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Sterol regulatory element-binding protein 1c (SREBP-1c) is a central regulator of lipogenesis whose activity is controlled by proteolytic cleavage. The metabolic factors that affect its processing are incompletely understood. Here, we show that dynamic changes in the acyl chain composition of ER phospholipids affect SREBP-1c maturation in physiology and disease. The abundance of polyunsaturated phosphatidylcholine in liver ER is selectively increased in response to feeding and in the setting of obesity-linked insulin resistance. Exogenous delivery of polyunsaturated phosphatidylcholine to ER accelerated SREBP-1c processing through a mechanism that required an intact SREBP cleavage-activating protein (SCAP) pathway. Furthermore, induction of the phospholipid-remodeling enzyme LPCAT3 in response to liver X receptor (LXR) activation promoted SREBP-1c processing by driving the incorporation of polyunsaturated fatty acids into ER. Conversely, LPCAT3 deficiency increased membrane saturation, reduced nuclear SREBP-1c abundance, and blunted the lipogenic response to feeding, LXR agonist treatment, or obesity-linked insulin resistance. Desaturation of the ER membrane may serve as an auxiliary signal of the fed state that promotes lipid synthesis in response to nutrient availability.

The role of specific phospholipids (PLs) in lipid transport has been difficult to assess due to an inability to selectively manipulate membrane composition in vivo. Here we show that the phospholipid remodeling enzyme lysophosphatidylcholine acyltransferase 3 (Lpcat3) is a critical determinant of triglyceride (TG) secretion due to its unique ability to catalyze the incorporation of arachidonate into membranes. Mice lacking Lpcat3 in the intestine fail to thrive during weaning and exhibit enterocyte lipid accumulation and reduced plasma TGs. Mice lacking Lpcat3 in the liver show reduced plasma TGs, hepatosteatosis, and secrete lipid-poor very low-density lipoprotein (VLDL) lacking arachidonoyl PLs. Mechanistic studies indicate that Lpcat3 activity impacts membrane lipid mobility in living cells, suggesting a biophysical basis for the requirement of arachidonoyl PLs in lipidating lipoprotein particles. These data identify Lpcat3 as a key factor in lipoprotein production and illustrate how manipulation of membrane composition can be used as a regulatory mechanism to control metabolic pathways. Living cells are surrounded by a membrane that forms a barrier between the cell and its external environment. This membrane is largely made up of a variety of molecules known as lipids. The particular lipid molecules found in a cell membrane strongly influence its mobility, flexibility and other physical properties. The liver and intestine can package lipids gained from the diet into molecules called lipoproteins, which are released into the bloodstream for use by the body. An enzyme known as Lpcat3 is found in high levels in the liver and intestine and it appears to be involved in the production of lipoproteins. Altering the amount of Lpcat3 in cells can change the types of lipids found in the cell membranes, connected to the production of lipoproteins. Rong et al. studied newborn mice that were missing the Lpcat3 protein in either the liver or intestine. Mice lacking Lpcat3 in the intestine had higher levels of lipids inside their intestine cells and grew more slowly than normal mice. Mice lacking Lpcat3 in the liver also accumulated lipids in their cells and their bloodstream had lower levels of lipids that contain a molecule called arachidonic acid than normal mice. Further experiments showed that the loss of Lpcat3 reduces the ability of lipids to move within the cell membrane. The experiments show that Lpcat3 plays a key role in attaching arachidonic acid to membrane lipids to promote the release of lipoproteins into the bloodstream. Rong et al.'s findings reveal that changing the type of lipids in the cell membrane plays an important role in regulating the levels of lipids in the blood.

