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Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease, characterized by excessive hepatic lipid accumulation. Recently, we demonstrated that Smad ubiquitination regulatory factor 1 (Smurf1) deficiency significantly alleviates mouse hepatic steatosis. However, the mechanism of Smurf1-regulating hepatic lipid accumulation requires further exploration and clarification. Hence, this study explores the potential mechanism of Smurf1 in hepatic steatosis. In this study, hepatic Smurf1 proteins in NAFLD patients and healthy individuals were determined using immunohistochemical staining. Control and NAFLD mouse models were established by feeding Smurf1-knockout (KO) and wild-type mice with either a high-fat diet (HFD) or a chow diet (CD) for eight weeks. Oleic acid (OA)-induced steatotic hepatocytes were used as the NAFLD mode cells. Lipid content in liver tissues was analyzed. Smurf1-MDM2 interaction, MDM2 and p53 ubiquitination, and p53 target genes expression in liver tissues and hepatocytes were analyzed. We found that hepatic Smurf1 is highly expressed in NAFLD patients and HFD-induced NAFLD mice. Its deletion attenuates hepatocyte steatosis. Mechanistically, Smurf1 interacts with and stabilizes mouse double minute 2 (MDM2), promoting p53 degradation. In Smurf1-deficient hepatocytes, an increase in p53 suppresses SREBP-1c expression and elevates the expression of both malonyl-CoA decarboxylase (MCD) and lipin1 (Lpin1), two essential proteins in lipid catabolism. Contrarily, the activities of these three proteins and hepatocyte steatosis are reversed by p53 knockdown in Smurf1-deficient hepatocytes. This study shows that Smurf1 is involved in the pathogenesis of NAFLD by balancing de novo lipid synthesis and lipolysis.

Objective Acute myocardial infarction (AMI) initiates pathological inflammation which aggravates tissue damage and causes heart failure. Lysophosphatidic acid (LPA), produced by autotaxin (ATX), promotes inflammation and the development of atherosclerosis. The role of ATX/LPA signaling nexus in cardiac inflammation and resulting adverse cardiac remodeling is poorly understood. Approach and results We assessed autotaxin activity and LPA levels in relation to cardiac and systemic inflammation in AMI patients and C57BL/6 (WT) mice. Human and murine peripheral blood and cardiac tissue samples showed elevated levels of ATX activity, LPA, and inflammatory cells following AMI and there was strong correlation between LPA levels and circulating inflammatory cells. In a gain of function model, lipid phosphate phosphatase-3 (LPP3) specific inducible knock out (Mx1-Plpp3Δ) showed higher systemic and cardiac inflammation after AMI compared to littermate controls (Mx1-Plpp3fl/fl); and a corresponding increase in bone marrow progenitor cell count and proliferation. Moreover, in Mx1- Plpp3Δ mice, cardiac functional recovery was reduced with corresponding increases in adverse cardiac remodeling and scar size (as assessed by echocardiography and Masson's Trichrome staining). To examine the effect of ATX/LPA nexus inhibition, we treated WT mice with the specific pharmacological inhibitor, PF8380, twice a day for 7 days post AMI. Inhibition of the ATX/LPA signaling nexus resulted in significant reduction in post-AMI inflammatory response, leading to favorable cardiac functional recovery, reduced scar size and enhanced angiogenesis. Conclusion ATX/LPA signaling nexus plays an important role in modulating inflammation after AMI and targeting this mechanism represents a novel therapeutic target for patients presenting with acute myocardial injury.

Lipin1 is important in lipid synthesis because of its phosphatidate phosphatase activity, and it also functions as transcriptional coactivators to regulate the expression of genes involved in lipid metabolism. We found that fld mice exhibit cognitive impairment, and it is related to the DAG-PKD-ERK pathway. We used fld mice to explore the relationship between lipin1 and cognitive function. Our results confirmed the presence of cognitive impairment in the hippocampus of lipin1-deficient mice. As shown in behavioral test, the spatial learning and memory ability of fld mice was much worse than that of wild-type mice. Electron microscopy results showed that the number of synapses in hippocampus of fld mice was significantly reduced. BDNF,SYP, PSD95 were significantly reduced. These results suggest that lipin1 impairs synaptic plasticity. Hence,a deficiency of lipin1 leads to decreased DAG levels and inhibits PKD activation, thereby affecting the phosphorylation of ERK and the CREB.

Lipin-2 is a phosphatidate phosphatase with key roles in regulating lipid storage and energy homeostasis. LPIN2-genetic deficiency is associated with an autoinflammatory disorder, underscoring its critical role in innate immune signaling; however, the regulatory mechanisms underlying protein stability remain unknown. Here, we demonstrate that Lipin-2 interacts with β-TRCP, a substrate receptor subunit of the SCFβ-TRCP E3 ligase, and undergoes ubiquitination and proteasomal degradation. β-TRCP-knockout in RAW264.7 macrophages resulted in Lipin-2 accumulation, leading to the suppression of LPS-induced MAPK activation and subsequent proinflammatory gene expression. Consistent with this, treatment with MLN4924, a Cullin-neddylation inhibitor that suppresses SCF E3 activity, increased Lipin-2 protein and concomitantly decreased Il1b expression. These findings suggested that β-TRCP-mediated Lipin-2 degradation affects macrophage-elicited proinflammatory responses and could lead to new therapeutic approaches to treat inflammatory diseases.

PLPPs (Phospholipid phosphatases) are widely expressed in different human tissues, regulate cell signal transduction, and are overexpressed in cancers such as gliomas, pancreatic adenocarcinoma, lung adenocarcinoma, and so on. As a member of the PLPP family, PLPP2 (phospholipid phosphatase 2) plays a vital role in the occurrence and development of breast cancer, but its mechanism is still unclear. Our research found that PLPP2 was overexpressed in breast cancer, and the higher expression level of PLPP2 showed a worse prognosis for breast cancer patients. Further analysis showed that overexpression of PLPP2 affected the expression of CDC34 (cell-division cycle 34), LSM7 (Like-Smith 7), and SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) through EMT (epigenetic-mesenchymal transition) related pathways to promote the occurrence and development of breast cancer. In vitro, silencing PLPP2 significantly reduced the proliferation, invasion, and migration abilities of human breast cancer cells MDA-MB-231. ER+ is a common subtype of breast cancer. Furthermore, we found that the overexpression of PLPP2 was significantly related to the poor prognosis of ER+ breast cancer. These results indicate that PLPP2 has value as a potential therapeutic target for breast cancer, especially for ER+ breast cancer.

