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1.  The Lipin Family: Mutations and Metabolism 
Current opinion in lipidology  2009;20(3):165-170.
Purpose of review
The family of three lipin proteins act as phosphatidate phosphatase (PAP) enzymes required for glycerolipid biosynthesis, and also as transcriptional coactivators that regulate expression of lipid metabolism genes. The genes for lipin-1, lipin-2 and lipin-3 are expressed in key metabolic tissues, including adipose tissue, skeletal muscle, and liver, but the physiological functions of each member of the family have not been fully elucidated. Here we examine the most recent studies that provide information about the roles of lipin proteins in metabolism and human disease.
Recent findings
Recent studies have identified mutations that cause lipin-1 or lipin-2 deficiency in humans, leading to acute myoglobinuria in childhood or the inflammatory disorder Majeed syndrome, respectively. The effects of lipin-1 deficiency appear to include both the loss of glycerolipid building blocks and the accumulation of lipid intermediates that disrupt cellular function. Several studies have demonstrated that polymorphisms in the LPIN1 and LPIN2 genes are associated with metabolic disease traits, including insulin sensitivity, diabetes, blood pressure, and response to thiazolidinedione drugs. Furthermore, lipin-1 expression levels in adipose tissue and/or liver are positively correlated with insulin sensitivity. Studies of lipin-1 in adipocytes have shed some light on its relationship with insulin sensitivity.
Summary
Lipin-1 and lipin-2 are required for normal lipid homeostasis, and have unique physiological roles. Future studies, for example using engineered mouse models, will be required to fully elucidate their specific roles in normal physiology and disease.
doi:10.1097/MOL.0b013e32832adee5
PMCID: PMC2875192  PMID: 19369868
triglyceride; phosphatidic acid phosphatase; transcriptional coactivator; lipodystrophy; obesity; insulin resistance; myopathy
2.  Lipin-1 and lipin-3 together determine adiposity in vivo☆ 
Molecular Metabolism  2013;3(2):145-154.
The lipin protein family of phosphatidate phosphatases has an established role in triacylglycerol synthesis and storage. Physiological roles for lipin-1 and lipin-2 have been identified, but the role of lipin-3 has remained mysterious. Using lipin single- and double-knockout models we identified a cooperative relationship between lipin-3 and lipin-1 that influences adipogenesis in vitro and adiposity in vivo. Furthermore, natural genetic variations in Lpin1 and Lpin3 expression levels across 100 mouse strains correlate with adiposity. Analysis of PAP activity in additional metabolic tissues from lipin single- and double-knockout mice also revealed roles for lipin-1 and lipin-3 in spleen, kidney, and liver, for lipin-1 alone in heart and skeletal muscle, and for lipin-1 and lipin-2 in lung and brain. Our findings establish that lipin-1 and lipin-3 cooperate in vivo to determine adipose tissue PAP activity and adiposity, and may have implications in understanding the protection of lipin-1-deficient humans from overt lipodystrophy.
doi:10.1016/j.molmet.2013.11.008
PMCID: PMC3953701  PMID: 24634820
Gene family; Knockout mouse; Adipogenesis; Triacylglycerol; Glycerolipid biosynthesis
3.  A Phosphatidic Acid Binding/Nuclear Localization Motif Determines Lipin1 Function in Lipid Metabolism and Adipogenesis 
Molecular Biology of the Cell  2010;21(18):3171-3181.
A polybasic motif in the metabolic regulator lipin1 is both a membrane anchor and a nuclear localization sequence required for lipin1 function in phospholipid metabolism and adipogenesis.
Lipins are phosphatidic acid phosphatases with a pivotal role in regulation of triglyceride and glycerophospholipid metabolism. Lipin1 is also an amplifier of PGC-1α, a nuclear coactivator of PPAR-α responsive gene transcription. Lipins do not contain recognized membrane-association domains, but interaction of these enzymes with cellular membranes is necessary for access to their phospholipid substrate. We identified a role for a conserved polybasic amino acid motif in an N-terminal domain previously implicated as a determinant of nuclear localization in selective binding of lipin1β to phosphatidic acid, using blot overlay assays and model bilayer membranes. Studies using lipin1β polybasic motif variants establish that this region is also critical for nuclear import and raise the possibility that nuclear/cytoplasmic shuttling of lipin1β is regulated by PA. We used pharmacological agents and lipin1β polybasic motif mutants to explore the role of PA-mediated membrane association and nuclear localization on lipin1β function in phospholipid metabolism and adipogenic differentiation. We identify a role for the lipin1 polybasic motif as both a lipid binding motif and a primary nuclear localization sequence. These two functions are necessary for full expression of the biological activity of the protein in intracellular lipid metabolism and transcriptional control of adipogenesis.
doi:10.1091/mbc.E10-01-0073
PMCID: PMC2938383  PMID: 20660155
4.  Lipins: Multifunctional Lipid Metabolism Proteins 
Annual review of nutrition  2010;30:257-272.