Background Metabolic reprogramming is one of the hallmarks of cancer which favours rapid energy production, biosynthetic capabilities and therapy resistance. In our previous study, we showed bitter melon extract (BME) prevents carcinogen induced mouse oral cancer. RNA sequence analysis from mouse tongue revealed a significant modulation in “Metabolic Process” by altering glycolysis and lipid metabolic pathways in BME fed group as compared to cancer group. In present study, we evaluated the effect of BME on glycolysis and lipid metabolism pathways in human oral cancer cells. Methods Cal27 and JHU022 cells were treated with BME. RNA and protein expression were analysed for modulation of glycolytic and lipogenesis genes by quantitative real-time PCR, western blot analyses and immunofluorescence. Lactate and pyruvate level was determined by GC/MS. Extracellular acidification and glycolytic rate were measured using the Seahorse XF analyser. Shotgun lipidomics in Cal27 and JHU022 cell lines following BME treatment was performed by ESI/ MS. ROS was measured by FACS. Results Treatment with BME on oral cancer cell lines significantly reduced mRNA and protein expression levels of key glycolytic genes SLC2A1 (GLUT-1), PFKP, LDHA, PKM and PDK3. Pyruvate and lactate levels and glycolysis rate were reduced in oral cancer cells following BME treatment. In lipogenesis pathway, we observed a significant reduction of genes involves in fatty acid biogenesis, ACLY, ACC1 and FASN, at the mRNA and protein levels following BME treatment. Further, BME treatment significantly reduced phosphatidylcholine, phosphatidylethanolamine, and plasmenylethanolamine, and reduced iPLA2 activity. Additionally, BME treatment inhibited lipid raft marker flotillin expression and altered its subcellular localization. ER-stress associated CHOP expression and generation of mitochondrial reactive oxygen species were induced by BME, which facilitated apoptosis. Conclusion Our study revealed that bitter melon extract inhibits glycolysis and lipid metabolism and induces ER and oxidative stress-mediated cell death in oral cancer. Thus, BME-mediated metabolic reprogramming of oral cancer cells will have important preventive and therapeutic implications along with conventional therapies. Graphical abstract

We previously demonstrated neutrophil MPO derived HOCl targets the vinyl ether bond of plasmalogens resulting in the Liberation of 2-chlorofatty aldehydes (2-ClFALDs) and their oxidation products, 2-chlorofatty acids (2-ClFAs), which elicit neutrophil extracellular trap (NET) formation. In this study, the click chemistry analog of 2-chlorohexadecanoic acid (2-ClHA) was utilized to identify 127 proteins covalently modified by 2-ClHA in human neutrophils. Bioinformatics revealed that multiple proteins modified by 2-ClHA are related to protein modification and binding as well as metabolite interconversion. Three key proteins involved in NET formation and function were modified by 2-ClHA including peptidyl arginine deiminase 4 (PAD4), neutrophil defensin alpha 3 (DEFA3), and neutrophil collagenase (MMP8). PAD4 activity was shown to be increased by 2-ClFA treatment. Further studies investigated 2-ClFA modified protein localization over time during NET formation. Initially PAD4 and 2-ClFA-modified proteins were extranuclear but over time they both localized to distinct nuclear regions. Following DNA release from neutrophils, 2-ClFA-modified proteins were found throughout the neutrophil and DNA strands. In summary, multiple neutrophil proteins are modified by 2-ClHA, including PAD4. 2-ClHA modification and activation of PAD4 is suggested as a key component of 2-ClHA elicited NET formation. Highlights • One hundred and twenty-seven neutrophil proteins are covalently modified by 2-ClFA. • 2-ClFA covalently modified human neutrophil proteins including neutrophil defensin alpha 3 (DEFA3), neutrophil collagenase (MMP8) and peptidyl arginine deiminase 4 (PAD4). • Co-localization of DEFA3 and PAD4 with 2-ClFA is maintained over time during NET formation. • Both 2-ClFA and PAD4 colocalize to the nucleus prior to the release of DNA strands during NET formation. • 2-ClFA activates PAD4. Graphical abstract