In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg

The PAH1-encoded phosphatidate phosphatase in Saccharomyces cerevisiae plays a major role in triacylglycerol synthesis and the control of phospholipid synthesis. For its catalytic function on the nuclear/endoplasmic reticulum membrane, Pah1 translocates to the membrane through its phosphorylation/dephosphorylation. Pah1 phosphorylation on multiple serine/threonine residues is complex and catalyzed by diverse protein kinases. In this work, we demonstrate that Pah1 is phosphorylated by the YCK1-encoded casein kinase I (CKI), regulating Pah1 catalytic activity and phosphorylation. Phosphoamino acid analysis coupled with phosphopeptide mapping of the CKI-phosphorylated Pah1 indicated that it is phosphorylated mainly on multiple serine residues. Using site-directed mutagenesis and phosphorylation analysis of Pah1, we identified eight serine residues (i.e. Ser-114, Ser-475, Ser-511, Ser-602, Ser-677, Ser-705, Ser-748, and Ser-774) as the target sites of CKI. Of these residues, Ser-475 and Ser-511 were specific for CKI, whereas the others were shared by casein kinase II (Ser-705), Cdc28-cyclin B (Ser-602), Pho85-Pho80 (Ser-114, Ser-602, and Ser-748), protein kinase A (Ser-667 and Ser-774), and protein kinase C (Ser-677). CKI-mediated phosphorylation of Pah1 stimulated both its phosphatidate phosphatase activity and its subsequent phosphorylation by casein kinase II. However, the CKI-mediated phosphorylation of Pah1 strongly inhibited its subsequent phosphorylation by Pho85-Pho80, protein kinase A, and protein kinase C. In a reciprocal analysis, Pah1 phosphorylation by Pho85-Pho80 inhibited subsequent phosphorylation by CKI. CKI-mediated Pah1 phosphorylation was also inhibited by a peptide containing the Pah1 residues 506-517, including the kinase-specific Ser-511 residue. These findings advance our understanding of how Pah1 catalytic activity and phosphorylation are regulated by multiple protein kinases.

Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids. Cell surface LPP3 can terminate the signaling actions of bioactive lysophosphatidic acid (LPA) and sphingosine 1 phosphate, which likely explains its role in developmental angiogenesis, vascular injury responses, and cell migration. Heritable variants in the final intron PLPP3 associate with interindividual variability in coronary artery disease risk that may result from disruption of enhancer sequences that normally act in cis to increase expression of the gene. However, the mechanisms regulating PLPP3 expression are not well understood. We show that the human PLPP3 promoter contains three functional NF-κB response elements. All of these are required for maximal induction of PLPP3 promoter activity in reporter assays. The identified sequences recruit RelA and RelB components of the NF-κB transcription complex to chromatin, and these transcription factors bind to the identified target sequences in two different cell types. LPA promotes binding of Rel family transcription factors to the PLPP3 promoter and increases PLPP3 gene expression through mechanisms that are attenuated by an NF-κB inhibitor, LPA receptor antagonists, and inhibitors of phosphoinositide 3 kinase. These findings indicate that up-regulation of PLPP3 during inflammation and atherosclerosis results from canonical activation of the NF-κB signaling cascade to increase PLPP3 expression through nuclear import and binding of RelA and RelB transcription factors to the PLPP3 promoter and suggest a mechanism by which the LPP3 substrate, LPA, can regulate PLPP3 expression.

Pah1 phosphatidate (PA) phosphatase plays a major role in triacylglycerol synthesis in Saccharomyces cerevisiae by producing its precursor diacylglycerol and concurrently regulates de novo phospholipid synthesis by consuming its precursor PA. The function of Pah1 requires its membrane localization, which is controlled by its phosphorylation state. Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, whereas its phosphorylation occurs by multiple known and unknown protein kinases. In this work, we show that Rim11, a yeast homolog of mammalian glycogen synthase kinase-3β, is a protein kinase that phosphorylates Pah1 on serine (Ser12, Ser602, and Ser818) and threonine (Thr163, Thr164, Thr522) residues. Enzymological characterization of Rim11 showed that its K

The PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol, controls the divergence of phosphatidate into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the nuclear/endoplasmic reticulum membrane as a dephosphorylated form by the Nem1-Spo7 protein phosphatase complex. The phosphorylation of Pah1 by protein kinases, which include casein kinases I and II, Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and B, controls its cellular location, catalytic activity, and susceptibility to proteasomal degradation. Nem1 (catalytic subunit) and Spo7 (regulatory subunit), which form a protein phosphatase complex catalyzing the dephosphorylation of Pah1 for its activation, are phosphorylated by protein kinases A and C. In this review, we discuss the functions and interrelationships of the protein kinases in the control of the Nem1-Spo7/Pah1 phosphatase cascade and lipid synthesis.

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the dephosphorylation of PA to produce diacylglycerol, controls the bifurcation of PA into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the membrane as a dephosphorylated form by the Nem1-Spo7 protein phosphatase. We show that the conserved Trp-637 residue of Pah1, located in the intrinsically disordered region, is required for normal synthesis of membrane phospholipids, sterols, triacylglycerol, and the formation of lipid droplets. Analysis of mutant Pah1-W637A showed that the tryptophan residue is involved in the phosphorylation-mediated/dephosphorylation-mediated membrane association of the enzyme and its catalytic activity. The endogenous phosphorylation of Pah1-W637A was increased at the sites of the N-terminal region but was decreased at the sites of the C-terminal region. The altered phosphorylation correlated with an increase in its membrane association. In addition, membrane-associated PA phosphatase activity in vitro was elevated in cells expressing Pah1-W637A as a result of the increased membrane association of the mutant enzyme. However, the inherent catalytic function of Pah1 was not affected by the W637A mutation. Prediction of Pah1 structure by AlphaFold shows that Trp-637 and the catalytic residues Asp-398 and Asp-400 in the haloacid dehalogenase-like domain almost lie in the same plane, suggesting that these residues are important to properly position the enzyme for substrate recognition at the membrane surface. These findings underscore the importance of Trp-637 in Pah1 regulation by phosphorylation, membrane association of the enzyme, and its function in lipid synthesis.

The Nem1–Spo7 protein phosphatase plays a role in lipid synthesis by controlling the membrane localization of Pah1, the diacylglycerol-producing phosphatidate (PA) phosphatase that is crucial for the synthesis of triacylglycerol in the yeast Saccharomyces cerevisiae. By dephosphorylating Pah1, Nem1–Spo7 facilitates its translocation to the nuclear/endoplasmic reticulum membrane for catalytic activity. Like its substrate Pah1, Nem1–Spo7 is phosphorylated in the cell, but the specific protein kinases involved remain to be identified. In this study, we demonstrate that the Nem1–Spo7 complex is phosphorylated by protein kinase A (PKA), which is associated with active cell growth, metabolic activity, and membrane phospholipid synthesis. In vitro phosphorylation of purified Nem1–Spo7 and of their synthetic peptides revealed that both subunits of the phosphatase complex are PKA substrates. Using phosphoamino acid and phosphopeptide-mapping analyses coupled with site-directed mutagenesis, we identified Ser-140 and Ser-210 of Nem1 and Ser-28 of Spo7 as PKA-targeted phosphorylation sites. Immunodetection of the phosphatase complex from the cell with anti-PKA substrate antibody confirmed the in vivo phosphorylations of Nem1 and Spo7 on the serine residues. Lipid-labeling analysis of cells bearing phosphorylation-deficient alleles of NEM1 and SPO7 indicated that the PKA phosphorylation of the phosphatase complex stimulates phospholipid synthesis and attenuates the synthesis of triacylglycerol. This work advances the understanding of how PKA-mediated posttranslational modifications of Nem1 and Spo7 regulate lipid synthesis in yeast.

