Phosphatidic Acid Phosphohydrolase Research Articles

Phosphatidic acid phosphohydrolase modulates glycerolipid synthesis in Marchantia polymorpha and is crucial for growth under both nutrient-replete and -deficient conditions

Main conclusionThe phosphatidic acid phosphohydrolase of Marchantia polymorpha modulates plastid glycolipid synthesis through the ER pathway and is essential for normal plant development regardless of nutrient availability.Membrane lipid remodeling is one of the strategies plant cells use to secure inorganic phosphate (Pi) for plant growth, but many aspects of the molecular mechanism and its regulation remain unclear. Here we analyzed membrane lipid remodeling using a non-vascular plant, Marchantia polymorpha. The lipid composition and fatty acid profile during Pi starvation in M. polymorpha revealed a decrease in phospholipids and an increase in both galactolipids and betaine lipids. In Arabidopsis thaliana, phosphatidic acid phosphohydrolase (PAH) is involved in phospholipid degradation and is crucial for tolerance to both Pi and nitrogen starvation. We produced two M. polymorpha PAH (MpPAH) knockout mutants (Mppah-1 and Mppah-2) and found that, unlike Arabidopsis mutants, Mppah impaired plant growth with shorter rhizoids compared with wild-type plants even under nutrient-replete conditions. Mutation of MpPAH did not significantly affect the mole percent of each glycerolipid among total membrane glycerolipids from whole plants under both Pi-replete and Pi-deficient conditions. However, the fatty acid composition of monogalactosyldiacylglycerol indicated that the amount of plastid glycolipids produced through the endoplasmic reticulum pathway was suppressed in Mppah mutants. Phospholipids accumulated in the mutants under N starvation. These results reveal that MpPAH modulates plastid glycolipid synthesis through the endoplasmic reticulum pathway more so than what has been observed for Arabidopsis PAH; moreover, unlike Arabidopsis, MpPAH is crucial for M. polymorpha growth regardless of nutrient availability.

Haploinsufficiency of Lipin3 leads to hypertriglyceridemia and obesity by disrupting the expression and nucleocytoplasmic localization of Lipin1.

Lipin proteins including Lipin 1-3 act as transcriptional co-activators and phosphatidic acid phosphohydrolase enzymes, which play crucial roles in lipid metabolism. However, little is known about the function of Lipin3 in triglyceride (TG) metabolism. Here, we identified a novel mutation (NM_001301860: p.1835A>T/p.D612V) of Lipin3 in a large family with hypertriglyceridemia (HTG) and obesity through whole-exome sequencing and Sanger sequencing. Functional studies revealed that the novel variant altered the half-life and stability of the Lipin3 protein. Hence, we generated Lipin3 heterozygous knockout (Lipin3-heKO) mice and cultured primary hepatocytes to explore the pathophysiological roles of Lipin3 in TG metabolism. We found that Lipin3-heKO mice exhibited obvious obesity, HTG, and non-alcoholic fatty liver disorder. Mechanistic study demonstrated that the haploinsufficiency of Lipin3 in primary hepatocytes may induce the overexpression and abnormal distribution of Lipin1 in cytosol and nucleoplasm. The increased expression of Lipin1 in cytosol may contribute to TG anabolism, and the decreased Lipin1 in nucleoplasm can reduce PGC1α, further leading to mitochondrial dysfunction and reduced TG catabolism. Our study suggested that Lipin3 was a novel disease-causing gene inducing obesity and HTG. We also established a relationship between Lipin3 and mitochondrial dysfunction.

LsSpt23p is a regulator of triacylglycerol synthesis in the oleaginous yeast Lipomyces starkeyi