The lipin proteins are evolutionarily conserved proteins with roles in lipid metabolism and disease. There are three lipin protein family members in mammals and one or two orthologs in plants, invertebrates, and single-celled eukaryotes. Studies in yeast and mouse led to the identification of two distinct molecular functions of lipin proteins. Lipin proteins have phosphatidate phosphatase activity and catalyze the formation of diacylglycerol in the glycerol-3-phosphate pathway, implicating them in the regulation of triglyceride and phospholipid biosynthesis. Mammalian lipin proteins also possess transcriptional coactivator activity and have been implicated in the regulation of metabolic gene expression. Here we review key findings in the field that demonstrate roles for lipin family members in metabolic homeostasis and in rare human diseases, and we examine evidence implicating genetic variations in lipin genes in common metabolic dysregulation such as obesity, hyperinsulinemia, hypertension, and type 2 diabetes.
doi:10.1146/annurev.nutr.012809.104729
PMCID: PMC3738581  PMID: 20645851
triglyceride; obesity; insulin resistance; phosphatidate phosphatase; transcriptional coactivator
5.  Inactivation of the Host Lipin Gene Accelerates RNA Virus Replication through Viral Exploitation of the Expanded Endoplasmic Reticulum Membrane 
PLoS Pathogens  2014;10(2):e1003944.
RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of PAH1 (phosphatidic acid phosphohydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in pah1Δ yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in pah1Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the Arabidopsis Pah2p in Nicotiana benthamiana decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.
Author Summary
Genetic diseases alter cellular pathways and they likely influence pathogen-host interactions as well. To test the relationship between a key cellular gene, whose mutation causes genetic diseases, and a pathogen, the authors have chosen the cellular lipins. Lipins are involved in a key cellular decision on using lipids for membrane biogenesis or for storage. Spontaneous mutations in the LIPIN1 gene in mammals, which cause impaired lipin-1 function, contribute to common metabolic dysregulation and several major diseases, such as obesity, hyperinsulinemia, type 2 diabetes, fatty liver distrophy and hypertension. In this work, the authors tested if tomato bushy stunt virus (TBSV), which, similar to many (+)RNA viruses, depends on host membrane biogenesis, is affected by deletion of the single lipin gene (PAH1) in yeast model host. They show that pah1Δ yeast supports increased replication of TBSV. They demonstrate that TBSV takes advantage of the expanded ER membranes in lipin-deficient yeast to efficiently assemble viral replicase complexes. Their findings suggest possible positive effect of a genetic disease caused by mutation on the replication of an infectious agent.
doi:10.1371/journal.ppat.1003944
PMCID: PMC3930575  PMID: 24586157
6.  Lipin Is a Central Regulator of Adipose Tissue Development and Function in Drosophila melanogaster ▿  
Molecular and Cellular Biology  2011;31(8):1646-1656.
Lipins are evolutionarily conserved proteins found from yeasts to humans. Mammalian and yeast lipin proteins have been shown to control gene expression and to enzymatically convert phosphatidate to diacylglycerol, an essential precursor in triacylglcerol (TAG) and phospholipid synthesis. Loss of lipin 1 in the mouse, but not in humans, leads to lipodystrophy and fatty liver disease. Here we show that the single lipin orthologue of Drosophila melanogaster (dLipin) is essential for normal adipose tissue (fat body) development and TAG storage. dLipin mutants are characterized by reductions in larval fat body mass, whole-animal TAG content, and lipid droplet size. Individual cells of the underdeveloped fat body are characterized by increased size and ultrastructural defects affecting cell nuclei, mitochondria, and autophagosomes. Under starvation conditions, dLipin is transcriptionally upregulated and functions to promote survival. Together, these data show that dLipin is a central player in lipid and energy metabolism, and they establish Drosophila as a genetic model for further studies of conserved functions of the lipin family of metabolic regulators.
doi:10.1128/MCB.01335-10
PMCID: PMC3126333  PMID: 21300783
7.  Temporal and Spatial Regulation of the Phosphatidate Phosphatases Lipin 1 and 2*S⃞ 
The Journal of Biological Chemistry  2008;283(43):29166-29174.
Lipins are the founding members of a novel family of Mg2+-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis. Despite their importance, there is still little information on how their activity is regulated. Here we demonstrate that the functions of lipin 1 and 2 are evolutionarily conserved from unicellular eukaryotes to mammals. The two lipins display distinct intracellular localization in HeLa M cells, with a pool of lipin 2 exhibiting a tight membrane association. Small interfering RNA-mediated silencing of lipin 1 leads to a dramatic decrease of the cellular PAP1 activity in HeLa M cells, whereas silencing of lipin 2 leads to an increase of lipin 1 levels and PAP1 activity. Consistent with their distinct functions in HeLa M cells, lipin 1 and 2 exhibit reciprocal patterns of protein expression in differentiating 3T3-L1 adipocytes. Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis. Finally, we show that the PAP1 activity of both lipins is inhibited by phosphorylation during mitosis, leading to a decrease in the cellular PAP1 activity during cell division. We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.
doi:10.1074/jbc.M804278200
PMCID: PMC2570901  PMID: 18694939
8.  Distinct Roles of the Phosphatidate Phosphatases Lipin 1 and 2 during Adipogenesis and Lipid Droplet Biogenesis in 3T3-L1 Cells* 
The Journal of Biological Chemistry  2013;288(48):34502-34513.