Enhancement of triacylglycerol (TAG) and oleic acid accumulation in cottonseed is desirable as it is world’s sixth largest source of plant oil in addition to its potential application in biofuel industry and material industry. In this two years study, economic N management technology, optimal plant density and simplified seedling raising technology were integrated to investigate the systematic advantage of integrated soil-cotton system management (ISSM1 and ISSM2) over conventional management practice (CM) on lipid and fatty acid profiles of cottonseed embryos under different soil available nutrition conditions. In cottonseed embryos, TAG constituted for 94.7% to 97.8% of the total lipids and the integrated soil-cotton system management resulted in the TAG yield and oleic acid increased by 32.5% and 4.5% in ISSM1 embryos, and by 19.8% and 3.8% in ISSM2 embryos. A high embryo weight compensates decreased TAG content resulted from increased N rate. Moreover, TAG yield was positively related to TAG accumulation rate, and the rapid TAG accumulation stage from 24 days to 35 days contributed the highest to the increased TAG accumulation rate (0.15 g–0.18 g per 100 embryos per day). Increased activities of glucose-6-phosphate and phosphatidate phosphatase were observed under ISSM1 and ISSM2, which reflect a quantity increase in carbon flux and energy production required for lipid biosynthesis. Moreover, increased phosphoenolpyruvate carboxylase not only regulates carbon partitioning but also enhances seed filling period and embryo weight. Taken our results suggest that the combination of simplified seedling raising technology, economic N management schedule and optimal plant density was capable of increasing carbon flux and energy production required for lipid biosynthesis and thus generate high levels of embryo TAG and oleic acid at different soil available nutrition conditions. Targeted improvement of cottonseed yield should not focus on TAG content alone but also embryo weight and seed filling period.

LPP3 is an integral membrane protein belonging to a family of enzymes (LPPs) that display broad substrate specificity and catalyse dephosphorylation of several lipid substrates, including lysophosphatidic acid and sphingosine-1-phosphate. In mammals, the LPP family consists of three enzymes named LPP1, LPP2 and LPP3, which are encoded by three independent genes, PLPP1, PLPP2 and PLPP3, respectively (formerly known as PPAP2A, PPAP2C, PPAP2B). These three enzymes, in vitro, do not seem to differ for catalytic activities and substrate preferences. However, in vivo targeted inactivation of the individual genes has indicated that the enzymes do not have overlapping functions and that LPP3, specifically, plays a crucial role in vascular development. In 2011, two genome-wide association studies have identified PLPP3 as a novel locus associated with coronary artery disease susceptibility. Shortly after these reports, tissue specific inactivation of PLPP3 in mice highlighted a specific role for LPP3 in vascular pathophysiology and, more recently, in atherosclerosis development. This review is aimed at providing an updated overview on the function of LPP3 in embryonic cardiovascular development and on the experimental and clinical evidences relating this enzyme to vascular cell functions and cardiovascular disease.

Introduction Lipin-1 deficiency is a major cause of rhabdomyolysis that are precipitated by febrile illness. The prognosis is poor, with one-third of patients dying from cardiac arrest during a crisis episode. Apart from acute rhabdomyolysis, most patients are healthy, showing normal clinical and cardiac ultrasound parameters. Patients and methods We report cardiac and exercise examinations of 8 children carrying two LPIN1 mutations. The examinations were performed outside of a myolysis episode, but one patient presented with fever during one examination. Results All but one patient displayed normal resting cardiac function, as determined by echocardiography. One patient exhibited slight left ventricular dysfunction at rest and a lack of increased stroke volume during cycle ramp exercise. During exercise, peripheral muscle adaptation was impaired in 2 patients compared to healthy controls: they presented an abnormal increase in cardiac output relative to oxygen uptake: dQ/dVO2 = 8.2 and 9.5 (> 2DS of controls population). One patient underwent 2 exercise tests; during one test, the patient was febrile, leading to acute rhabdomyolysis in the following hours. He exhibited changes in recovery muscle reoxygenation parameters and an increased dQ/dVO2 during exercise compared with that under normothermia (7.9 vs 6), which did not lead to acute rhabdomyolysis. The four patients assessed by cardiac 1H-magnetic resonance spectroscopy exhibited signs of intracardiac steatosis. Conclusion We observed abnormal haemodynamic profiles during exercise in 3/8 patients with lipin-1 deficiency, suggesting impaired muscle oxidative phosphorylation during exercise. Fever appeared to be an aggravating factor. One patient exhibited moderate cardiac dysfunction, which was possibly related to intracardiac stored lipid toxicity.

FXR agonists are used to treat non-alcoholic fatty liver disease (NAFLD), in part because they reduce hepatic lipids. Here, we show that FXR activation with the FXR agonist GSK2324 controls hepatic lipids via reduced absorption and selective decreases in fatty acid synthesis. Using comprehensive lipidomic analyses, we show that FXR activation in mice or humans specifically reduces hepatic levels of mono- and polyunsaturated fatty acids (MUFA and PUFA). Decreases in MUFA are due to FXR-dependent repression of Scd1, Dgat2, and Lpin1 expression, which is independent of SHP and SREBP1c. FXR-dependent decreases in PUFAs are mediated by decreases in lipid absorption. Replenishing bile acids in the diet prevented decreased lipid absorption in GSK2324-treated mice, suggesting that FXR reduces absorption via decreased bile acids. We used tissue-specific FXR KO mice to show that hepatic FXR controls lipogenic genes, whereas intestinal FXR controls lipid absorption. Together, our studies establish two distinct pathways by which FXR regulates hepatic lipids.

Lipins 1, 2, and 3 are Mg2+-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA in vitro. The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation in vitro.

PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol at the endoplasmic reticulum membrane, plays a major role in controlling the utilization of phosphatidate for the synthesis of triacylglycerol or membrane phospholipids. The conserved N-LIP and haloacid dehalogenase-like domains of Pah1 are required for phosphatidate phosphatase activity and the in vivo function of the enzyme. Its non-conserved regions, which are located between the conserved domains and at the C terminus, contain sites for phosphorylation by multiple protein kinases. Truncation analyses of the non-conserved regions showed that they are not essential for the catalytic activity of Pah1 and its physiological functions (e.g. triacylglycerol synthesis). This analysis also revealed that the C-terminal region contains a previously unrecognized WRDPLVDID domain (residues 637-645) that is conserved in yeast, mice, and humans. The deletion of this domain had no effect on the catalytic activity of Pah1 but caused the loss of its in vivo function. Site-specific mutational analyses of the conserved residues within WRDPLVDID indicated that Trp-637 plays a crucial role in Pah1 function. This work also demonstrated that the catalytic activity of Pah1 is required but is not sufficient for its in vivo functions.

