotope precursors, d3-serine and d4-ethanolamine, and analyzed by high performance liquid chromatography-mass spectrometry. The major conclusions from this study are that (i) in both McA-RH7777 and Chinese hamster ovary K1 cells, the CDP-ethanolamine pathway was favored over PtdSer decarboxylation, and (ii) both pathways for PtdEtn synthesis are able to produce all diacyl-PtdEtn species, but most of these species were preferentially made by one pathway. For example, the CDP-ethanolamine pathway preferentially synthesized phospholipids with mono- or di-unsaturated fatty acids on the sn-2 position (e.g. (16:0-18:2)PtdEtn and (18:1-18:2)PtdEtn), whereas PtdSer decarboxylation generated species with mainly polyunsaturated fatty acids on the sn-2 position (e.g. (18:0-20:4)PtdEtn and (18:O-20:5)PtdEtn in McArdle and (18:0-20:4)PtdEtn and (18:0-22:6)PtdEtn in Chinese hamster ovary K1 cells). (iii) The main PtdEtn species newly synthesized from the Kennedy pathway in the microsomal fraction appeared to equilibrate rapidly between the endoplasmic reticulum and mitochondria. (iv) Newly synthesized PtdEtn species preferably formed in the mitochondria, which is at least in part due to the substrate specificity of the phosphatidylserine decarboxylase, seemed to be retained in this organelle. Our data suggest a potentially essential role of the PtdSer decarboxylation pathway in mitochondrial functioning. [ABSTRACT FROM AUTHOR]

Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets., (© 2021 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.)

Hepatic stellate cell (HSC) activation is a critical step in the development of chronic liver disease. During activation, HSCs lose their lipid droplets (LDs) containing triacylglycerol (TAG), cholesteryl esters (CEs), and retinyl esters (REs). Here we aimed to investigate which enzymes are involved in LD turnover in HSCs during activation in vitro. Targeted deletion of the Atgl gene in mice HSCs had little effect on the decrease of the overall TAG, CE, and RE levels during activation. However, ATGL-deficient HSCs specifically accumulated TAG species enriched in PUFAs and degraded new TAG species more slowly. TAG synthesis and levels of PUFA-TAGs were lowered by the diacylglycerol acyltransferase (DGAT)1 inhibitor, T863. The lipase inhibitor, Atglistatin, increased the levels of TAG in both WT and ATGL-deficient mouse HSCs. Both Atglistatin and T863 inhibited the induction of activation marker, α-smooth muscle actin, in rat HSCs, but not in mouse HSCs. Compared with mouse HSCs, rat HSCs have a higher turnover of new TAGs, and Atglistatin and the DGAT1 inhibitor, T863, were more effective. Our data suggest that ATGL preferentially degrades newly synthesized TAGs, synthesized by DGAT1, and is less involved in the breakdown of preexisting TAGs and REs in HSCs. Furthermore a large change in TAG levels has modest effect on rat HSC activation., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

The degree of fatty acid unsaturation, that is, the ratio of unsaturated versus saturated fatty acyl chains, determines membrane fluidity. Regulation of expression of the fatty acid desaturase Ole1p was hitherto the only known mechanism governing the degree of fatty acid unsaturation in Saccharomyces cerevisiae. We report a novel mechanism for the regulation of fatty acid desaturation that is based on competition between Ole1p and the glycerol-3-phosphate acyltransferase Sct1p/Gat2p for the common substrate C16:0-CoA. Deletion of SCT1 decreases the content of saturated fatty acids, whereas overexpression of SCT1 dramatically decreases the desaturation of fatty acids and affects phospholipid composition. Whereas overexpression of Ole1p increases desaturation, co-overexpression of Ole1p and Sct1p results in a fatty acid composition intermediate between those obtained upon overexpression of the enzymes separately. On the basis of these results, we propose that Sct1p sequesters C16:0-CoA into lipids, thereby shielding it from desaturation by Ole1p. Ta-king advantage of the growth defect conferred by overexpressing SCT1, we identified the acyltransferase Cst26p/Psi1p as a regulator of Sct1p activity by affecting the phosphorylation state and overexpression level of Sct1p. The level of Sct1p phosphorylation is increased when cells are supplemented with saturated fatty acids, demonstrating the physiological relevance of our findings.

The mutant Chinese hamster ovary cell line MT58 contains a thermosensitive mutation in CTP:phosphocholine cytidylyltransferase, the regulatory enzyme in the CDP-choline pathway. As a result, MT58 cells have a 50% decrease in their phosphatidylcholine (PC) level within 24 h when cultured at the nonpermissive temperature (40 degrees C). This is due to a relative rapid breakdown of PC that is not compensated for by the inhibition of de novo PC synthesis. Despite this drastic decrease in cellular PC content, cells are viable and can proliferate by addition of lysophosphatidylcholine. By [(3)H]oleate labeling, we found that the FA moiety of the degraded PC is recovered in triacylglycerol. In accordance with this finding, an accumulation of lipid droplets is seen in MT58 cells. Analysis of PC-depleted MT58 cells by electron and fluorescence microscopy revealed a partial dilation of the rough endoplasmic reticulum, resulting in spherical structures on both sites of the nucleus, whereas the morphology of the plasma membrane, mitochondria, and Golgi complex was unaffected. In contrast to these morphological observations, protein transport from the ER remains intact. Surprisingly, protein transport at the level of the Golgi complex is impaired. Our data suggest that the transport processes at the Golgi complex are regulated by distal changes in lipid metabolism.

