The prevalence of obesity in the western world has focused attention on factors that influence triglyceride biosynthesis, storage, and utilization. Members of the lipin protein family have a newly discovered enzymatic role in triglyceride and phospholipid biosynthesis as a phosphatidate phosphatase, and also act as an inducible transcriptional coactivator in conjunction with PGC-1α and PPARα. Through these activities, the founding member of the family, lipin-1, influences lipid metabolism and glucose homeostasis in diverse tissues including adipose tissue, skeletal muscle, and liver. The physiological roles of lipin-2 and lipin-3 are less well defined, but are likely to carry out similar functions in glycerolipid biosynthesis and gene expression in a distinct tissue distribution.
adipose tissue; ipodystrophy; obesity; triaclyglycerol; phosphatidate phosphatase; transcriptional coactivator
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.
triglyceride; obesity; insulin resistance; phosphatidate phosphatase; transcriptional coactivator
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.
Lipin; phosphatidic acid phosphatase; lipid droplets; brain
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.
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.
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.
Adipocyte; Lipids; Mouse; Phosphatase; Phosphatidate; Triacylglycerol; Lipin
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.
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.
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.
Seipin; Adipogenesis; Lipodystrophy; Lipin; Endoplasmic reticulum
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.
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.
lipin; PGC-1α; metabolism; heart failure
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.
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.
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.
lipin; oligomer; FRET; phosphatidic acid phosphatase
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.
Alcoholic fatty liver; signal transduction; lipid metabolism; acetylation; sumoylation
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.
Phosphatidate phosphatase (PAP) enzymes catalyze the dephosphorylation of phosphatidate, yielding diacylglycerol and inorganic phosphate. In eukaryotic cells, PAP activity has a central role in the synthesis of phospholipids and triacylglycerol through its product diacylglycerol, and it also generates and/or degrades lipid-signaling molecules that are related to phosphatidate. There are two types of PAP enzyme, Mg2+ dependent (PAP1) and Mg2+ independent (PAP2), but only genes encoding PAP2 enzymes had been identified until recently, when a gene (PAH1) encoding a PAP1 enzyme was found in Saccharomyces cerevisiae. This discovery has revealed a molecular function of the mammalian protein lipin, a deficiency of which causes lipodystrophy in mice. With molecular information now available for both types of PAP, the specific roles of these enzymes in lipid metabolism are being clarified.
Human lipin1 catalyzes the highly regulated conversion of phosphatidic acids to diacylglycerides. Lipin’s cellular location, protein partners, and biological function are directed by phosphorylation/dephosphorylation events catalyzed by the phosphoserine phosphatase dullard. To define the determinants of dullard substrate recognition and catalysis, and hence, lipin regulation, steady-state kinetic analysis was performed on phosphoserine-bearing nonapeptides based on the phosphorylation sites of lipin. The results demonstrate that dullard shows specificity toward the peptide corresponding to the insulin-dependent phosphorylation site (Ser106) of lipin with kcat/Km = 1 × 104 M−1 s−1. These results are consistent with a coil/loop structure for the insulin-dependent phosphorylation site on human lipin1 and make the requirement for an adaptor protein to confer activity such as that proposed for the yeast homologue, unlikely.
protein phosphoserine phosphatase; HAD superfamily; phosphatidic acid phosphatase
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.
In humans, disruption of the gene BSCL2, encoding the protein seipin, causes congenital generalised lipodystrophy (CGL) with severe insulin resistance and dyslipidaemia. While the causative gene has been known for over a decade, the molecular functions of seipin are only now being uncovered. Most pathogenic mutations in BSCL2 represent substantial disruptions including significant deletions and frameshifts. However, several more subtle mutations have been reported that cause premature stop codons or single amino acid substitutions. Here we have examined these mutant forms of seipin to gain insight into how they may cause CGL.
We generated constructs expressing mutant seipin proteins and determined their expression and localisation. We also assessed their capacity to recruit the key adipogenic phosphatidic acid phosphatase lipin 1, a recently identified molecular role of seipin in developing adipocytes. Finally, we used atomic force microscopy to define the oligomeric structure of seipin and to determine whether this is affected by the mutations.
We show that the R275X mutant of seipin is not expressed in pre-adipocytes. While the other premature stop mutant forms fail to bind lipin 1 appropriately, the point mutants T78A, L91P and A212P all retain this capacity. We demonstrate that wild-type human seipin forms oligomers of 12 subunits in a circular configuration but that the L91P and A212P mutants of seipin do not.