In yeast, PAH1 plays an important role in cell homeostasis and lipid biosynthesis. PAH1 encodes for the PA phosphatase, Pah1p, which is responsible for de novo TAG and phospholipid synthesis. It has been suggested that the lack of Pah1p causes irregular vacuolar morphology and dysfunctional V-ATPase pump activity. However, the molecular connection between Pah1p and V-ATPase activity has remained unclear. Through real-time PCR, we have shown that PAH1 is maximally induced at the stationary stage in the presence of inositol. We also found that vacuoles were less fragmented when PAH1 is maximally expressed. Subsequently, we observed that vacuoles from pah1Δ cells were more acidic than those in WT cells. Furthermore, V-ATPase genes were upregulated in the absence of Pah1p. These results suggest that Pah1p plays an important role in vacuolar activity by negatively regulating the expression of V-ATPase genes. As such, we provide evidence to show the role of Pah1p in vacuolar acidification and fragmentation.

The PAH1-encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1-encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP–diacylglycerol pathway. The lack of PAP in the pah1Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1. Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1Δ mutant is induced through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2–Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UASINO mutation suppressed pah1Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1-encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis.

Protein kinase C in Saccharomyces cerevisiae, i.e., Pkc1, is an enzyme that plays an important role in signal transduction and the regulation of lipid metabolic enzymes. Pkc1 is structurally similar to its counterparts in higher eukaryotes, but its requirement of phosphatidylserine (PS) and diacylglycerol (DAG) for catalytic activity has been unclear. In this work, we examined the role of these lipids in Pkc1 activity with protein and peptide substrates. In agreement with previous findings, yeast Pkc1 did not require PS and DAG for its activity on the peptide substrates derived from lipid metabolic proteins such as Pah1 [phosphatidate (PA) phosphatase], Nem1 (PA phosphatase phosphatase), and Spo7 (protein phosphatase regulatory subunit). However, the lipids were required for Pkc1 activity on the protein substrates Pah1, Nem1, and Spo7. Compared with DAG, PS had a greater effect on Pkc1 activity, and its dose-dependent interaction with the protein kinase was shown by the liposome binding assay. The Pkc1-mediated degradation of Pah1 was attenuated in the cho1Δ mutant, which is deficient in PS synthase, supporting the notion that the phospholipid regulates Pkc1 activity in vivo.

Saponins are a structurally diverse family of triterpenes that are widely found as main constituents in many traditional plant-based medicines and often have bioactivities of industrial interest. The heterologous production of triterpene saponins in microbes remains challenging and only limited successful pathway engineering endeavors have been reported. To improve the production capacities of a Saccharomyces cerevisiae saponin production platform, we assessed the effects of several hitherto unexplored gene knockout targets on the heterologous production of triterpenoids. Here, we show that the disruption of the phosphatidic acid phosphatase-encoding PAH1 through CRISPR/Cas9 results in a dramatic expansion of the endoplasmic reticulum (ER), which stimulated the production of recombinant triterpene biosynthesis enzymes and ultimately boosted triterpenoid and triterpene saponin accumulation. Compared to the wild-type starter strain, accumulation of the oleanane-type sapogenin β-amyrin, of its oxidized derivative medicagenic acid, and its glucosylated version medicagenic-28-O-glucoside was respectively increased by eight-, six- and 16-fold in the pah1 strain. A positive effect of pah1 could also be observed for the production of other terpenoids depending on ER-associated enzymes for their biosynthesis, such as the sesquiterpenoid artemisinic acid, which increased by twofold relative to the wild-type strain. Hence, this report demonstrates that pathway engineering in yeast through transforming the subcellular morphology rather than altering metabolic fluxes is a powerful strategy to increase yields of bioactive plant-derived products in heterologous hosts.

Summary Germ cell death occurs in many species [1–3] and has been proposed as a mechanism by which the fittest, strongest, or least damaged germ cells are selected for transmission to the next generation. However, little is known about how the choice is made between germ cell survival and death. Here, we focus on the mechanisms that regulate germ cell survival during embryonic development in Drosophila . We find that the decision to die is a germ cell-intrinsic process linked to quantitative differences in germ plasm inheritance, such that higher germ plasm inheritance correlates with higher primordial germ cell (PGC) survival probability. We demonstrate that the maternal factor lipid phosphate phosphatase Wunen-2 (Wun2) regulates PGC survival in a dose-dependent manner. Since wun2 mRNA levels correlate with the levels of other maternal determinants at the single-cell level, we propose that Wun2 is used as a readout of the overall germ plasm quantity, such that only PGCs with the highest germ plasm quantity survive. Furthermore, we demonstrate that Wun2 and p53, another regulator of PGC survival, have opposite yet independent effects on PGC survival. Since p53 regulates cell death upon DNA damage and various cellular stresses, we hypothesize that together they ensure selection of the PGCs with highest germ plasm quantity and least cellular damage.

The conversion of the glycerophospholipid phosphatidic acid (PA) into diacylglycerol (DAG) is essential for the biosynthesis of membrane phospholipids and storage fats. Importantly, both PA and DAG can also serve signaling functions in the cell. The dephosphorylation of PA that yields DAG can be executed by two different classes of enzymes, Mg(2+)-dependent lipins and Mg(2+)-independent lipid phosphate phosphatases. Here, I will discuss the current status of research directed at understanding the roles of these enzymes in insect development and metabolism. Special emphasis will be given to studies in the model organism Drosophila melanogaster.

PPARγ agonists induced obesity in animal models as a side effect. Microarray experiments reveal that PPARγ agonist upregulates the expression of lipin-1 and this upregulation is correlated with the activity of the agonists. Lipin-1 induced by PPARγ agonists decreased the levels of PPARγ and ERK1/2 phosphorylation through direct interaction with these proteins in 3T3-L1 cells. In PPARγ agonist-treated 3T3-L1 preadipocytes, the knockdown of lipin-1 expression by small interfering RNA inhibited the adipogenesis that was induced by PPARγ agonists. In contrast, PPARγ2 expression was increased, and lipid droplets were accumulated in lipin-1-overexpressing 3T3-L1 adipocytes. Rosiglitazone (RGZ), a strong PPARγ agonist, synergistically promoted PPARγ dephosphorylation and adipogenesis in lipin-1-overexpressing 3T3-L1 preadipocytes. Therefore, lipin-1 has dual functions as a transcriptional cofactor and phosphatidate phosphatase (PAP) in the differentiation of preadipocyte cells induced by strong PPARγ agonists. In addition, the adipogenesis of 3T3-L1 cells was markedly upregulated by diacylglycerol (DAG), which was produced by lipin-1. Therefore, lipin-1 induction by PPARγ agonists might be an important factor in understanding the biological mechanism of the agonists’ adverse effects, and this information may be valuable in the development of type-2 diabetes mellitus (T2DM) therapeutics with reduced adverse effects and greater tolerability.