Background: Lipins are phosphatidate phosphatases that generate diacylglycerol for lipid synthesis.
Results: Lipin 1 or lipin 2 depletion has distinct effects on differentiating adipocytes. Cells depleted of both lipins after initiation of adipogenesis accumulate triacylglycerol but display lipid droplet fragmentation.
Conclusion: Lipins have a role in lipid droplet biogenesis after initiation of adipogenesis.
Significance: Lipins play multiple roles during adipocyte differentiation.
Lipins are evolutionarily conserved Mg2+-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.
doi:10.1074/jbc.M113.488445
PMCID: PMC3843065  PMID: 24133206
Adipocyte; Lipids; Mouse; Phosphatase; Phosphatidate; Triacylglycerol; Lipin
9.  Cardiac lipin 1 expression is regulated by the peroxisome proliferator activated receptor γ coactivator 1α/estrogen related receptor axis 
Lipin family proteins (lipin 1, 2, and 3) are bifunctional intracellular proteins that regulate metabolism by acting as coregulators of DNA-bound transcription factors and also dephosphorylate phosphatidate to form diacylglycerol [phosphatidate phosphohydrolase activity] in the triglyceride synthesis pathway. Herein, we report that lipin 1 is enriched in heart and that hearts of mice lacking lipin 1 (fld mice) exhibit accumulation of phosphatidate. We also demonstrate that the expression of the gene encoding lipin 1 (Lpin1) is under the control of the estrogen-related receptors (ERRs) and their coactivator the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). PGC-1α, ERRα, or ERRγ overexpression increased Lpin1 transcription in cultured ventricular myocytes and the ERRs were associated with response elements in the first intron of the Lpin1 gene. Concomitant RNAi-mediated knockdown of ERRα and ERRγ abrogated the induction of lipin 1 expression by PGC-1α overexpression. Consistent with these data, 3-fold overexpression of PGC-1α in intact myocardium of transgenic mice increased cardiac lipin 1 and ERRα/γ expression. Similarly, injection of the β2-adrenergic agonist clenbuterol induced PGC-1α and lipin 1 expression, and the induction in lipin 1 after clenbuterol occurred in a PGC-1α-dependent manner. In contrast, expression of PGC-1α, ERRα, ERRγ, and lipin 1 was down-regulated in failing heart. Cardiac phosphatidic acid phosphohydrolase activity was also diminished, while cardiac phosphatidate content was increased, in failing heart. Collectively, these data suggest that lipin 1 is the principal lipin protein in the myocardium and is regulated in response to physiologic and pathologic stimuli that impact cardiac metabolism.
doi:10.1016/j.yjmcc.2011.04.009
PMCID: PMC3104300  PMID: 21549711
lipin; PGC-1α; metabolism; heart failure
10.  Lipin 1 Represses NFATc4 Transcriptional Activity in Adipocytes To Inhibit Secretion of Inflammatory Factors ▿ †  
Molecular and Cellular Biology  2010;30(12):3126-3139.
Lipin 1 is a bifunctional protein that regulates gene transcription and, as a Mg2+-dependent phosphatidic acid phosphatase (PAP), is a key enzyme in the biosynthesis of phospholipids and triacylglycerol. We describe here the functional interaction between lipin 1 and the nuclear factor of activated T cells c4 (NFATc4). Lipin 1 represses NFATc4 transcriptional activity through protein-protein interaction, and lipin 1 is present at the promoters of NFATc4 transcriptional targets in vivo. Catalytically active and inactive lipin 1 can suppress NFATc4 transcriptional activity, and this suppression may involve recruitment of histone deacetylases to target promoters. In fat pads from mice deficient for lipin 1 (fld mice) and in 3T3-L1 adipocytes depleted of lipin 1 there is increased expression of several NFAT target genes including tumor necrosis factor alpha, resistin, FABP4, and PPARγ. Finally, both lipin 1 protein and total PAP activity are decreased with increasing adiposity in the visceral, but not subcutaneous, fat pads of ob/ob mice. These observations place lipin 1 as a potentially important link between triacylglycerol synthesis and adipose tissue inflammation.
doi:10.1128/MCB.01671-09
PMCID: PMC2876672  PMID: 20385772
11.  Lipin proteins form homo- and hetero-oligomers 
The Biochemical journal  2010;432(1):65-76.