Our study represents the most comprehensive analysis so far of mutants of seipin causing lipodystrophy and reveals several different molecular mechanisms by which these mutations may cause disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-013-3029-3) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
Adipogenesis; Adipose tissue; BSCL2; Lipin; Lipodystrophy; Seipin
Three lipid phosphate phosphatases (LPPs) regulate cell signaling by modifying the concentrations of a variety of lipid phosphates versus their dephosphorylated products. In particular, the LPPs are normally considered to regulate signaling by the phospholipase D (PLD) pathway by converting phosphatidate (PA) to diacylglycerol (DAG). LPP activities do modulate the accumulations of PA and DAG following PLD activation, but this could also involve an effect upstream of PLD activation. The active sites of the LPPs are on the exterior surface of plasma membranes, or on the luminal surface of internal membranes. Consequently, the actions of the LPPs in metabolizing PA formed by PLD1 or PLD2 should depend on the access of this substrate to the active site of the LPPs. Alternatively, PA generated on the cytosolic surface of membranes should be readily accessible to the family of specific phosphatidate phosphatases, namely the lipins. Presently, there is only indirect evidence for the lipins participating in cell signaling following PLD activation. So far, we know relatively little about how individual LPPs and specific phosphatidate phosphatases (lipins) modulate cell signaling through controlling the turnover of bioactive lipids that are formed after PLD activation.
Diacylglycerol; lysophosphatidate; phosphatidate; phospholipase D; triacylglycerol synthesis
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.
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.
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.
The nutrient- and growth factor-responsive kinase, mTOR Complex 1 (mTORC1) regulates many processes that control growth including protein synthesis, autophagy, and lipogenesis. Through unknown mechanisms, mTORC1 promotes the function of SREBP, a master regulator of lipo- and sterolgenic gene transcription. Here, we demonstrate that mTORC1 regulates SREBP by controlling the nuclear entry of lipin 1, a phosphatidic acid phosphatase. Dephosphorylated, nuclear, catalytically active lipin 1 promotes nuclear remodeling and mediates the effects of mTORC1 on SREBP target gene, SREBP promoter activity, and nuclear SREBP protein abundance. Inhibition of mTORC1 in the liver significantly impairs SREBP function and makes mice resistant, in a lipin 1-dependent fashion, to the hepatic steatosis and hypercholesterolemia induced by a high fat and cholesterol diet. These findings establish lipin 1 as a key component of the mTORC1-SREBP pathway.
Lipin 1 controls fatty acid metabolism in the nucleus as a transcriptional regulator and in the cytosol as an enzyme catalyzing the penultimate step in phosphoglycerol triacylglyceride (TAG) synthesis. We sought to evaluate the effects of lipin 1 on hepatic TAG synthesis and secretion by gain-of-function and loss-of-function approaches.
Methods and Results
Rates of TAG synthesis were not impaired in hepatocytes isolated from adult lipin 1—deficient (fld) mice and were actually increased in 14-day-old fld mice. Additionally, compared to littermate controls, VLDL-TAG secretion rates were markedly increased in fld mice of both ages. Lipin 1 overexpression did not alter TAG synthesis rates but significantly suppressed VLDL-TAG secretion. The lipin 1-mediated suppression of VLDL-TAG secretion was linked to the peptide motif mediating its transcriptional-regulatory effects. However, the expression of candidate genes required for VLDL assembly and secretion was unaltered by lipin 1 activation or deficiency. Finally, the hepatic expression of lipin 1 was diminished in obese insulin-resistant mice, whereas adenoviral-mediated overexpression of lipin 1 in liver of these mice inhibits VLDL-TAG secretion and improves hepatic insulin signaling.
Collectively, these studies reveal new and unexpected effects of lipin 1 on hepatic TAG metabolism and obesity-related hepatic insulin resistance.
lipin 1; liver; triglyceride; VLDL secretion; dyslipidemia
Phosphorylation of the conserved lipin Pah1p/Smp2p in
Saccharomyces cerevisiae was previously shown to control
transcription of phospholipid biosynthetic genes and nuclear structure by
regulating the amount of membrane present at the nuclear envelope (Santos-Rosa,
H., Leung, J., Grimsey, N., Peak-Chew, S., and Siniossoglou, S. (2005)
EMBO J. 24, 1931–1941). A recent report
identified Pah1p as a Mg2+-dependent phosphatidate (PA)
phosphatase that regulates de novo lipid synthesis (Han G.-S.,
Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210
–9218). In this work we use a combination of mass spectrometry and
systematic mutagenesis to identify seven Ser/Thr-Pro motifs within Pah1p that
are phosphorylated in vivo. We show that phosphorylation on
these sites is required for the efficient transcriptional dere-pression of key
enzymes involved in phospholipid biosynthesis. The phosphorylation-deficient
Pah1p exhibits higher PA phosphatase-specific activity than the wild-type Pah1p,
indicating that phosphorylation of Pah1p controls PA production. Opi1p is a
transcriptional repressor of phospholipid biosynthetic genes, responding to PA
levels. Genetic analysis suggests that Pah1p regulates transcription of these
genes through both Opi1p-dependent and -independent mechanisms. We also provide
evidence that derepression of phos-pholipid biosynthetic genes is not sufficient
to induce the nuclear membrane expansion shown in the