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Lipid metabolism in obesity The function of the endoplasmic reticulum (ER) changes during obesity: in the liver, ER-associated protein synthesis slows down, and genes involved in lipid metabolism are switched on. ER stress is an important factor in obesity, insulin resistance and type 2 diabetes. A possible mechanism for this link has now been identified. Perturbation of fatty acid and lipid metabolism in the ER inhibits the activity of SERCA, the main ER calcium importer. Changing the lipid composition or increasing the amount of SERCA in the ER is shown to relieve the stress and improve glucose homeostasis in vivo . The endoplasmic reticulum (ER) is the main site of protein and lipid synthesis, membrane biogenesis, xenobiotic detoxification and cellular calcium storage, and perturbation of ER homeostasis leads to stress and the activation of the unfolded protein response 1 . Chronic activation of ER stress has been shown to have an important role in the development of insulin resistance and diabetes in obesity 2 . However, the mechanisms that lead to chronic ER stress in a metabolic context in general, and in obesity in particular, are not understood. Here we comparatively examined the proteomic and lipidomic landscape of hepatic ER purified from lean and obese mice to explore the mechanisms of chronic ER stress in obesity. We found suppression of protein but stimulation of lipid synthesis in the obese ER without significant alterations in chaperone content. Alterations in ER fatty acid and lipid composition result in the inhibition of sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and ER stress. Correcting the obesity-induced alteration of ER phospholipid composition or hepatic Serca overexpression in vivo both reduced chronic ER stress and improved glucose homeostasis. Hence, we established that abnormal lipid and calcium metabolism are important contributors to hepatic ER stress in obesity.

Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

Phospholipid metabolism, including phosphatidylcholine (PC) biosynthesis, is crucial for various biological functions and is associated with longevity. Phosphatidylethanolamine N-methyltransferase (PEMT) is a protein that catalyzes the biosynthesis of PC, the levels of which change in various organs such as the brain and kidneys during aging. However, the role of PEMT for systemic PC supply is not fully understood. To address how PEMT affects aging-associated energy metabolism in tissues responsible for nutrient absorption, lipid storage, and energy consumption, we employed NMR-based metabolomics to study the liver, plasma, intestine (duodenum, jejunum, and ileum), brown/white adipose tissues (BAT and WAT), and skeletal muscle of young (9–10 weeks) and old (91–132 weeks) wild-type (WT) and PEMT knockout (KO) mice. We found that the effect of PEMT-knockout was tissue-specific and age-dependent. A deficiency of PEMT affected the metabolome of all tissues examined, among which the metabolome of BAT from both young and aged KO mice was dramatically changed in comparison to the WT mice, whereas the metabolome of the jejunum was only slightly affected. As for aging, the absence of PEMT increased the divergence of the metabolome during the aging of the liver, WAT, duodenum, and ileum and decreased the impact on skeletal muscle. Overall, our results suggest that PEMT plays a previously underexplored, critical role in both aging and energy metabolism.

The major facilitator superfamily domain 2a protein was identified recently as a lysophosphatidylcholine (LPC) symporter with high affinity for LPC species enriched with DHA (LPC-DHA). To test the hypothesis that reproductive state and choline intake influence plasma LPC-DHA, we performed a post hoc analysis of samples available through 10 weeks of a previously conducted feeding study, which provided two doses of choline (480 and 930 mg/d) to non-pregnant ( n 21), third-trimester pregnant ( n 26), and lactating ( n 24) women; all participants consumed 200 mg of supplemental DHA and 22 % of their daily choline intake as 2 H-labelled choline. The effects of reproductive state and choline intake on total LPC-DHA (expressed as a percentage of LPC) and plasma enrichments of labelled LPC and LPC-DHA were assessed using mixed and generalised linear models. Reproductive state interacted with time ( P  = 0·001) to influence total LPC-DHA, which significantly increased by week 10 in non-pregnant women, but not in pregnant or lactating women. Contrary to total LPC-DHA, patterns of labelled LPC-DHA enrichments were discordant between pregnant and lactating women ( P  < 0·05), suggestive of unique, reproductive state-specific mechanisms that result in reduced production and/or enhanced clearance of LPC-DHA during pregnancy and lactation. Regardless of the reproductive state, women consuming 930 v . 480 mg choline per d exhibited no change in total LPC-DHA but higher d3-LPC-DHA ( P  = 0·02), indicating that higher choline intakes favour the production of LPC-DHA from the phosphatidylethanolamine N -methyltransferase pathway of phosphatidylcholine biosynthesis. Our results warrant further investigation into the effect of reproductive state and dietary choline on LPC-DHA dynamics and its contribution to DHA status.

Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.

Phosphatidylethanolamine N-methyltransferase (Pemt) catalyzes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) mainly in the liver. Under an obese state, the upregulation of Pemt induces endoplasmic reticulum (ER) stress by increasing the PC/PE ratio in the liver. We targeted the Pemt gene in mice to explore the therapeutic impact of Pemt on the progression of diabetic nephropathy and diabetes, which was induced by the injection of streptozotocin (STZ). Although the blood glucose levels were similar in STZ-induced diabetic Pemt+/+ and Pemt-/-mice, the glomerular hypertrophy and albuminuria in Pemt-/- mice were significantly reduced. Pemt deficiency reduced the intraglomerular F4/80-positive macrophages, hydroethidine fluorescence, tubulointerstitial fibrosis and tubular atrophy. The expression of glucose-regulated protein-78 (GRP78) was enriched in the renal tubular cells in STZ-induced diabetic mice, and this was ameliorated by Pemt deficiency. In mProx24 renal proximal tubular cells, the treatment with ER-stress inducers, tunicamycin and thapsigargin, increased the expression of GRP78, which was reduced by transfection of a shRNA lentivirus for Pemt (shRNA-Pemt). The number of apoptotic cells in the renal tubules was significantly reduced in Pemt-/- diabetic mice, and shRNA-Pemt upregulated the phosphorylation of Akt and decreased the cleavage of caspase 3 and 7 in mProx24 cells. Taken together, these findings indicate that the inhibition of Pemt activity ameliorates the ER stress associated with diabetic nephropathy in a model of type 1 diabetes and corrects the functions of the three major pathways downstream of ER stress, i.e. oxidative stress, inflammation and apoptosis.

Plasmodium knowlesi is recognised as the main cause of human malaria in Southeast Asia. The disease is often misdiagnosed as P. falciparum or P. malariae infections by microscopy, and the disease is difficult to eliminate due to its presence in both humans and monkeys. P. knowlesi infections can rapidly cause severe disease and require prompt diagnosis and treatment. No protein biomarker exists for the rapid diagnostic test (RDT) detection of P. knowlesi infections. Plasmodium knowlesi infections can be diagnosed by PCR. Phosphoethanolamine-N-methyltransferase (PMT) is involved in malaria lipid biosynthesis and is not found in the human host. The P. falciparum, P. vivax and P. knowlesi PMT proteins were recombinantly expressed in BL21(DE3) Escherichia coli host cells, affinity purified and used to raise antibodies in chickens. Antibodies against each recombinant PMT protein all detected all three recombinant proteins and the native 29 kDa P. falciparum PMT protein on western blots and in ELISA. Antibodies against a PMT epitope (PLENNQYTDEGVKC) common to all three PMT orthologues detected all three proteins. Antibodies against unique peptides from each orthologue of PMT, PfCEVEHKYLHENKE, PvVYSIKEYNSLKDC, PkLYPTDEYNSLKDC detected only the parent protein in western blots and P. falciparum infected red blood cell lysates or blood lysates spiked with the respective proteins. Similar concentrations of PfPMT and the control, PfLDH, were detected in the same parasite lysate. The recombinant PfPMT protein was detected by a human anti-malaria antibody pool. PMT, like the pan-specific LDH biomarker used in RDT tests, is both soluble, present at comparable concentrations in the parasite and constitutes a promising antimalarial drug target. PMT is absent from the human proteome. PMT has the potential as a biomarker for human malaria and in particular as the first P. knowlesi specific protein with diagnostic potential for the identification of a P. knowlesi infection.

