Search

Lipin 1 regulates cellular lipid homeostasis through roles in glycerolipid synthesis (through phosphatidic acid phosphatase activity) and transcriptional coactivation. Lipin 1-deficient individuals exhibit episodic disease symptoms that are triggered by metabolic stress, such as stress caused by prolonged fasting. We sought to identify critical lipin 1 activities during fasting. We determined that lipin 1 deficiency induces widespread alternative mRNA splicing in liver during fasting, much of which is normalized by refeeding. The role of lipin 1 in mRNA splicing was largely independent of its enzymatic function. We identified interactions between lipin 1 and spliceosome proteins, as well as a requirement for lipin 1 to maintain homeostatic levels of spliceosome small nuclear RNAs and specific RNA splicing factors. In fasted Lpin1-/- liver, we identified a correspondence between alternative splicing of phospholipid biosynthetic enzymes and dysregulated phospholipid levels; splicing patterns and phospholipid levels were partly normalized by feeding. Thus, lipin 1 influences hepatic lipid metabolism through mRNA splicing, as well as through enzymatic and transcriptional activities, and fasting exacerbates the deleterious effects of lipin 1 deficiency on metabolic homeostasis.

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

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

Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for treating male infertility, which afflicts >100 million men world-wide. While much has been learned about rodent SSCs, human SSCs remain poorly understood. Here, we molecularly characterize human SSCs and define conditions favoring their culture. To achieve this, we first identified a cell-surface protein, PLPPR3, that allowed purification of human primitive undifferentiated spermatogonia (uSPG) highly enriched for SSCs. Comparative RNA-sequencing analysis of these enriched SSCs with differentiating SPG (KIT+ cells) revealed the full complement of genes that shift expression during this developmental transition, including genes encoding key components in the TGF-β, GDNF, AKT, and JAK-STAT signaling pathways. We examined the effect of manipulating these signaling pathways on cultured human SPG using both conventional approaches and single-cell RNA-sequencing analysis. This revealed that GDNF and BMP8B broadly support human SPG culture, while activin A selectively supports more advanced human SPG. One condition-AKT pathway inhibition-had the unique ability to selectively support the culture of primitive human uSPG. This raises the possibility that supplementation with an AKT inhibitor could be used to culture human SSCs in vitro for therapeutic applications.

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and C-Lip regions that are separated by 500-residues in humans. To understand how the N-Lip and C-Lip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and C-Lip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the C-Lip to form an immunoglobulin-like domain and the remaining C-Lip forms a HAD-like catalytic domain. An N-Lip C-Lip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies.

The regulation of cellular lipid storage and membrane lipid composition plays a critical role in metabolic homeostasis, and dysregulation may contribute to disorders such as obesity, fatty liver, type 2 diabetes, and cardiovascular disease. The mammalian lipin proteins (lipin 1, lipin 2, and lipin 3) are phosphatidic acid phosphatase (PAP) enzymes that modulate levels of cellular triacylglycerols and phospholipids, and also regulate lipid intermediates in cellular signaling pathways. Lipin proteins also have the ability to coactivate/corepress transcription. In humans and mice, lipin gene mutations cause severe metabolic phenotypes including rhabdomyolysis (lipin 1), autoinflammatory disease (lipin 2), and impaired intestinal lipoprotein assembly (lipin 2/lipin 3). Characterization of these diseases has revealed roles for lipin PAP activity in fundamental cellular processes such as autophagy, inflammasome activation, and lipoprotein assembly. Lipin protein activity is regulated at pre- and posttranscriptional levels, which suggests a need for their ordered response to specific physiological stimuli. Challenges for the future include better elucidation of the unique biochemical and physiological properties of individual lipin family members and determination of lipin protein structure-function relationships. Further research may propel exploration of lipin proteins as viable therapeutic targets in metabolic or inflammatory disorders.

The lipin phosphatidic acid phosphatase (PAP) enzymes are required for triacylglycerol (TAG) synthesis from glycerol 3-phosphate in most mammalian tissues. The 3 lipin proteins (lipin 1, lipin 2, and lipin 3) each have PAP activity, but have distinct tissue distributions, with lipin 1 being the predominant PAP enzyme in many metabolic tissues. One exception is the small intestine, which is unique in expressing exclusively lipin 2 and lipin 3. TAG synthesis in small intestinal enterocytes utilizes 2-monoacylglycerol and does not require the PAP reaction, making the role of lipin proteins in enterocytes unclear. Enterocyte TAGs are stored transiently as cytosolic lipid droplets or incorporated into lipoproteins (chylomicrons) for secretion. We determined that lipin enzymes are critical for chylomicron biogenesis, through regulation of membrane phospholipid composition and association of apolipoprotein B48 with nascent chylomicron particles. Lipin 2/3 deficiency caused phosphatidic acid accumulation and mammalian target of rapamycin complex 1 (mTORC1) activation, which were associated with enhanced protein levels of a key phospholipid biosynthetic enzyme (CTP:phosphocholine cytidylyltransferase α) and altered membrane phospholipid composition. Impaired chylomicron synthesis in lipin 2/3 deficiency could be rescued by normalizing phospholipid synthesis levels. These data implicate lipin 2/3 as a control point for enterocyte phospholipid homeostasis and chylomicron biogenesis.