PgpB belongs to the lipid phosphate phosphatase protein family and is one of three bacterial integral membrane phosphatases catalyzing dephosphorylation of phosphatidylglycerol phosphate (PGP) to generate phosphatidylglycerol. Although the structure of its apo form became recently available, the mechanisms of PgpB substrate binding and catalysis are still unclear. We found that PgpB was inhibited by phosphatidylethanolamine (PE) in a competitive mode in vitro. Here we report the crystal structure of the lipid-bound form of PgpB. The structure shows that a PE molecule is stabilized in a membrane-embedded tunnel formed by TM3 and the “PSGH” fingerprint peptide near the catalytic site, providing structural insight into PgpB substrate binding mechanism. Noteworthy, in silico docking of varied lipid phosphates exhibited similar substrate binding modes to that of PE, and the residues in the lipid tunnel appear to be important for PgpB catalysis. The catalytic triad in the active site is essential for dephosphorylating substrates lysophosphatidic acid, phosphatidic acid, or sphingosine-1-phosphate but surprisingly not for the native substrate PGP. Remarkably, residue His-207 alone is sufficient to hydrolyze PGP, indicating a specific catalytic mechanism for PgpB in PG biosynthesis. We also identified two novel sensor residues, Lys-93 and Lys-97, on TM3. Our data show that Lys-97 is essential for the recognition of lyso-form substrates. Modification at the Lys-93 position may alter substrate specificity of lipid phosphate phosphatase proteins in prokaryotes versus eukaryotes. These studies reveal new mechanisms of lipid substrate selection and catalysis by PgpB and suggest that the enzyme rests in a PE-stabilized state in the bilayer.

Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 phosphatase complex.

The expansion of lipid droplets (LDs) and the differentiation of preadipocytes are two important aspects of mammalian lipid storage. In this study, we examined the role of CDP-diacylglycerol (DAG) synthases (CDSs), encoded by CDS1 and CDS2 genes in mammals, in lipid storage. CDS enzymes catalyze the formation of CDP-DAG from phosphatidic acid (PA). Knocking down either CDS1 or CDS2 resulted in the formation of giant or supersized LDs in cultured cells. Moreover, depleting CDS1 almost completely blocked the differentiation of 3T3-L1 preadipocytes, whereas depleting CDS2 had a moderate inhibitory effect on adipocyte differentiation. The levels of many PA species were significantly increased upon knocking down CDS1. In contrast, only a small number of PA species were increased upon depleting CDS2. Importantly, the amount of PA in the endoplasmic reticulum was dramatically increased upon knocking down CDS1 or CDS2. Our results suggest that the changes in PA level and localization may underlie the formation of giant LDs as well as the block in adipogenesis in CDS-deficient cells. We have therefore identified CDS1 and CDS2 as important novel regulators of lipid storage, and these results highlight the crucial role of phospholipids in mammalian lipid storage.

14 Páginas; 9 Figuras, Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its derivatives diphosphoinositol phosphates (DPIPs) play key signaling and regulatory roles. However, a direct function of these molecules in lipid and membrane homeostasis remains obscure. Here, we have studied the cold tolerance phenotype of yeast cells lacking the Inp51-mediated phosphoinositide-5-phosphatase. Genetic and biochemical approaches showed that increased metabolism of PI(4,5)P2 reduces the activity of the Pho85 kinase by increasing the levels of the DPIP isomer 1-IP7. This effect was key in the cold tolerance phenotype. Indeed, pho85 mutant cells grew better than the wild-type at 15 °C, and lack of this kinase abolished the inp51-mediated cold phenotype. Remarkably, reduced Pho85 function by loss of Inp51 affected the activity of the Pho85-regulated target Pah1, the yeast phosphatidate phosphatase. Cells lacking Inp51 showed reduced Pah1 abundance, derepression of an INO1-lacZ reporter, decreased content of triacylglycerides and elevated levels of phosphatidate, hallmarks of the pah1 mutant. However, the inp51 phenotype was not associated to low Pah1 activity since deletion of PAH1 caused cold sensitivity. In addition, the inp51 mutant exhibited features not shared by pah1, including a 40%-reduction in total lipid content and decreased membrane fluidity. These changes may influence the activity of membrane-anchored and/or associated proteins since deletion of INP51 slows down the transit to the vacuole of the fluorescent dye FM4-64. In conclusion, our work supports a model in which changes in the PI(4,5)P2 pool affect the 1-IP7 levels modulating the activity of Pho85, Pah1 and likely additional Pho85-controlled targets, and regulate lipid composition and membrane properties., This research has been supported by the Comisión Interministerial de Ciencia y Tecnología Project (AGL2010-17516) from the Spanish Ministry of Economy and Competitiveness (MINECO). IC-S was supported by a pre-doctoral fellowship within the “FPI” Program (MINECO).

The plasticity-related gene 1 (PRG1) modulates bioactive lipids at the postsynaptic density and is a novel player in neuronal plasticity and regulation of glutamatergic transmission at principal neurons. PRG1, a neuronal molecule, is highly expressed during development and regeneration processes at the postsynaptic density, modulates synaptic lysophosphatidic acid (LPA) levels and is related to epilepsy and brain injury. In the present study, we analyzed the interaction between the synaptic molecules PRG1 and LPA2R with other plasticity-related molecules the neurotrophins. The protein levels of NGF, BDNF and NT-3 were measured using ELISA in hippocampal tissue of homozygous (PRG(-/-)) and heterozygous (PRG(+/-)) PRG1 deficient mice and compared to their wild type (PRG(+/+)/WT) littermates. In the hippocampus, protein levels of NT-3 were significantly increased in PRG(-/-) mice (compared to WT-litters) while protein levels of NGF and BDNF were not affected. Since PRG1 deficiency leads to increased neuronal excitability and higher hippocampal network activity, which may well influence neurotrophin levels, we further assessed PRG1 deficient mice on an LPA2-receptor (LPA2R) deficient background, reported to normalize hippocampal over-excitability in PRG1(-/-) mice. However, on an LPA2R deficient background, protein levels of NT-3 in PRG1(-/-) mice (PRG1(-/-)/LPA2R(-/-)) were not significantly different when compared to WT animals. Since PRG1 deficient mice showed over-excitability in glutamatergic neurons. This was normalized by additional LPA2R deletion, and we conclude the increased NT3-levels were directly or indirectly attributable to increased hippocampal network activity, possibly exerting a protective effect against over-excitability.