SUMMARY
Lipin family members (lipin 1, 2, 3) are bi-functional proteins that dephosphorylate phosphatidic acid (PA) to produce diacylglycerol (DAG) and act in the nucleus to regulate gene expression. Although other components of the triglyceride synthesis pathway can form oligomeric complexes, it is unknown whether lipin proteins also exist as oligomers. In this study, by using various approaches, we revealed that lipin 1 formed stable homo-oligomers with itself and hetero-oligomers with lipin 2/3. Both the N- and C-terminal regions of lipin 1 mediate its oligomerization in a head-to-head/tail-to-tail manner. We also show that lipin 1 subcellular localization can be influenced through oligomerization, and the individual lipin 1 monomers in the oligomer function independently in catalyzing dephosphorylation of PA. This study provides evidence that lipin proteins function as oligomeric complexes and that the three mammalian lipin isoforms can form combinatorial units.
doi:10.1042/BJ20100584
PMCID: PMC3117669  PMID: 20735359
lipin; oligomer; FRET; phosphatidic acid phosphatase
12.  Dietary Cholesterol Reduces Plasma Triacylglycerol in Apolipoprotein E-Null Mice: Suppression of Lipin-1 and -2 in the Glycerol-3-Phosphate Pathway 
PLoS ONE  2011;6(8):e22917.
Background
Cholesterol metabolism is tightly regulated by both cholesterol and its metabolites in the mammalian liver, but the regulatory mechanism of triacylglycerol (TG) synthesis remains to be elucidated. Lipin, which catalyzes the conversion of phosphatidate to diacylglycerol, is a key enzyme involved in de novo TG synthesis in the liver via the glycerol-3-phosphate (G3P) pathway. However, the regulatory mechanisms for the expression of lipin in the liver are not well understood.
Methodology/Principal Findings
Apolipoprotein E-knock out (apoE-KO) mice were fed a chow supplemented with 1.25% cholesterol (high-Chol diet). Cholesterol and bile acids were highly increased in the liver within a week. However, the amount of TG in very low-density lipoprotein (VLDL), but not in the liver, was reduced by 78%. The epididymal adipose tissue was almost eradicated in the long term. DNA microarray and real-time RT-PCR analyses revealed that the mRNA expression of all the genes in the G3P pathway in the liver was suppressed in the high-Chol diet apoE-KO mice. In particular, the mRNA and protein expression of lipin-1 and lipin-2 was markedly decreased, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), which up-regulates the transcription of lipin-1, was also suppressed. In vitro analysis using HepG2 cells revealed that the protein expression of lipin-2 was suppressed by treatment with taurocholic acid.
Conclusions/Significance
These data using apoE-KO mice indicate that cholesterol and its metabolites are involved in regulating TG metabolism through a suppression of lipin-1 and lipin-2 in the liver. This research provides evidence for the mechanism of lipin expression in the liver.
doi:10.1371/journal.pone.0022917
PMCID: PMC3153461  PMID: 21857965
13.  Regulation of Hepatic Lipin-1 by Ethanol: Role of AMPK-SREBP-1 Signaling 
Hepatology (Baltimore, Md.)  2011;55(2):437-446.
Lipin-1 is a protein that exhibits dual functions as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride synthesis pathways and a transcriptional co-regulator. Our previous studies have shown that ethanol causes fatty liver by activation of sterol regulatory element-binding protein 1 (SREBP-1) and inhibition of hepatic AMP-activated kinase (AMPK) in mice. Here, we tested the hypothesis that AMPK-SREBP-1 signaling may be involved in ethanol-mediated up-regulation of lipin-1 gene expression. The effects of ethanol on lipin-1 were investigated in cultured hepatic cells and in the livers of chronic ethanol-fed mice. Ethanol exposure robustly induced activity of a mouse lipin-1 promoter, promoted cytoplasmic localization of lipin-1 and caused excess lipid accumulation both in cultured hepatic cells and in mouse livers. Mechanistic studies showed that ethanol-mediated induction of lipin-1 gene expression was inhibited by a known activator of AMPK or overexpression of a constitutively active form of AMPK. Importantly, overexpression of processed nuclear form of SREBP-1c (nSREBP-1c) abolished the ability of AICAR to suppress ethanol-mediated induction of lipin-1 gene expression level. Chromatin immunoprecipitation (ChIP) assays further revealed that ethanol exposure significantly increased association of acetylated Histone H3 at lysine 9 (Lys9) with the SRE-containing region in the promoter of the lipin-1 gene. In conclusion, ethanol-induced up-regulation of lipin-1 gene expression is mediated through inhibition of AMPK and activation of SREBP-1.
doi:10.1002/hep.24708
PMCID: PMC3253249  PMID: 21953514
Alcoholic fatty liver; signal transduction; lipid metabolism; acetylation; sumoylation
14.  Sumoylation Regulates Nuclear Localization of Lipin-1α in Neuronal Cells 
PLoS ONE  2009;4(9):e7031.