Neutrophils are the most abundant white blood cells recruited to the sites of infection and inflammation. During neutrophil activation, myeloperoxidase (MPO) is released and converts hydrogen peroxide to hypochlorous acid (HOCl). HOCl reacts with plasmalogen phospholipids to liberate 2-chlorofatty aldehyde (2-ClFALD), which is metabolized to 2-chlorofatty acid (2-ClFA). 2-ClFA and 2-ClFALD are linked with inflammatory diseases and induce endothelial dysfunction, neutrophil extracellular trap formation (NETosis) and neutrophil chemotaxis. Here we examine the neutrophil-derived chlorolipid production in the presence of pathogenic E. coli strain CFT073 and non-pathogenic E. coli strain JM109. Neutrophils cocultured with CFT073 E. coli strain and JM109 E. coli strain resulted in 2-ClFALD production. 2-ClFA was elevated only in CFT073 coculture. NETosis is more prevalent in CFT073 cocultures with neutrophils compared to JM109 cocultures. 2-ClFA and 2-ClFALD were both shown to have significant bactericidal activity, which is more severe in JM109 E. coli . 2-ClFALD metabolic capacity was 1000-fold greater in neutrophils compared to either strain of E. coli . MPO inhibition reduced chlorolipid production as well as bacterial killing capacity. These findings indicate the chlorolipid profile is different in response to these two different strains of E. coli bacteria.

Bile secretion is essential for whole body sterol homeostasis. Loss‐of‐function mutations in specific canalicular transporters in the hepatocyte disrupt bile flow and result in cholestasis. We show that two of these transporters, ABCB11 and ATP8B1, are functional targets of miR‐33, a micro‐RNA that is expressed from within an intron of SREBP‐2 . Consequently, manipulation of miR‐33 levels in vivo with adenovirus or with antisense oligonucleotides results in changes in bile secretion and bile recovery from the gallbladder. Using radiolabelled cholesterol, we show that systemic silencing of miR‐33 leads to increased sterols in bile and enhanced reverse cholesterol transport in vivo . Finally, we report that simvastatin causes, in a dose‐dependent manner, profound hepatotoxicity and lethality in mice fed a lithogenic diet. These latter results are reminiscent of the recurrent cholestasis found in some patients prescribed statins. Importantly, pretreatment of mice with anti‐miR‐33 oligonucleotides rescues the hepatotoxic phenotype. Therefore, we conclude that miR‐33 mediates some of the undesired, hepatotoxic effects of statins. →See accompanying article http://dx.doi.org/10.1002/emmm.201201565

The liver is the major organ responsible for lipid biosynthesis. Sterol regulatory element-binding proteins (SREBP) are major transcription factors that regulate the expression of genes regulating fatty acid and cholesterol biosynthesis. Here we show that elaidic acid upregulates hepatic de-novo fatty acid and cholesterol synthesis in HuH-7 cells. To define the molecular mechanism involved in this unique regulation on hepatic lipogenesis, luciferase reporter gene assays were performed in HEK293 cells to compare the regulation of sterol regulatory element (SRE) that is present in SREBP-target promoter by elaidic acid and oleic acid. The results show that elaidic acid potently induced SRE-luciferase activity, whereas oleic acid inhibited this activity. Furthermore, elaidic acid increased SREBP-1c mRNA, while oleic acid did not alter it. Oleic acid inhibited mature form of SREBP-1 protein level, while elaidic acid did not show inhibitory effects. In addition, elaidic acid was also found to increase several selected lipogenic genes that are involved in fatty acids and sterol synthesis. These data demonstrate a unique role of elaidic acid, the most abundant trans fatty acid, in modulating hepatic lipogenesis.

Omega (n)-3 polyunsaturated fatty acids (PUFA) are converted to bioactive lipid components that are important mediators in metabolic and physiological pathways; however, which bioactive compounds are metabolically active, and their mechanisms of action are still not clear. We investigated using lipidomic techniques, the effects of diets high in n-3 PUFA on the fatty acid composition of various bioactive lipids in plasma and liver. Female C57BL/6 mice were fed semi-purified diets (20% w/w fat) containing varying amounts of n-3 PUFA before mating, during gestation and lactation, and until weaning. Male offspring were continued on their mothers' diets for 16 weeks. Hepatic and plasma lipids were extracted in the presence of non-naturally occurring internal standards, and tandem electrospray ionization mass spectrometry methods were used to measure the fatty acyl compositions. There was no significant difference in total concentrations of phospholipids in both groups. However, there was a significantly higher concentration of eicosapentaenoic acid containing phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and cholesteryl esters (CE) (p < 0.01) in the high n-3 PUFA group compared to the low n-3 PUFA group in both liver and plasma. Plasma and liver from the high n-3 PUFA group also had a higher concentration of free n-3 PUFA (p < 0.05). There were no significant differences in plasma concentrations of different fatty acyl species of phosphatidylethanolamine, triglycerides, sphingomyelin and ceramides. Our findings reveal for the first time that a diet high in n-3 PUFA caused enrichment of n-3 PUFA in PC, LPC, CE and free fatty acids in the plasma and liver of C57BL/6 mice. PC, LPC, and unesterified free n-3 PUFA are important bioactive lipids, thus altering their fatty acyl composition will have important metabolic and physiological roles.