The oleaginous yeast Lipomyces starkeyi has considerable potential in industrial application, since it can accumulate a large amount of triacylglycerol (TAG), which is produced from sugars under nitrogen limitation condition. However, the regulation of lipogenesis in L. starkeyi has not been investigated in depth. In this study, we compared the genome sequences of wild-type and mutants with increased TAG productivity, and identified a regulatory protein, LsSpt23p, which contributes to the regulation of TAG synthesis in L. starkeyi. L. starkeyi mutants overexpressing LsSPT23 had increased TAG productivity compared with the wild-type strain. Quantitative real-time PCR analysis showed that LsSpt23p upregulated the expression of GPD1, which encodes glycerol 3-phosphate dehydrogenase; the Kennedy pathway genes SCT1, SLC1, PAH1, DGA1, and DGA2; the citrate-mediated acyl-CoA synthesis pathway-related genes ACL1, ACL2, ACC1, FAS1, and FAS2; and OLE1, which encodes ∆9 fatty acid desaturase. Chromatin immunoprecipitation-quantitative PCR assays indicated that LsSpt23p acts as a direct regulator of SLC1 and PAH1, all the citrate-mediated acyl-CoA synthesis pathway-related genes, and OLE1. These results indicate that LsSpt23p regulates TAG synthesis. Phosphatidic acid is a common substrate of phosphatidic acid phosphohydrolase, which is used for TAG synthesis, and phosphatidate cytidylyltransferase 1 for phospholipid synthesis in the Kennedy pathway. LsSpt23p directly regulated PAH1 but did not affect the expression of CDS1, suggesting that the preferred route of carbon is the Pah1p-mediated TAG synthesis pathway under nitrogen limitation condition. The present study contributes to understanding the regulation of TAG synthesis, and will be valuable in future improvement of TAG productivity in oleaginous yeasts. KEY POINTS: LsSpt23p was identified as a positive regulator of TAG biosynthesis LsSPT23 overexpression enhanced TAG biosynthesis gene expression and TAG production LsSPT23M1108T overexpression mutant showed fivefold higher TAG production than control.

Lipin-1 stability and its adipogenesis functions are regulated in contrasting ways by AKT1 and LKB1.

Lipin-1 is a protein that plays a critical role in many cellular functions. At molecular level, it acts as a phosphatidic acid phosphohydrolase and a transcriptional coactivator. The functions of lipin-1 are largely dependent upon its subcellular localization, post-translational modifications like phosphorylation and acetylation, and also on its interaction with other proteins such as 14-3-3. However, the kinases and phosphatases that are responsible for these post translational modifications are not entirely known. Using bioinformatics and other biochemical approaches, we demonstrate lipin-1 as a novel target for AKT1 and LKB1. While AKT1 stabilizes lipin-1, LKB1 causes its degradation. Interestingly, our findings further show that lipin-1 enhances AKT1 activity as can be seen by increased phosphorylation of the substrates of AKT1. Taken together, our results suggest that lipin-1 plays an important role in the regulation of PI3K-AKT-mTOR pathway, which is dysregulated in majority of cancers. Therefore, understating the role of lipin-1 may provide new and important insights into the regulation and functions of the PI3K-mTOR pathway, which is one of the major targets for anti-cancer drug development strategies.

LPIN1 promotes triglycerides synthesis and is transcriptionally regulated by PPARG in buffalo mammary epithelial cells

Studies on 3T3-L1 cells and HepG2 hepatocytes have shown that phosphatidic acid phosphohydrolase1 (LPIN1) plays a key role in adipogenesis, acting as a co-activator of peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a) to regulate fatty acid metabolism. However, the functional role and regulatory mechanism of LPIN1 gene in milk fat synthesis of buffalo are still unknown. In this study, overexpression of buffalo LPIN1 gene transfected with recombinant fusion expression vector significantly increased the expression of AGPAT6, DGAT1, DGAT2, GPAM and BTN1A1 genes involved in triglyceride (TAG) synthesis and secretion, as well as PPARG and SREBF1 genes regulating fatty acid metabolism in the buffalo mammary epithelial cells (BMECs), while the lentivirus-mediated knockdown of buffalo LPIN1 dramatically decreased the relative mRNA abundance of these genes. Correspondingly, total cellular TAG content in the BMECs increased significantly after LPIN1 overexpression, but decreased significantly after LPIN1 knockdown. In addition, the overexpression or knockdown of PPARG also enhanced or reduced the expression of LPIN1 and the transcriptional activity of its promoter. The core region of buffalo LPIN1 promoter spans from − 666 bp to + 42 bp, and two PPAR response elements (PPREs: PPRE1 and PPRE2) were identified in this region. Site mutagenesis analysis showed that PPARG directly regulated the transcription of buffalo LPIN1 by binding to the PPRE1 and PPRE2 on its core promoter. The results here reveal that the LPIN1 gene is involved in the milk fat synthesis of BMECs, and one of the important pathways is to participate in this process through direct transcriptional regulation of PPARG, which in turn significantly affects the content of TAG in BMECs.