Lipin-1 is a protein that has dual functions as a phosphatidic acid phosphohydrolase (PAP) and a nuclear transcriptional coactivator. It remains unknown how the nuclear localization and coactivator functions of lipin-1 are regulated. Here, we show that lipin-1 (including both the alpha and beta isoforms) is modified by sumoylation at two consensus sumoylation sites. We are unable to detect sumoylation of the related proteins lipin-2 and lipin-3. Lipin-1 is sumoylated at relatively high levels in brain, where lipin-1α is the predominant form. In cultured embryonic cortical neurons and SH-SY5Y neuronal cells, ectopically expressed lipin-1α is localized in both the nucleus and the cytoplasm, and the nuclear localization is abrogated by mutating the consensus sumyolation motifs. The sumoylation site mutant of lipin-1α loses the capacity to coactivate the transcriptional (co-) activators PGC-1α and MEF2, consistent with its nuclear exclusion. Thus, these results show that sumoylation facilitates the nuclear localization and transcriptional coactivator behavior of lipin-1α that we observe in cultured neuronal cells, and suggest that lipin-1α may act as a sumoylation-regulated transcriptional coactivator in brain.
doi:10.1371/journal.pone.0007031
PMCID: PMC2737637  PMID: 19753306
15.  Lipin-1 gamma isoform is a novel lipid droplet-associated protein highly expressed in the brain 
FEBS letters  2011;585(12):1979-1984.
Lipin-1 proteins are phosphatidic acid phosphatases catalyzing the conversion from phosphatidic acid to diacylglycerol. Two alternative splicing isoforms, lipin-1α and -1β, are localized at different subcellular compartments. A third splicing isoform, lipin-1γ was recently cloned and its subcellular localization is unknown. Here, we demonstrate that lipin-1γ is localized to lipid droplets, an association mediated by a hydrophobic, lipin-1γ-specific domain. Additional expression of lipin-1γ altered lipid droplet morphology without affecting the triacylglycerol level. In human tissues, lipin-1γ is the main lipin-1 isoform expressed in normal human brain, suggesting a specialized role in regulating brain lipid metabolism.
doi:10.1016/j.febslet.2011.05.035
PMCID: PMC3117272  PMID: 21616074
Lipin; phosphatidic acid phosphatase; lipid droplets; brain
16.  Dual Function Lipin Proteins and Glycerolipid Metabolism 
Lipin family proteins are emerging as critical regulators of lipid metabolism. In triglyceride synthesis, lipins act as lipid phosphatase enzymes at the endoplasmic reticular membrane, catalyzing the dephosphorylation of phosphatidic acid to form diacylglycerol, which is the penultimate step in this process. However, lipin proteins are not integral membrane proteins and can rapidly translocate within the cell. In fact, emerging evidence suggests that lipins also play critical roles in the nucleus as transcriptional regulatory proteins. Thus, lipins are poised to regulate cellular lipid metabolism at multiple regulatory nodal points. This review summarizes the history of lipin proteins and discusses the current state of our understanding of lipin biology.
doi:10.1016/j.tem.2011.02.006
PMCID: PMC3118913  PMID: 21470873
17.  Complex Interplay between the Lipin 1 and the Hepatocyte Nuclear Factor 4 α (HNF4α) Pathways to Regulate Liver Lipid Metabolism 
PLoS ONE  2012;7(12):e51320.
Lipin 1 is a bifunctional protein that serves as a metabolic enzyme in the triglyceride synthesis pathway and regulates gene expression through direct protein-protein interactions with DNA-bound transcription factors in liver. Herein, we demonstrate that lipin 1 is a target gene of the hepatocyte nuclear factor 4α (HNF4α), which induces lipin 1 gene expression in cooperation with peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) through a nuclear receptor response element in the first intron of the lipin 1 gene. The results of a series of gain-of-function and loss-of-function studies demonstrate that lipin 1 coactivates HNF4α to activate the expression of a variety of genes encoding enzymes involved in fatty acid catabolism. In contrast, lipin 1 reduces the ability of HNF4α to induce the expression of genes encoding apoproteins A4 and C3. Although the ability of lipin to diminish HNF4α activity on these promoters required a direct physical interaction between the two proteins, lipin 1 did not occupy the promoters of the repressed genes and enhances the intrinsic activity of HNF4α in a promoter-independent context. Thus, the induction of lipin 1 by HNF4α may serve as a mechanism to affect promoter selection to direct HNF4α to promoters of genes encoding fatty acid oxidation enzymes.
doi:10.1371/journal.pone.0051320
PMCID: PMC3517414  PMID: 23236470
18.  The human lipodystrophy protein seipin is an ER membrane adaptor for the adipogenic PA phosphatase lipin 1 
Molecular Metabolism  2012;2(1):38-46.