In yeast and higher eukaryotes, phospholipids and triacylglycerol are derived from phosphatidate at the nuclear/endoplasmic reticulum membrane. In de novo biosynthetic pathways, phosphatidate is channeled into membrane phospholipids via its conversion to CDP-diacylglycerol. Its dephosphorylation to diacylglycerol is required for the synthesis of triacylglycerol as well as for the synthesis of phosphatidylcholine and phosphatidylethanolamine via the Kennedy pathway. In addition to the role of phosphatidate as a precursor, it is a regulatory molecule in the transcriptional control of phospholipid synthesis genes via the Henry regulatory circuit. Pah1 phosphatidate phosphatase and Dgk1 diacylglycerol kinase are key players that function counteractively in the control of the phosphatidate level at the nuclear/endoplasmic reticulum membrane. Loss of Pah1 phosphatidate phosphatase activity not only affects triacylglycerol synthesis but also disturbs the balance of the phosphatidate level, resulting in the alteration of lipid synthesis and related cellular defects. The pah1Δ phenotypes requiring Dgk1 diacylglycerol kinase exemplify the importance of the phosphatidate level in the misregulation of cellular processes. The catalytic function of Pah1 requires its translocation from the cytoplasm to the nuclear/endoplasmic reticulum membrane, which is regulated through its phosphorylation in the cytoplasm by multiple protein kinases as well as through its dephosphorylation by the membrane-associated Nem1-Spo7 protein phosphatase complex. This article is part of a Special Issue entitled Endoplasmic reticulum platforms for lipid dynamics edited by Shamshad Cockcroft and Christopher Stefan.

Yeast Fld1 and Ldb16 resemble mammalian seipin, implicated in neutral lipid storage. Both proteins form a complex at the endoplasmic reticulum-lipid droplet (LD) interface. Malfunction of this complex either leads to LD clustering or to the generation of supersized LD (SLD) in close vicinity to the nuclear envelope, in response to altered phospholipid (PL) composition. We show that similar to mutants lacking Fld1, deletion of LDB16 leads to abnormal proliferation of a subdomain of the nuclear envelope, which is tightly associated with clustered LD. The human lipin-1 ortholog, the PAH1 encoded phosphatidic acid (PA) phosphatase, and its activator Nem1 are highly enriched at this site. The specific accumulation of PA-binding marker proteins indicates a local enrichment of PA in the fld1 and ldb16 mutants. Furthermore, we demonstrate that clustered LD in fld1 or ldb16 mutants are transformed to SLD if phosphatidylcholine synthesis is compromised by additional deletion of the phosphatidylethanolamine methyltransferase, Cho2. Notably, treatment of wild-type cells with oleate induced a similar LD clustering and nuclear membrane proliferation phenotype as observed in fld1 and ldb16 mutants. These data suggest that the Fld1–Ldb16 complex affects PA homeostasis at an LD-forming subdomain of the nuclear envelope. Lack of Fld1–Ldb16 leads to locally elevated PA levels that induce an abnormal proliferation of nER membrane structures and the clustering of associated LD. We suggest that the formation of SLD is a consequence of locally altered PL metabolism at this site.

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries and the underlying cause of cardiovascular disease, a major cause of mortality worldwide. The over-accumulation of modified cholesterol-containing low-density lipoproteins (e.g. oxLDL) in the artery wall and the subsequent recruitment and activation of macrophages contributes to the development of atherosclerosis. The excessive uptake of modified-LDL by macrophages leads to a lipid-laden "foamy" phenotype and pro-inflammatory cytokine production. Modified-LDLs promote foam cell formation in part by stimulating de novo lipid biosynthesis. However, it is unknown if lipid biosynthesis directly regulates foam cell pro-inflammatory mediator production. Lipin-1, a phosphatidate phosphohydrolase required for the generation of diacylglycerol during glycerolipid synthesis has recently been demonstrated to contribute to bacterial-induced pro-inflammatory responses by macrophages. In this study we present evidence demonstrating the presence of lipin-1 within macrophages in human atherosclerotic plaques. Additionally, reducing lipin-1 levels in macrophages significantly inhibits both modified-LDL-induced foam cell formation in vitro, as observed by smaller/fewer intracellular lipid inclusions, and ablates modified-LDL-elicited production of the pro-atherogenic mediators tumor necrosis factor-α, interleukin-6, and prostaglandin E2. These findings demonstrate a critical role for lipin-1 in the regulation of macrophage inflammatory responses to modified-LDL. These data begin to link the processes of foam cell formation and pro-inflammatory cytokine production within macrophages.

We previously identified DS1 plants that showed resistance to compatible Ralstonia solanacearum with accelerated defense responses. Here, we describe activation mechanisms of defense responses in DS1 plants. After inoculation with incompatible R. solanacearum 8107, DS1 plants showed hyperinduction of hypersensitive response (HR) and reactive oxygen species (ROS) generation. Transient expression of PopP1 and AvrA induced hyperinduction of HR and ROS generation. Furthermore, Pseudomonas cichorii (Pc) and a type III secretion system (TTSS)-deficient mutant of P. cichorii showed accelerated induction of HR and ROS generation. Chitin and flg22 did not induce either HR or ROS hyperaccumulation; however, INF1 accelerated HR and ROS in DS1 plants. Activation of these defense responses was closely associated with increased phosphatidic acid (PA) content. Our results show that DS1 plants exhibit PA-mediated sensitization of plant defenses and that cell death-inducing stress is required to achieve full activation of defense responses.

Local modulation of glucocorticoid action in adipocytes regulates adiposity and systemic insulin sensitivity. However, the specific cofactors that mediate glucocorticoid receptor (GR) action in adipocytes remain unclear. Here we show that the silencing mediator of retinoid and thyroid hormone receptors (SMRT) is recruited to GR in adipocytes and regulates ligand-dependent GR function. Decreased SMRT expression in adipocytes in vivo increases expression of glucocorticoid-responsive genes. Moreover, adipocytes with decreased SMRT expression exhibit altered glucocorticoid regulation of lipolysis. We conclude that SMRT regulates the metabolic functions of GR in adipocytes in vivo. Modulation of GR-SMRT interactions in adipocytes represents a novel approach to control the local degree of glucocorticoid action and thus influence adipocyte metabolic function.

Pah1 is the phosphatidate phosphatase in the yeast Saccharomyces cerevisiae that produces diacylglycerol for triacylglycerol synthesis and concurrently controls the levels of phosphatidate used for phospholipid synthesis. Phosphorylation and dephosphorylation of Pah1 regulate its subcellular location and phosphatidate phosphatase activity. Compared with its phosphorylation by multiple protein kinases, Pah1 is dephosphorylated by a protein phosphatase complex consisting of Nem1 (catalytic subunit) and Spo7 (regulatory subunit). In this work, we characterized the Nem1-Spo7 phosphatase complex for its enzymological, kinetic, and regulatory properties with phosphorylated Pah1. The dephosphorylation of Pah1 by Nem1-Spo7 phosphatase resulted in the stimulation (6-fold) of phosphatidate phosphatase activity. For Pah1 phosphorylated by the Pho85-Pho80 kinase complex, maximum Nem1-Spo7 phosphatase activity required Mg(2+) ions (8 mm) and Triton X-100 (0.25 mm) at pH 5.0. The energy of activation for the reaction was 8.4 kcal/mol, and the enzyme was thermally labile at temperatures above 40 °C. The enzyme activity was inhibited by sodium vanadate, sodium fluoride, N-ethylmaleimide, and phenylglyoxal but was not significantly affected by lipids or nucleotides. Nem1-Spo7 phosphatase activity was dependent on the concentrations of Pah1 phosphorylated by Pho85-Pho80, Cdc28-cyclin B, PKA, and PKC with kcat and Km values of 0.29 s(-1) and 81 nm, 0.11 s(-1) and 127 nm, 0.10 s(-1) and 46 nm, and 0.02 s(-1) and 38 nm, respectively. Its specificity constant (kcat/Km) for Pah1 phosphorylated by Pho85-Pho80 was 1.6-, 4-, and 6-fold higher, respectively, than that phosphorylated by PKA, Cdc28-cyclin B, and PKC.