Myeloid-associated lipin-1 transcriptional co-regulatory activity is atheroprotective

Background and aimsAtherosclerosis is the most prominent underlying cause of cardiovascular disease (CVD). It is initiated by cholesterol deposition in the arterial intima, which causes macrophage recruitment and proinflammatory responses that promote plaque growth, necrotic core formation, and plaque rupture. Lipin-1 is a phosphatidic acid phosphohydrolase for glycerolipid synthesis. We have shown that lipin-1 phosphatase activity promotes macrophage pro-inflammatory responses when stimulated with modified low-density lipoprotein (modLDL) and accelerates atherosclerosis. Lipin-1 also independently acts as a transcriptional co-regulator where it enhances the expression of genes involved in β-oxidation. In hepatocytes and adipocytes, lipin-1 augments the activity of transcription factors such as peroxisome proliferator-activated receptor (PPARs). PPARs control the expression of anti-inflammatory genes in macrophages and slow or reduce atherosclerotic progression. Therefore, we hypothesize myeloid-derived lipin-1 transcriptional co-regulatory activity reduces atherosclerosis. MethodsWe used myeloid-derived lipin-1 knockout (lipin-1mKO) and littermate control mice and AAV8-PCSK9 along with high-fat diet to elicit atherosclerosis. ResultsLipin-1mKO mice had larger aortic root plaques than littermate control mice after 8 and 12 weeks of a high-fat diet. Lipin-1mKO mice also had increased serum proinflammatory cytokine concentrations, reduced apoptosis in plaques, and larger necrotic cores in the plaques compared to control mice. ConclusionsCombined, the data suggest lipin-1 transcriptional co-regulatory activity in myeloid cells is atheroprotective.

Myocardial Lipin 1 knockout in mice approximates cardiac effects of human LPIN1 mutations.

Lipin 1 is a bifunctional protein that is a transcriptional regulator and has phosphatidic acid (PA) phosphohydrolase activity, which dephosphorylates PA to generate diacylglycerol. Human lipin 1 mutations lead to episodic rhabdomyolysis, and some affected patients exhibit cardiac abnormalities, including exercise-induced cardiac dysfunction and cardiac triglyceride accumulation. Furthermore, lipin 1 expression is deactivated in failing heart, but the effects of lipin 1 deactivation in myocardium are incompletely understood. We generated mice with cardiac-specific lipin 1 KO (cs-Lpin1–/–) to examine the intrinsic effects of lipin 1 in the myocardium. Cs-Lpin1–/– mice had normal systolic cardiac function but mild cardiac hypertrophy. Compared with littermate control mice, PA content was higher in cs-Lpin1–/– hearts, which also had an unexpected increase in diacylglycerol and triglyceride content. Cs-Lpin1–/– mice exhibited diminished cardiac cardiolipin content and impaired mitochondrial respiration rates when provided with pyruvate or succinate as metabolic substrates. After transverse aortic constriction–induced pressure overload, loss of lipin 1 did not exacerbate cardiac hypertrophy or dysfunction. However, loss of lipin 1 dampened the cardiac ionotropic response to dobutamine and exercise endurance in association with reduced protein kinase A signaling. These data suggest that loss of lipin 1 impairs cardiac functional reserve, likely due to effects on glycerolipid homeostasis, mitochondrial function, and protein kinase A signaling.

Uncovering the Role of Eaf1 in the Delicate Balance of Lipid Droplet Synthesis and Membrane Composition in Saccharomyces cerevisiae