Disruption of the gene BSCL2 causes a severe, generalised lipodystrophy, demonstrating the critical role of its protein product, seipin, in human adipose tissue development. Seipin is essential for adipocyte differentiation, whilst the study of seipin in non-adipose cells has suggested a role in lipid droplet formation. However, its precise molecular function remains poorly understood. Here we demonstrate that seipin can inducibly bind lipin 1, a phosphatidic acid (PA) phosphatase important for lipid synthesis and adipogenesis. Knockdown of seipin during early adipogenesis decreases the association of lipin 1 with membranes and increases the accumulation of its substrate PA. Conversely, PA levels are reduced in differentiating cells by overexpression of wild-type seipin but not by expression of a mutated seipin that is unable to bind lipin 1. Together our data identify lipin as the first example of a seipin-interacting protein and reveals a novel molecular function for seipin in developing adipocytes.
doi:10.1016/j.molmet.2012.11.002
PMCID: PMC3757660  PMID: 24024128
Seipin; Adipogenesis; Lipodystrophy; Lipin; Endoplasmic reticulum
19.  Glucose-6-phosphate isomerase deficiency results in mTOR activation, failed translocation of lipin 1α to the nucleus and hypersensitivity to glucose: Implications for the inherited glycolytic disease 
Biochimica et biophysica acta  2011;1812(11):1393-1402.
Inherited glucose-6-phosphate isomerase (GPI) deficiency is the second most frequent glycolytic erythroenzymopathy in humans. Patients present with non-spherocytic anemia of variable severity and with neuromuscular dysfunction. We previously described Chinese hamster (CHO) cell lines with mutations in GPI and loss of GPI activity. This resulted in a temperature sensitivity and severe reduction in the synthesis of glycerolipids due to a reduction in phosphatidate phosphatase (PAP). In the current article we attempt to describe the nature of this pleiotropic effect. We cloned and sequenced the CHO lipin 1 cDNA, a gene that codes for PAP activity. Overexpression of lipin 1 in the GPI-deficient cell line, GroD1 resulted in increased PAP activity, however it failed to restore glycerolipid biosynthesis. Fluorescent microscopy showed a failure of GPI-deficient cells to localize lipin 1α to the nucleus. We also found that glucose-6-phosphate levels in GroD1 cells were 10-fold over normal. Lowering glucose levels in the growth medium partially restored glycerolipid biosynthesis and nuclear localization of lipin 1α. Western blot analysis of the elements within the mTOR pathway, which influences lipin 1 activity, was consistent with an abnormal activation of this system. Combined, these data suggest that GPI deficiency results in an accumulation of glucose-6-phosphate, and possibly other glucose-derived metabolites, leading to activation of mTOR and sequestration of lipin 1 to the cytosol, preventing its proper functioning. These results shed light on the mechanism underlying the pathologies associated with inherited GPI deficiency and the variability in the severity of the symptoms observed in these patients.
doi:10.1016/j.bbadis.2011.07.007
PMCID: PMC3185221  PMID: 21787864
20.  Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p 
Molecular Biology of the Cell  2013;24(13):2124-2133.
Binding and dephosphorylation of the yeast lipin Pah1p by its phosphatase Nem1p-Spo7p is essential for its membrane targeting and is mediated by a C-terminal acidic stretch on Pah1p. This results in the recruitment of Pah1p to the vicinity of lipid droplets, where it controls triglyceride and droplet biogenesis in an acidic tail–dependent manner.
Lipins are evolutionarily conserved phosphatidate phosphatases that perform key functions in phospholipid, triglyceride, and membrane biogenesis. Translocation of lipins on membranes requires their dephosphorylation by the Nem1p-Spo7p transmembrane phosphatase complex through a poorly understood mechanism. Here we identify the carboxy-terminal acidic tail of the yeast lipin Pah1p as an important regulator of this step. Deletion or mutations of the tail disrupt binding of Pah1p to the Nem1p-Spo7p complex and Pah1p membrane translocation. Overexpression of Nem1p-Spo7p drives the recruitment of Pah1p in the vicinity of lipid droplets in an acidic tail–dependent manner and induces lipid droplet biogenesis. Genetic analysis shows that the acidic tail is essential for the Nem1p-Spo7p–dependent activation of Pah1p but not for the function of Pah1p itself once it is dephosphorylated. Loss of the tail disrupts nuclear structure, INO1 gene expression, and triglyceride synthesis. Similar acidic sequences are present in the carboxy-terminal ends of all yeast lipin orthologues. We propose that acidic tail–dependent binding and dephosphorylation of Pah1p by the Nem1p-Spo7p complex is an important determinant of its function in lipid and membrane biogenesis.
doi:10.1091/mbc.E13-01-0021
PMCID: PMC3694796  PMID: 23657815
21.  Endoplasmic Reticulum Stress Promotes LIPIN2-Dependent Hepatic Insulin Resistance 
Diabetes  2011;60(4):1072-1081.
OBJECTIVE
Diet-induced obesity (DIO) is linked to peripheral insulin resistance—a major predicament in type 2 diabetes. This study aims to identify the molecular mechanism by which DIO-triggered endoplasmic reticulum (ER) stress promotes hepatic insulin resistance in mouse models.