Recent evidences suggest that NAD(+) depletion leads to abnormal hepatic lipid metabolism in high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD); however, the contributing mechanism is not well understood. Our previous study showed that troxerutin, a trihydroxyethylated derivative of natural bioflavonoid rutin, effectively inhibited obesity, and normalized hyperglycemia and hyperlipidemia in high-cholesterol diet-induced diabetic mice. Here we investigated whether troxerutin improved hepatic lipid metabolism via preventing NAD(+) depletion in HFD-induced NAFLD mouse model and the mechanisms underlying these effects. Our results showed that troxerutin markedly prevented obesity, liver steatosis and injury in HFD-fed mice. Troxerutin largely suppressed oxidative stress-mediated NAD(+)-depletion by increasing nicotinamide phosphoribosyltransferase (NAMPT) protein expression and decreasing poly (ADP-ribose) polymerase-1 (PARP1) protein expression and activity in HFD-treated mouse livers. Consequently, troxerutin remarkably restored Silent mating type information regulation 2 homolog1 (SirT1) protein expression and activity in HFD-treated mouse livers. Therefore, troxerutin promoted SirT1-mediated AMP-activated protein kinase (AMPK) activation to inhibit mammalian target of rapamycin complex 1 (mTORC1) signaling, which enhanced nuclear lipin 1 localization, lowered cytoplasmic lipin 1 localization and the ratio of hepatic Lpin 1β/α. Ultimately, troxerutin improved lipid homeostasis by enhancing fatty acid oxidation and triglyceride secretion, and suppressing lipogenesis in HFD-fed mouse livers. In conclusion, troxerutin displayed beneficial effects on hepatic lipid homeostasis in HFD-induced NAFLD by blocking oxidative stress to restore NAD(+)-depletion-mediated dysfunction of lipin 1 signaling. This study provides novel mechanistic insights into NAFLD pathogenesis and indicates that troxerutin is a candidate for pharmacological intervention of NAFLD via restoring NAD(+) levels.

Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1-3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins.

Background & Aims Sirtuin (SIRT1) is a nicotinamide adenine dinucleotide−dependent protein deacetylase that regulates hepatic lipid metabolism by modifying histones and transcription factors. Ethanol exposure disrupts SIRT1 activity and contributes to alcoholic liver disease in rodents, but the exact pathogenic mechanism is not clear. We compared mice with liver-specific deletion of Sirt1 (Sirt1LKO) mice with their LOX littermates (controls). Methods We induced alcoholic liver injury in male Sirt1LKO and control mice, placing them on Lieber-DeCarli ethanol-containing diets for 10 days and then administering a single dose of ethanol (5 g/kg body weight) via gavage. Liver and serum samples were collected. We also measured messenger RNA levels of SIRT1, SFRS10, and lipin-1β and lipin-1α in liver samples from patients with alcoholic hepatitis and individuals without alcoholic hepatitis (controls). Results On the ethanol-containing diet, livers of Sirt1LKO mice accumulated larger amounts of hepatic lipid and expressed higher levels of inflammatory cytokines than control mice; serum of Sirt1LKO mice had increased levels of alanine aminotransferase and aspartate aminotransferase. Hepatic deletion of SIRT1 exacerbated ethanol-mediated defects in lipid metabolism, mainly by altering the function of lipin-1, a transcriptional regulator of lipid metabolism. In cultured mouse AML-12 hepatocytes, transgenic expression of SIRT1 prevented fat accumulation in response to ethanol exposure, largely by reversing the aberrations in lipin-1 signaling induced by ethanol. Liver samples from patients with alcoholic hepatitis had reduced levels of SIRT1 and a higher ratio of Lpin1β/α messenger RNAs than controls. Conclusions In mice, hepatic deletion of Sirt1 promotes steatosis, inflammation, and fibrosis in response to ethanol challenge. Ethanol-mediated impairment of hepatic SIRT1 signaling via lipin-1 contributes to development of alcoholic steatosis and inflammation. Reagents designed to increase SIRT1 regulation of lipin-1 can be developed to treat patients with alcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a complex disease. The considerable variability in the natural history of the disease suggests an important role for genetic variants in the disease development and progression. There is evidence based on genome-wide association studies and/or candidate gene studies that genetic polymorphisms underlying insulin signaling, lipid metabolism, oxidative stress, fibrogenesis, and inflammation can predispose individuals to NAFLD. This review highlights some of the genetic variants in NAFLD.

Lipin-1 deficiency is associated with massive rhabdomyolysis episodes in humans, precipitated by febrile illnesses. Despite well-known roles of lipin-1 in lipid biosynthesis and transcriptional regulation, the pathogenic mechanisms leading to rhabdomyolysis remain unknown. Here we show that primary myoblasts from lipin-1-deficient patients exhibit a dramatic decrease in LPIN1 expression and phosphatidic acid phosphatase 1 activity, and a significant accumulation of lipid droplets (LD). The expression levels of LPIN1-target genes [peroxisome proliferator-activated receptors delta and alpha (PPARδ, PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), acyl-coenzyme A dehydrogenase, very long (ACADVL), carnitine palmitoyltransferase IB and 2 (CPT1B and CPT2)] were not affected while lipin-2 protein level, a closely related member of the family, was increased. Microarray analysis of patients' myotubes identified 19 down-regulated and 51 up-regulated genes, indicating pleiotropic effects of lipin-1 deficiency. Special attention was paid to the up-regulated ACACB (acetyl-CoA carboxylase beta), a key enzyme in the fatty acid synthesis/oxidation balance. We demonstrated that overexpression of ACACB was associated with free fatty acid accumulation in patients' myoblasts whereas malonyl-carnitine (as a measure of malonyl-CoA) and CPT1 activity were in the normal range in basal conditions accordingly to the normal daily activity reported by the patients. Remarkably ACACB invalidation in patients' myoblasts decreased LD number and size while LPIN1 invalidation in controls induced LD accumulation. Further, pro-inflammatory treatments tumor necrosis factor alpha+Interleukin-1beta(TNF1α+IL-1ß) designed to mimic febrile illness, resulted in increased malonyl-carnitine levels, reduced CPT1 activity and enhanced LD accumulation, a phenomenon reversed by dexamethasone and TNFα or IL-1ß inhibitors. Our data suggest that the pathogenic mechanism of rhabdomyolysis in lipin-1-deficient patients combines the predisposing constitutive impairment of lipid metabolism and its exacerbation by pro-inflammatory cytokines.