The yeast lysine acetyltransferase, NuA4, is known to be critical important to regulating a variety of cell functions through acetylation of protein targets, resulting in changes in localization, function and/or abundance of its target proteins. Most recently, we have discovered a fascinating role for NuA4 in establishing the balance between lipid droplet formation and phospholipid availability for organelle and cell membranes. This was first discovered upon deletion of the main scaffolding subunit of NuA4 which resulting a strange extension of the nuclear membrane, commonly known as a nuclear flare. This occurs in over 50% of eaf1 cells, compared to only 4% in wild type (WT) cells. Under normal conditions, nuclear flares are primarily detected during mitotic arrest where nuclear membrane production continues unchecked, resulting in excess membrane and nuclear flares. However, nuclear flares in eaf1 cells are detected throughout the cell cycle, suggesting a gross dysregulation of lipid phospholipid production in the absence of NuA4. In addition to nuclear flares, the loss of the NuA4 complex causes significant effects to the vacuole. I have seen experimentally that 60% of all eaf1 cells exhibited severe defects in vacuole fusion, containing more than 10 vacuolar lobes, compared to 7% in WT cells. To further understand this regulation, we set out to find a potential protein target of NuA4-dependant acetylation that could be the potential link between NuA4 and lipid production. Sitting at the cross-roads between lipid droplet formation and membrane phospholipid production, phosphatidic acid phosphohydrolase 1 (Pah1) acts as a regulator for lipid biosynthesis in S. cerevisiae. Specifically, Pah1 converts a key metabolite, phosphatidic acid (PA) into diacylglycerol (DAG), which is then subsequently processed to form TAG and stored in lipid droplets. Additionally, it has recently been shown that NuA4 acetylates Pah1, which provides a direct mechanism behind this regulation. We hypothesize that acetylation of Pah1 is required to target the protein to the nuclear and vacuolar membrane. We have seen that upon deletion of EAF1, Pah1 3xGFP has an increase abundance in the cytoplasm and becomes localized to distinct punctate structures in the cell. Suggesting a dramatic change in its localization in the absence of acetylation. Additionally, through use of DAG biosensors, we have seen a drastic movement of DAG pools from the vacuolar membrane to the cytosol and plasma membrane in eaf1 cells. This could explain the defects in vacuole fusion. In its entirety, it has become clear that NuA4 is critical to regulating lipid availability for membranes, and determining the resulting membrane composition. We hope to further characterize the specific relationship between NuA4 and Pah1, in order to determine the how acetylation by NuA4 can affect lipid production for droplets and membranes.

Lipin 1 deficiency causes adult-onset myasthenia with motor neuron dysfunction in humans and neuromuscular junction defects in zebrafish.

Lipin 1 is an intracellular protein acting as a phosphatidic acid phosphohydrolase enzyme controlling lipid metabolism. Human recessive mutations in LPIN1 cause recurrent, early-onset myoglobinuria, a condition normally associated with muscle pain and weakness. Whether and how lipin 1 deficiency in humans leads to peripheral neuropathy is yet unclear. Herein, two novel compound heterozygous mutations in LPIN1 with neurological disorders, but no myoglobinuria were identified in an adult-onset syndromic myasthenia family. The present study sought to explore the pathogenic mechanism of LPIN1 in muscular and neural development.Methods: The clinical diagnosis of the proband was compared to the known 48 cases of LPIN1 recessive homozygous mutations. Whole-exome sequencing was carried out on the syndromic myasthenia family to identify the causative gene. The pathogenesis of lipin 1 deficiency during somitogenesis and neurogenesis was investigated using the zebrafish model. Whole-mount in situ hybridization, immunohistochemistry, birefringence analysis, touch-evoke escape response and locomotion assays were performed to observe in vivo the changes in muscles and neurons. The conservatism of the molecular pathways regulated by lipin 1 was evaluated in human primary glioblastoma and mouse myoblast cells by siRNA knockdown, drug treatment, qRT-PCR and Western blotting analysis.Results: The patient exhibited adult-onset myasthenia accompanied by muscle fiber atrophy and nerve demyelination without myoglobinuria. Two novel heterozygous mutations, c.2047A>C (p.I683L) and c.2201G>A (p.R734Q) in LPIN1, were identified in the family and predicted to alter the tertiary structure of LPIN1 protein. Lipin 1 deficiency in zebrafish embryos generated by lpin1 morpholino knockdown or human LPIN1 mutant mRNA injections reproduced the myotomes defects, a reduction both in primary motor neurons and secondary motor neurons projections, morphological changes of post-synaptic clusters of acetylcholine receptors, and myelination defects, which led to reduced touch-evoked response and abnormalities of swimming behaviors. Loss of lipin 1 function in zebrafish and mammalian cells also exhibited altered expression levels of muscle and neuron markers, as well as abnormally enhanced Notch signaling, which was partially rescued by the specific Notch pathway inhibitor DAPT.Conclusions: These findings pointed out that the compound heterozygous mutations in human LPIN1 caused adult-onset syndromic myasthenia with peripheral neuropathy. Moreover, zebrafish could be used to model the neuromuscular phenotypes due to the lipin 1 deficiency, where a novel pathological role of over-activated Notch signaling was discovered and further confirmed in mammalian cell lines.

Regulation of Signaling and Metabolism by Lipin-mediated Phosphatidic Acid Phosphohydrolase Activity.