One of the hallmarks of cancer is metabolic reprogramming which controls cellular homeostasis and therapy resistance. Here, we investigated the effect of momordicine-I (M-I), a key bioactive compound from Momordica charantia (bitter melon), on metabolic pathways in human head and neck cancer (HNC) cells and a mouse HNC tumorigenicity model. We found that M-I treatment on HNC cells significantly reduced the expression of key glycolytic molecules, SLC2A1 (GLUT-1), HK1 , PFKP , PDK3 , PKM , and LDHA at the mRNA and protein levels. We further observed reduced lactate accumulation, suggesting glycolysis was perturbed in M-I treated HNC cells. Metabolomic analyses confirmed a marked reduction in glycolytic and TCA cycle metabolites in M-I-treated cells. M-I treatment significantly downregulated mRNA and protein expression of essential enzymes involved in de novo lipogenesis, including ACLY , ACC1 , FASN , SREBP1 , and SCD1 . Using shotgun lipidomics, we found a significant increase in lysophosphatidylcholine and phosphatidylcholine loss in M-I treated cells. Subsequently, we observed dysregulation of mitochondrial membrane potential and significant reduction of mitochondrial oxygen consumption after M-I treatment. We further observed M-I treatment induced autophagy, activated AMPK and inhibited mTOR and Akt signaling pathways and leading to apoptosis. However, blocking autophagy did not rescue the M-I-mediated alterations in lipogenesis, suggesting an independent mechanism of action. M-I treated mouse HNC MOC2 cell tumors displayed reduced Hk1, Pdk3, Fasn, and Acly expression. In conclusion, our study revealed that M-I inhibits glycolysis, lipid metabolism, induces autophagy in HNC cells and reduces tumor volume in mice. Therefore, M-I-mediated metabolic reprogramming of HNC has the potential for important therapeutic implications. Graphical Abstract

The liver plays a central role in regulating glucose and lipid metabolism. Aberrant insulin action in the liver is a major driver of selective insulin resistance, in which insulin fails to suppress glucose production but continues to activate lipogenesis in the liver, resulting in hyperglycemia and hypertriglyceridemia. The underlying mechanisms of selective insulin resistance are not fully understood. Here It is shown that hepatic membrane phospholipid composition controlled by lysophosphatidylcholine acyltransferase 3 (LPCAT3) regulates insulin signaling and systemic glucose and lipid metabolism. Hyperinsulinemia induced by high‐fat diet (HFD) feeding augments hepatic Lpcat3 expression and membrane unsaturation. Loss of Lpcat3 in the liver improves insulin resistance and blunts lipogenesis in both HFD‐fed and genetic ob/ob mouse models. Mechanistically, Lpcat3 deficiency directly facilitates insulin receptor endocytosis, signal transduction, and hepatic glucose production suppression and indirectly enhances fibroblast growth factor 21 (FGF21) secretion, energy expenditure, and glucose uptake in adipose tissue. These findings identify hepatic LPCAT3 and membrane phospholipid composition as a novel regulator of insulin sensitivity and provide insights into the pathogenesis of selective insulin resistance. Hepatic phospholipid (PL) saturation, mediated by depletion of a PL remodeling enzyme LPCAT3, protects mice from diet‐induced obesity through FGF21 and elevated energy expenditure. Ablation of Lpcat3 in the liver improves insulin sensitivity by facilitating insulin receptor endocytosis into early endosomes and glucose uptake in brown adipose tissue.