RESEARCH DESIGN AND METHODS
C57BL/6 mice and primary hepatocytes were used to evaluate the role of LIPIN2 in ER stress-induced hepatic insulin resistance. Tunicamycin, thapsigargin, and lipopolysaccharide were used to invoke acute ER stress conditions. To promote chronic ER stress, mice were fed with a high-fat diet for 8–12 weeks. To verify the role of LIPIN2 in hepatic insulin signaling, adenoviruses expressing wild-type or mutant LIPIN2, and shRNA for LIPIN2 were used in animal studies. Plasma glucose, insulin levels as well as hepatic free fatty acids, diacylglycerol (DAG), and triacylglycerol were assessed. Additionally, glucose tolerance, insulin tolerance, and pyruvate tolerance tests were performed to evaluate the metabolic phenotype of these mice.
RESULTS
LIPIN2 expression was enhanced in mouse livers by acute ER stress–inducers or by high-fat feeding. Transcriptional activation of LIPIN2 by ER stress is mediated by activating transcription factor 4, as demonstrated by LIPIN2 promoter assays, Western blot analyses, and chromatin immunoprecipitation assays. Knockdown of hepatic LIPIN2 in DIO mice reduced fasting hyperglycemia and improved hepatic insulin signaling. Conversely, overexpression of LIPIN2 impaired hepatic insulin signaling in a phosphatidic acid phosphatase activity–dependent manner.
CONCLUSIONS
These results demonstrate that ER stress–induced LIPIN2 would contribute to the perturbation of hepatic insulin signaling via a DAG-protein kinase C ε–dependent manner in DIO mice.
doi:10.2337/db10-1046
PMCID: PMC3064081  PMID: 21357464
22.  Cell Autonomous Lipin 1 Function Is Essential for Development and Maintenance of White and Brown Adipose Tissue 
Molecular and Cellular Biology  2012;32(23):4794-4810.
Through analysis of mice with spatially and temporally restricted inactivation of Lpin1, we characterized its cell autonomous function in both white (WAT) and brown (BAT) adipocyte development and maintenance. We observed that the lipin 1 inactivation in adipocytes of aP2Cre/+/LpfEx2-3/fEx2-3 mice resulted in lipodystrophy and the presence of adipocytes with multilocular lipid droplets. We further showed that time-specific loss of lipin 1 in mature adipocytes in aP2Cre-ERT2/+/LpfEx2-3/fEx2-3 mice led to their replacement by newly formed Lpin1-positive adipocytes, thus establishing a role for lipin 1 in mature adipocyte maintenance. Importantly, we observed that the presence of newly formed Lpin1-positive adipocytes in aP2Cre-ERT2/+/LpfEx2-3/fEx2-3 mice protected these animals against WAT inflammation and hepatic steatosis induced by a high-fat diet. Loss of lipin 1 also affected BAT development and function, as revealed by histological changes, defects in the expression of peroxisome proliferator-activated receptor alpha (PPARα), PGC-1α, and UCP1, and functionally by altered cold sensitivity. Finally, our data indicate that phosphatidic acid, which accumulates in WAT of animals lacking lipin 1 function, specifically inhibits differentiation of preadipocytes. Together, these observations firmly demonstrate a cell autonomous role of lipin 1 in WAT and BAT biology and indicate its potential as a therapeutical target for the treatment of obesity.
doi:10.1128/MCB.00512-12
PMCID: PMC3497595  PMID: 23028044
23.  NF-E2-Related Factor 1 (Nrf1) Serves as a Novel Regulator of Hepatic Lipid Metabolism through Regulation of the Lipin1 and PGC-1β Genes 
Molecular and Cellular Biology  2012;32(14):2760-2770.
Hepatic lipid metabolism is under elaborate regulation, and perturbations in this regulatory process at the transcriptional level lead to pathological conditions. NF-E2-related factor 1 (Nrf1) is a member of the cap'n'collar (CNC) transcription factor family. Hepatocyte-specific Nrf1 gene conditional-knockout mice are known to develop hepatic steatosis, but it remains unclear how Nrf1 contributes to the lipid homeostasis. Therefore, in this study we examined the gene expression profiles of Nrf1-deficient mouse livers. A pathway analysis based on the profiling results revealed that the levels of expression of the genes related to lipid metabolism, amino acid metabolism, and mitochondrial respiratory function were decreased in Nrf1-deficient mouse livers, indicating the profound effects that the Nrf1 deficiency conferred to various metabolic pathways. We discovered that the Nrf1 deficiency leads to the reduced expression of the transcriptional coactivator genes Lipin1 and PGC-1β (for peroxisome proliferator-activated receptor γ coactivator 1β). Chromatin immunoprecipitation analyses showed that Nrf1 binds to the antioxidant response elements (AREs) in regulatory regions of the Lipin1 and PGC-1β genes and the binding of Nrf1 to the AREs activates reporter gene transcription. These results thus identified Nrf1 to be a novel regulator of the Lipin1 and PGC-1β genes, providing new insights into the Nrf1 function in hepatic lipid metabolism.
doi:10.1128/MCB.06706-11
PMCID: PMC3416188  PMID: 22586274
24.  Identification of Two Legionella pneumophila Effectors that Manipulate Host Phospholipids Biosynthesis 
PLoS Pathogens  2012;8(11):e1002988.