For an insight regarding the control of PtdEtn (phosphatidylethanolamine) synthesis via the CDPethanolamine pathway, rat liver cDNA encoding ECT (CTP:phosphoethanolamine cytidylyltransferase) was transiently or stably transfected in Chinese-hamster ovary cells and a rat liver-derived cell line (McA-RH7777), resulting in a maximum of 26- and 4-fold increase in specific activity of ECT respectively. However, no effect of ECT overexpression on the rate of [3H]ethanolamine incorporation into PtdEtn was detected in both cell lines. This was explored further in cells overexpressing four times ECT activity (McA-ECT1). The rate of PtdEtn breakdown and PtdEtn mass were not changed in McA-ECT1 cells in comparison with control-transfected cells. Instead, an accumulation of CDPethanolamine (label and mass) was observed, suggesting that in McA-ECT1 cells the ethanolaminephosphotransferase-catalysed reaction became rate-limiting. However, overexpression of the human choline/ethanolaminephosphotransferase in McA-ECT1 and control-transfected cells had no effect on PtdEtn synthesis. To investigate whether the availability of DAG (diacylglycerol) limited PtdEtn synthesis in these cells, intracellular DAG levels were increased using PMA or phospholipase C. Exposure of cells to PMA or phospholipase C stimulated PtdEtn synthesis and this effect was much more pronounced in McA-ECT1 than in control-transfected cells. In line with this, the DAG produced after PMA exposure was consumed more rapidly in McA-ECT1 cells and the CDPethanolamine level decreased accordingly. In conclusion, our results suggest that the supply of CDPethanolamine, via the expression level of ECT, is an important factor governing the rate of PtdEtn biosynthesis in mammalian cells, under the condition that the amount of DAG is not limiting.

Inhibition of de novo synthesis of phosphatidylcholine (PC) by some anti-cancer drugs such as hexadecylphosphocholine leads to apoptosis in various cell lines. Likewise, in MT58, a mutant Chinese hamster ovary (CHO) cell line containing a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), an important regulatory enzyme in the CDP-choline pathway, inhibition of PC synthesis causes PC depletion. Cellular perturbations like metabolic insults and unfolded proteins can be registered by the endoplasmic reticulum (ER) and result in ER stress responses, which can lead eventually to apoptosis. In this study we investigated the effect of PC depletion on the ER stress response and ER-related proteins. Shifting MT58 cells to the non-permissive temperature of 40 degrees C resulted in PC depletion via an inhibition of CT within 24 h. Early apoptotic features appeared in several cells around 30 h, and most cells were apoptotic within 48 h. The temperature shift in MT58 led to an increase of pro-apoptotic CCAAT/enhancer-binding protein-homologous protein (CHOP; also known as GADD153) after 16 h, to a maximum at 24 h. Incubation of wild-type CHO-K1 or CT-expressing MT58 cells at 40 degrees C did not induce differences in CHOP protein levels in time. In contrast, expression of the ER chaperone BiP/GRP78, induced by an increase in misfolded/unfolded proteins, and caspase 12, a protease specifically involved in apoptosis that results from stress in the ER, did not differ between MT58 and CHO-K1 cells in time when cultured at 40 degrees C. Furthermore, heat-shock protein 70, a protein that is stimulated by accumulation of abnormal proteins and heat stress, displayed similar expression patterns in MT58 and K1 cells. These results suggest that PC depletion in MT58 induces the ER-stress-related protein CHOP, without raising a general ER stress response.

The present study was undertaken to study the role of AMP-activated kinase (AMPK) in the biosynthesis of two major membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of AMPK, produced dose-dependent inhibition of the incorporation of [(3)H]choline and [(3)H]ethanolamine into PC and PE, respectively. Determination of the cellular uptake of choline and ethanolamine showed that the reduced synthesis of PC and PE did not result from impaired uptake of these two precursors. The decreased synthesis of PC was not mirrored by a reduction in the activities of the enzymes of the CDP-choline pathway. The diminution of PE biosynthesis, however, was paralleled by a depressed activity of CTP:phosphoethanolamine cytidylyltransferase (ET), the pace-setting enzyme of the CDP-ethanolamine pathway. AICAR treatment of hepatocytes stimulated the conversion of choline into betaine, indicating that reduced PC synthesis most probably resulted from a decrease in the availability of choline. In addition, AICAR induced a 50% reduction in the cellular level of diacylglycerols, which may further impair the synthesis of PC and PE. The results thus indicate that AICAR inhibits the biosynthesis of PC and PE and that the effect is exerted at different sites in the two pathways. Increased oxidation of choline to betaine is the main target of AICAR in the PC pathway, whereas inhibition of ET activity is the locus of AICAR action in the PE pathway.