ABSTRACT Legionella species synthesize phosphatidylcholine (PC) in two independent pathways: the three-step methylation of phosphatidylethanolamine PMT pathway and the one-step PCS pathway, in which the Pcs enzyme catalyzes the reaction between choline and CDP-diacylglycerol to form PC. Legionella pcs genes encode highly hydrophobic proteins with phosphatidylcholine synthase activity, which contain up to eight transmembrane helices with N- and C-termini located inside the bacterial cell. The comparative analysis of nucleotide sequences of pcs showed that these genes share high sequence identity among members of the Legionellaceae family. Legionella pmtA genes involved in the PMT pathway encoded small cytosolic proteins with putative phosphatidylethanolamine N-methyltransferase activity. The pmtA genes identified in Legionella species had lower sequence identity to each other than the pcs genes. The phylogenetic tree constructed based on the pcs and pmtA gene sequences showed phylogenetic relatedness between Legionella spp. and other bacteria. The utilization of extracellular choline by the four Legionella species leads to changes not only in the lipid components but also in proteins, and the interactions between these components lead to changes in cell surface properties, which result in a decline in induction of proinflammatory cytokines (TNF-α and IL-6). Utilization of exogenous choline by Legionella spp. affects the production of pro-inflammatory cytokines.

Background Hemerocallis citrina Baroni (daylily) is a horticultural ornamental plant and vegetable with various applications as a raw material in traditional Chinese medicine and as a flavouring agent. Daylily contains many functional substances and is rich in lecithin, which is mostly composed of glycerophospholipids. To study the comprehensive dynamic changes in glycerophospholipid during daylily flowering and the underlying signalling mechanisms, we performed comprehensive, time-resolved lipidomic and transcriptomic analyses of ‘Datong Huanghua 6’ daylily. Results Labelling with PKH67 fluorescent antibodies clearly and effectively helped visualise lipid changes in daylily, while relative conductivity and malonaldehyde content detection revealed that the early stages of flowering were controllable processes; however, differences became non-significant after 18 h, indicating cellular damage. In addition, phospholipase D (PLD) and lipoxygenase (LOX) activities increased throughout the flowering process, suggesting that lipid hydrolysis and oxidation had intensified. Lipidomics identified 558 lipids that changed during flowering, with the most different lipids found 12 h before and 12 h after flowering. Transcriptome analysis identified 13 key functional genes and enzymes in the glycerophospholipid metabolic pathway. The two-way orthogonal partial least squares analysis showed that diacylglycerol diphosphate phosphatase correlated strongly and positively with phosphatidic acid (PA)(22:0/18:2), PA(34:2), PA(34:4), and diacylglycerol(18:2/21:0) but negatively with phospholipase C. In addition, ethanolamine phosphotransferase gene and phospholipid-N-methyltransferase gene correlated positively with phosphatidylethanolamine (PE)(16:0/18:2), PE(16:0/18:3), PE(33:2), and lysophosphatidylcholine (16:0) but negatively with PE(34:1). Conclusions Overall, this study elucidated changes in the glycerophospholipid metabolism pathway during the daylily flowering process, as well as characteristic genes, thus providing a basis for future studies of glycerophospholipids and signal transduction in daylilies.

The endoplasmic reticulum is the key organelle which controls protein folding, lipid biogenesis, and calcium (Ca²⁺) homeostasis. Cd exposure in Saccharomyces cerevisiae activated the unfolded protein response and was confirmed by the increased Kar2p expression. Cd exposure in wild-type (WT) cells increased PC levels and the PC biosynthetic genes. Deletion of the two phospholipid methyltransferases CHO2 and OPI3 modulated PC, TAG levels and the lipid droplets with cadmium exposure. Interestingly, we noticed an increase in the calcium levels upon Cd exposure in the mutant cells. This study concluded that Cd interrupted calcium homeostasis-induced lipid dysregulation leading to ER stress.