The intracellular pathogen Legionella pneumophila translocates a large number of effector proteins into host cells via the Icm/Dot type-IVB secretion system. Some of these effectors were shown to cause lethal effect on yeast growth. Here we characterized one such effector (LecE) and identified yeast suppressors that reduced its lethal effect. The LecE lethal effect was found to be suppressed by the over expression of the yeast protein Dgk1 a diacylglycerol (DAG) kinase enzyme and by a deletion of the gene encoding for Pah1 a phosphatidic acid (PA) phosphatase that counteracts the activity of Dgk1. Genetic analysis using yeast deletion mutants, strains expressing relevant yeast genes and point mutations constructed in the Dgk1 and Pah1 conserved domains indicated that LecE functions similarly to the Nem1-Spo7 phosphatase complex that activates Pah1 in yeast. In addition, by using relevant yeast genetic backgrounds we examined several L. pneumophila effectors expected to be involved in phospholipids biosynthesis and identified an effector (LpdA) that contains a phospholipase-D (PLD) domain which caused lethal effect only in a dgk1 deletion mutant of yeast. Additionally, LpdA was found to enhance the lethal effect of LecE in yeast cells, a phenomenon which was found to be dependent on its PLD activity. Furthermore, to determine whether LecE and LpdA affect the levels or distribution of DAG and PA in-vivo in mammalian cells, we utilized fluorescent DAG and PA biosensors and validated the notion that LecE and LpdA affect the in-vivo levels and distribution of DAG and PA, respectively. Finally, we examined the intracellular localization of both LecE and LpdA in human macrophages during L. pneumophila infection and found that both effectors are localized to the bacterial phagosome. Our results suggest that L. pneumophila utilize at least two effectors to manipulate important steps in phospholipids biosynthesis.
Author Summary
Legionella pneumophila is an intracellular pathogen that causes a severe pneumonia known as Legionnaires' disease. Following infection, the bacteria use a Type-IVB secretion system to translocate multiple effector proteins into macrophages and generate the Legionella-containing vacuole (LCV). The formation of the LCV involves the recruitment of specific bacterial effectors and host cell factors to the LCV as well as changes in its lipids composition. By screening L. pneumophila effectors for yeast growth inhibition, we have identified an effector, named LecE, that strongly inhibits yeast growth. By using yeast genetic tools, we found that LecE activates the yeast lipin homolog – Pah1, an enzyme that catalyzes the conversion of diacylglycerol to phosphatidic acid, these two molecules function as bioactive lipid signaling molecules in eukaryotic cells. In addition, by using yeast deletion mutants in genes relevant to lipids biosynthesis, we have identified another effector, named LpdA, which function as a phospholipase-D enzyme. Both effectors were found to be localized to the LCV during infection. Our results reveal a possible mechanism by which an intravacuolar pathogen might change the lipid composition of the vacuole in which it resides, a process that might lead to the recruitment of specific bacterial and host cell factors to the vacoule.
doi:10.1371/journal.ppat.1002988
PMCID: PMC3486869  PMID: 23133385
25.  Identification of a novel lipin homologue from the parasitic protozoan Trypanosoma brucei 
BMC Microbiology  2013;13:101.
Background
Arginine methylation is a post-translational modification that expands the functional diversity of proteins. Kinetoplastid parasites contain a relatively large group of protein arginine methyltransferases (PRMTs) compared to other single celled eukaryotes. Several T. brucei proteins have been shown to serve as TbPRMT substrates in vitro, and a great number of proteins likely to undergo methylation are predicted by the T. brucei genome. This indicates that a large number of proteins whose functions are modulated by arginine methylation await discovery in trypanosomes. Here, we employed a yeast two-hybrid screen using as bait the major T. brucei type I PRMT, TbPRMT1, to identify potential substrates of this enzyme.
Results
We identified a protein containing N-LIP and C-LIP domains that we term TbLpn. These domains are usually present in a family of proteins known as lipins, and involved in phospholipid biosynthesis and gene regulation. Far western and co-immunoprecipitation assays confirmed the TbPRMT1-TbLpn interaction. We also demonstrated that TbLpn is localized mainly to the cytosol, and is methylated in vivo. In addition, we showed that, similar to mammalian and yeast proteins with N-LIP and C-LIP domains, recombinant TbLpn exhibits phosphatidic acid phosphatase activity, and that two conserved aspartic acid residues present in the C-LIP domain are critical for its enzymatic activity.
Conclusions
This study reports the characterization of a novel trypanosome protein and provides insight into its enzymatic activity and function in phospholipid biosynthesis. It also indicates that TbLpn functions may be modulated by arginine methylation.
doi:10.1186/1471-2180-13-101
PMCID: PMC3654991  PMID: 23656927
Kinetoplastid; Lipin; Arginine methylation; Phosphatidic acid phosphatase

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