Phosphatidic acid (PA) is a glycerophospholipid intermediate in the triglyceride synthesis pathway that has incredibly important structural functions as a component of cell membranes and dynamic effects on intracellular and intercellular signaling pathways. Although there are many pathways to synthesize and degrade PA, a family of PA phosphohydrolases (lipin family proteins) that generate diacylglycerol constitute the primary pathway for PA incorporation into triglycerides. Previously, it was believed that the pool of PA used to synthesize triglyceride was distinct, compartmentalized, and did not widely intersect with signaling pathways. However, we now know that modulating the activity of lipin 1 has profound effects on signaling in a variety of cell types. Indeed, in most tissues except adipose tissue, lipin-mediated PA phosphohydrolase activity is far from limiting for normal rates of triglyceride synthesis, but rather impacts critical signaling cascades that control cellular homeostasis. In this review, we will discuss how lipin-mediated control of PA concentrations regulates metabolism and signaling in mammalian organisms.

Regulation of PLPP3 gene expression by NF-κB family transcription factors

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.

Macrophage-associated lipin-1 enzymatic activity promotes inflammation in vitro and in vivo

Abstract During atherosclerotic progression, macrophage lipid handling dictates macrophage responses. As macrophages process modified low-density lipoproteins (modLDLs), glycerolipid synthesis is turned on to process lipids into storage sites for excess cholesterol. The key regulatory enzyme in this pathway is lipin-1, a phosphatidic acid phosphohydrolase. In vitro studies provide evidence that lipin-1 enzymatic activity initiates a PKCα/βII-ERK1/2-cJun signaling cascade that primes the macrophage to be hyper-responsive to subsequent pro-inflammatory stimulus. Our in vivo data demonstrated that mice lacking lipin-1 enzymatic activity in myeloid-derived cells (lipin-1mEnzyKO) had decreased atherosclerosis. However, it is currently unknown if the loss of lipin-1 and as a result the decreased inflammatory response is responsible for the decreased severity of atherosclerosis observed in our lipin-1mEnzyKO mouse model. We defined the transcriptome regulated by lipin-1 within modLDL/lipopolysaccharide stimulated wild-type and lipin-1mEnzyKO macrophages. The loss of lipin-1 led to a reduction of pro-inflammatory transcripts. To link the decrease in inflammation in the lipin-1mEnzyKO BMDMs with the decrease in atherosclerosis severity seen in vivo, we collected RNA from the aortic arch of lipin-1mEnzyKO and wild-type mice. Analysis of the RNA using an Atherosclerosis RT2 Profiler Array indicated that the lipin-1mEnzyKO mice showed increases in anti-apoptotic gene transcripts and decreases in transcripts related to extracellular matrix degradation suggesting lipin-1mEnzyKO mice may have more stable plaques.

Glycerolipid intermediates as signaling mediators in physiology and disease

Lipids have a number of essential biological roles that, despite a wealth of knowledge, somehow remain understudied. Glycerolipids are a major form of stored energy, play important structural functions as hydrophobic components of cell membranes, and have dynamic effects on signaling mediators within the cell and to distant tissues. Generally, it was believed that the pathways that synthesized lipids for these functions were distinct, compartmentalized, and did not widely intersect. For example, the main pathways for triglyceride storage as an energy source were not thought to play major roles in regulating the synthesis of lipids for signaling cascades even though many intermediates play roles as activators of intracellular kinases. However, it is now better appreciated that some of these storage intermediates also have signaling functions. In most cells, a pathway utilizing glycerol‐3‐phosphate as its initial substrate is the major route of triglyceride synthesis. Glycerol‐3‐phosphate undergoes two sequential acylations and then is dephosphorylated by lipin proteins to form diacylglycerol. Modulating the activity of lipin 1, a phosphatidic acid phosphohydrolase, has profound effects on skeletal muscle, adipose tissue, cardiac, and hepatic signaling by regulating the cellular abundance of phosphatidic acid. Evidence will be presented that lipin 1‐mediated signaling effects regulate metabolic flux, mitochondrial homeostasis, autophagy, cardiac and skeletal myocyte function, and impact cell death processes. These studies demonstrate that lipin 1 mediated effects on the concentrations of these intermediates are not important for storing energy, but also impact critical signaling cascades that control cellular homeostasis.Support or Funding InformationSupported by NIH grants R01 HL119225 and R01 DK78187This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adipose-Specific Lipin-1 Overexpression Renders Hepatic Ferroptosis and Exacerbates Alcoholic Steatohepatitis in Mice.