Members of the lipin protein family are phosphatidate phosphatase (PAP) enzymes, which catalyze the dephosphorylation of phosphatidic acid to diacylglycerol, the penultimate step in TAG synthesis. Lipins are unique among the glycerolipid biosynthetic enzymes in that they also promote fatty acid oxidation through their activity as co-regulators of gene expression by DNA-bound transcription factors. Lipin function has been evolutionarily conserved from a single ortholog in yeast to the mammalian family of three lipin proteins-lipin-1, lipin-2, and lipin-3. In mice and humans, the levels of lipin activity are a determinant of TAG storage in diverse cell types, and humans with deficiency in lipin-1 or lipin-2 have severe metabolic diseases. Recent work has highlighted the complex physiological interactions between members of the lipin protein family, which exhibit both overlapping and unique functions in specific tissues. The analysis of "lipinopathies" in mouse models and in humans has revealed an important role for lipin activity in the regulation of lipid intermediates (phosphatidate and diacylglycerol), which influence fundamental cellular processes including adipocyte and nerve cell differentiation, adipocyte lipolysis, and hepatic insulin signaling. The elucidation of lipin molecular and physiological functions could lead to novel approaches to modulate cellular lipid storage and metabolic disease.

Peroxisome proliferator-activated receptor α (PPARα) and PPARγ coactivator 1α (PGC-1α) are crucial transcriptional regulators for genes involved in FA oxidation. Lipin-1 is essential for this increased capacity for β-oxidation in fasted livers, and it is also a phosphatidate phosphatase involved in triacylglycerol and phospholipid synthesis. Little is known about the regulation of these proteins in the heart during fasting, where there is increased FA esterification and oxidation. Lipin-1, lipin-2, lipin-3, carnitine palmitoyltransferase-1b (Cpt1b), and PGC-1α-b mRNA were increased by glucocorticoids and cAMP in neonatal rat cardiomyocytes. However, Cpt1b upregulation was caused by increased PPARα activation rather than expression. By contrast, the effects of PPARα in fasted livers are mediated through increased expression. During fasting, the expressions of PGC-1α-b and PGC-1α-c are increased in mouse hearts, and this is explained by increased cAMP-dependent signaling. By contrast, PGC-1α-a expression is increased in liver. Contrary to our expectations, lipin-1 expression was decreased and lipin-2 remained unchanged in hearts compared with increases in fasted livers. Our results identify novel differences in the regulation of lipins, PPARα, and PGC-1α splice variants during fasting in heart versus liver, even though the ultimate outcome in both tissues is to increase FA turnover and oxidation.

Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA)response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [32P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being Specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase e that allows the production of 18:2-DGPP from 18:2-PA e is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation Fil: Villasuso, Ana Laura. Universidad Nacional de Rio Cuarto; Argentina Fil: Di Palma, María Albana. Universidad Nacional de Rio Cuarto; Argentina Fil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina Fil: Pasquaré, Susana J.. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina Fil: Racagni, Graciela. Universidad Nacional de Rio Cuarto; Argentina Fil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina Fil: Machado, Estela E.. Universidad Nacional de Rio Cuarto; Argentina

Here we cloned chicken Lpin1-β, conducted the temporal and spatial expression pattern analysis of chicken Lpin1 isoforms by real-time PCR, and studied the 5′ flanking region variation and the potential effect. It was found that chicken Lpin1-α and Lpin1-β exhibited distinct tissue expression profiling, with prominent expression in the ovary and muscle tissues respectively. Chicken Lpin1 presented a tissue-specific temporal expression pattern in postnatal development (0–16 weeks). Energy restriction significantly elevated the mRNA level of total Lpin1 by increasing the expression of Lpin1-α and Lpin1-β in a nearly same magnitude. Eight variants/four haplotypes among six breeds were detected from the 5′ flanking region of chicken Lpin1, one multiple-nucleotide length polymorphism (g.258M > N) was found and predicted causing the change of 31 transcription factor binding sites including MyoD et al. Both g.258M > N and g. 65C > T variants showed significant association with muscle fiber traits, which suggested one novel role of Lpin1 on muscle fiber development.

Lipid phosphate phosphatases (LPP) are integral membrane proteins with broad substrate specificity that dephosphorylate lipid substrates including phosphatidic acid, lysophosphatidic acid, ceramide 1-phosphate, sphingosine 1-phosphate, and diacylglycerol pyrophosphate. Although the three mammalian enzymes (LPP1-3) demonstrate overlapping catalytic activities and substrate preferences in vitro, the phenotypes of mice with targeted inactivation of the Ppap2 genes encoding the LPP enzymes reveal nonredundant functions. A specific role for LPP3 in vascular development has emerged from studies of mice lacking Ppap2b. A meta-analysis of multiple, large genome-wide association studies identified a single nucleotide polymorphism in PPAP2B as a novel predictor of coronary artery disease. In this review, we will discuss the evidence that links LPP3 to vascular development and disease and evaluate potential molecular mechanisms. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Phosphatidate phosphatase (PAP) plays diverse roles in lipid metabolism and cell signaling.A novel yeast PAP is identified as the actin patch protein encoded by APP1.APP1 and other known genes (PAH1, DPP1, LPP1) are responsible for all detectable PAP activity in yeast.Identification of App1p as a PAP enzyme will facilitate the understanding of its cellular function. Phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. In the yeast Saccharomyces cerevisiae, PAP is encoded by PAH1, DPP1, and LPP1. The presence of PAP activity in the pah1Δ dpp1Δ lpp1Δ triple mutant indicated another gene(s) encoding the enzyme. We purified PAP from the pah1Δ dpp1Δ lpp1Δ triple mutant by salt extraction of mitochondria followed by chromatography with DE52, Affi-Gel Blue, phenyl-Sepharose, MonoQ, and Superdex 200. Liquid chromatography/tandem mass spectrometry analysis of a PAP-enriched sample revealed multiple putative phosphatases. By analysis of PAP activity in mutants lacking each of the proteins, we found that APP1, a gene whose molecular function has been unknown, confers ~30% PAP activity of wild type cells. The overexpression of APP1 in the pah1Δ dpp1Δ lpp1Δ mutant exhibited a 10-fold increase in PAP activity. The PAP activity shown by App1p heterologously expressed in Escherichia coli confirmed that APP1 is the structural gene for the enzyme. Introduction of the app1Δ mutation into the pah1Δ dpp1Δ lpp1Δ triple mutant resulted in a complete loss of PAP activity, indicating that distinct PAP enzymes in S. cerevisiae are encoded by APP1, PAH1, DPP1, and LPP1. Lipid analysis of cells lacking the PAP genes, singly or in combination, showed that Pah1p is the only PAP involved in the synthesis of triacylglycerol as well as in the regulation of phospholipid synthesis. App1p, which shows interactions with endocytic proteins, may play a role in vesicular trafficking through its PAP activity.

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.