Lipin‐1 is a Mg2+‐dependent phosphatidic acid phosphohydrolase involved in the generation of diacylglycerol during synthesis of phospholipids and triglycerides. Ethanol‐mediated inhibitory effects on adipose‐specific lipin‐1 expression were associated with experimental steatohepatitis in rodents. In the present study, using an adipose‐specific lipin‐1 overexpression transgenic (Lpin1‐Tg) mouse model, we tested a hypothesis that adipose‐specific lipin‐1 overexpression in mice might dampen ethanol‐induced liver damage. Experimental alcoholic steatohepatitis was induced by pair‐feeding ethanol to Lpin1‐Tg and wild‐type (WT) mice using the chronic‐plus‐binge ethanol feeding protocol. Unexpectedly, following the chronic‐plus‐binge ethanol challenge, Lpin1‐Tg mice exhibited much more pronounced steatosis, exacerbated inflammation, augmented elevation of serum liver enzymes, hepatobiliary damage, and fibrogenic responses compared with the WT mice. Mechanistically, overexpression of adipose lipin‐1 in mice facilitated the onset of hepatic ferroptosis, which is an iron‐dependent form of cell death, and subsequently induced ferroptotic liver damage in mice under ethanol exposure. Concurrently, adipose lipin‐1 overexpression induced defective adiponectin signaling pathways in ethanol‐fed mice. Conclusion: We identified ferroptosis as a mechanism in mediating the detrimental effects of adipose‐specific lipin‐1 overexpression in mice under chronic‐plus‐binge ethanol exposure. Our present study sheds light on potential therapeutic approaches for the prevention and treatment of human alcoholic steatohepatitis.

Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Lipin 1 regulates glycerolipid homeostasis by acting as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride-synthesis pathway and by regulating transcription factor activity. Mutations in human lipin 1 are a common cause of recurrent rhabdomyolysis in children. Mice with constitutive whole-body lipin 1 deficiency have been used to examine mechanisms connecting lipin 1 deficiency to myocyte injury. However, that mouse model is confounded by lipodystrophy not phenocopied in people. Herein, 2 muscle-specific mouse models were studied: 1) Lpin1 exon 3 and 4 deletion, resulting in a hypomorphic protein without PAP activity, but which preserved transcriptional coregulatory function; and 2) Lpin1 exon 7 deletion, resulting in total protein loss. In both models, skeletal muscles exhibited a chronic myopathy with ongoing muscle fiber necrosis and regeneration and accumulation of phosphatidic acid and, paradoxically, diacylglycerol. Additionally, lipin 1-deficient mice had abundant, but abnormal, mitochondria likely because of impaired autophagy. Finally, these mice exhibited increased plasma creatine kinase following exhaustive exercise when unfed. These data suggest that mice lacking lipin 1-mediated PAP activity in skeletal muscle may serve as a model for determining the mechanisms by which lipin 1 deficiency leads to myocyte injury and for testing potential therapeutic approaches.-Schweitzer, G. G., Collier, S. L., Chen, Z., McCommis, K. S., Pittman, S. K., Yoshino, J., Matkovich, S. J., Hsu, F.-F., Chrast, R., Eaton, J. M., Harris, T. E., Weihl, C. C., Finck, B. N. Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis.

Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection.

Unconditional and conditional QTL analyses of seed fatty acid composition in Brassica napus L.

BackgroundThe fatty acid composition of B. napus’ seeds determines the oil’s nutritional and industrial values, and affects seed germination. Many studies have reported correlations among C16:0, C18:0, C18:1, C18:2 and C18:3 based on phenotypic data; however, the genetic basis of the fatty acid composition in B. napus is still not well understood.ResultsIn this study, unconditional and conditional quantitative trail locus (QTL) mapping analyses were conducted using a recombinant inbred line in six environments. In total, 21 consensus QTLs each for C16:0, C18:0 and C18:2, 16 for C18:1 and 22 for C18:3 were detected by unconditional mapping. The QTLs with overlapping confidence intervals were integrated into 71 pleiotropically unique QTLs by meta-analysis. Two major QTLs, uuqA5–6 and uuqA5–7, simultaneously affected the fatty acids, except C18:0, in most of environments, with the homologous genes fatty acid desaturase 2 (FAD2) and glycerol-3-phosphate sn-2-acyltransferase 5 (GPAT5) occurring in the confidence interval of uuqA5–6, while phosphatidic acid phosphohydrolase 1 (PAH1) was assigned to uuqA5–7. Moreover, 49, 30, 48, 60 and 45 consensus QTLs were detected for C16:0, C18:0, C18:1, C18:2 and C18:3, respectively, by the conditional mapping analysis. In total, 128 unique QTLs were subsequently integrated from the 232 conditional consensus QTLs. A comparative analysis revealed that 63 unique QTLs could be identified by both mapping methodologies, and 65 additional unique QTLs were only identified in conditional mapping.ConclusionsThus, conditional QTL mapping for fatty acids may uncover numerous additional QTLs that were inhibited by the effects of other traits. These findings provide useful information for better understanding the genetic relationships among fatty acids at the QTL level.

Altered global microRNA expression in hepatic stellate cells LX-2 by angiotensin-(1–7) and miRNA-1914-5p identification as regulator of pro-fibrogenic elements and lipid metabolism

The development of new therapeutic strategies to control or reverse hepatic fibrosis requires thorough knowledge about its molecular and cellular basis. It is known that the heptapeptide angiotensin-(1-7) [ang-(1-7)] can reduce hepatic fibrosis and steatosis in vivo; therefore, it is important to uncover the mechanisms regulating its activity and cellular model of investigation. Ang-(1-7) is a peptide of the renin-angiotensin system (RAS), and here we investigated its modulatory effect on the expression pattern of microRNAs (miRNAs) in hepatic stellate cells (HSCs) LX-2, which transdifferentiate into fibrogenic and proliferative cells. We compared the miRNA profiles between quiesced, activated and ang-(1-7)-treated activated HSCs to identify miRNAs that may regulate their transdifferentiation. Thirteen miRNAs were pointed, and cellular and molecular analyses identified miRNA-1914-5p as a molecule that contributes to the effects of ang-(1-7) on lipid metabolism and on the pro-fibrotic environment control. In our cellular model, we also analyzed the regulators of fatty acid metabolism. Specifically, miRNA-1914-5p regulates the expression of malonyl-CoA decarboxylase (MLYCD) and phosphatidic acid phosphohydrolase (PAP or Lipin-1). Additionally, Lipin-1 was closely correlated with mRNA expression of peroxisome proliferator-activated receptors (PPAR)-α and -γ, which also contribute to lipid homeostasis and to the reduction of TGF-β1 expression. These findings provide a novel link between RAS and lipid metabolism in controlling HSCs activation.

Cyclin-dependent kinase activity enhances phosphatidylcholine biosynthesis in Arabidopsis by repressing phosphatidic acid phosphohydrolase activity.

SummaryCoordination of endomembrane biogenesis with cell cycle progression is considered to be important in maintaining cell function during growth and development. We previously showed that the disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) activity in Arabidopsis thaliana stimulates biosynthesis of the major phospholipid phosphatidylcholine (PC) and causes expansion of the endoplasmic reticulum. Here we show that PC biosynthesis is repressed by disruption of the core cell cycle regulator CYCLIN‐DEPENDENT KINASE A;1 (CDKA;1) and that this repression is reliant on PAH. Furthermore, we show that cyclin‐dependent kinases (CDKs) phosphorylate PAH1 at serine 162, which reduces both its activity and membrane association. Expression of a CDK‐insensitive version of PAH1 with a serine 162 to alanine substitution represses PC biosynthesis and also reduces the rate of cell division in early leaf development. Together our findings reveal a physiologically important mechanism that couples the rate of phospholipid biosynthesis and endomembrane biogenesis to cell cycle progression in Arabidopsis.

Role of lipin-1 in the pathogenesis of alcoholic fatty liver disease

Lipin-1 can promote the synthesis of triglyceride and fatty acid oxidation during lipid metabolism. Ethanol can up-regulate the expression of lipin-1 via the AMPK-SREBP-1-lipin-1 signaling pathway and increase lipin1β/αratio via the SITR1-SFRS10-LPIN1 signaling pathway. In the cytoplasm, lipin-1βfunctions as a Mg2+-dependent phosphatidic acid phosphohydrolase to accelerate the synthesis of triglyceride. In the nucleus, lipin-1αacts as a transcriptional coactivator and reduces mitochondrial fatty acid oxidation by inhibiting the activities of PPARαand PGC-1α, and therefore promotes lipid deposition in hepatocytes and causes hepatic steatosis.