Triacylglycerols (TAG) serve as the predominant form of energy storage in mammalian cells, and TAG synthesis influences conditions such as obesity, fatty liver, and insulin resistance. In most tissues, the glycerol 3-phosphate pathway enzymes are responsible for TAG synthesis, and the regulation and function of these enzymes is therefore important for metabolic homeostasis. Here we review the sites and regulation of glycerol-3-phosphate acyltransferase (GPAT), acylglycerol-3-phosphate acyltransferase (AGPAT), lipin phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferase (DGAT) enzyme action. We highlight the critical roles that these enzymes play in human health by reviewing Mendelian disorders that result from mutation in the corresponding genes. We also summarize the valuable insights that genetically engineered mouse models have provided into the cellular and physiological roles of GPATs, AGPATs, lipins and DGATs. Finally, we comment on the status and feasibility of therapeutic approaches to metabolic disease that target enzymes of the glycerol 3-phosphate pathway. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

The regulation of glycerolipid biosynthesis is critical for homeostasis of cellular lipid stores and membranes. Here we review the role of lipin phosphatidic acid phosphatase enzymes in glycerolipid synthesis. Lipin proteins are unique among glycerolipid biosynthetic enzymes in their ability to transit among cellular membranes, rather than remain membrane tethered. We focus on the mechanisms that underlie lipin protein interactions with membranes and the versatile roles of lipins in several organelles, including the endoplasmic reticulum, mitochondria, endolysosomes, lipid droplets, and nucleus. We also review the corresponding physiological roles of lipins, which have been uncovered by the study of genetic lipin deficiencies. We propose that the growing body of knowledge concerning the biochemical and cellular activities of lipin proteins will be valuable for understanding the physiological functions of lipin proteins in health and disease. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

The Toxoplasma inner membrane complex (IMC) is a specialized organelle underlying the parasite's plasma membrane that consists of flattened rectangular membrane sacs that are sutured together and positioned atop a supportive cytoskeleton. We have previously identified a novel class of proteins localizing to the transverse and longitudinal sutures of the IMC, which we named IMC sutures components (ISCs). Here, we have used proximity-dependent biotin identification at the sutures to better define the composition of this IMC subcompartment. Using ISC4 as bait, we demonstrate biotin-dependent labeling of the sutures and have uncovered two new ISCs. We also identified five new proteins that exclusively localize to the transverse sutures that we named transverse sutures components (TSCs), demonstrating that components of the IMC sutures consist of two groups: those that localize to the transverse and longitudinal sutures (ISCs) and those residing only in the transverse sutures (TSCs). In addition, we functionally analyze the ISC protein ISC3 and demonstrate that ISC3-null parasites have morphological defects and reduced fitness in vitro. Most importantly, Δisc3 parasites exhibit a complete loss of virulence in vivo. These studies expand the known composition of the IMC sutures and highlight the contribution of ISCs to the ability of the parasite to proliferate and cause disease.

Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production.

RationalePhosPhatidic Acid Phosphatase type 2B (PPAP2B), an integral membrane protein known as lipid phosphate phosphatase (LPP3) that inactivates lysophosphatidic acid, was implicated in coronary artery disease (CAD) by genome-wide association studies. However, it is unclear whether genome-wide association studies-identified coronary artery disease genes, including PPAP2B, participate in mechanotransduction mechanisms by which vascular endothelia respond to local atherorelevant hemodynamics that contribute to the regional nature of atherosclerosis.ObjectiveTo establish the critical role of PPAP2B in endothelial responses to hemodynamics.Methods and resultsReduced PPAP2B was detected in vivo in mouse and swine aortic arch (AA) endothelia exposed to chronic disturbed flow, and in mouse carotid artery endothelia subjected to surgically induced acute disturbed flow. In humans, PPAP2B was reduced in the downstream part of carotid plaques where low shear stress prevails. In culture, reduced PPAP2B was measured in human aortic endothelial cells under atherosusceptible waveform mimicking flow in human carotid sinus. Flow-sensitive microRNA-92a and transcription factor KLF2 were identified as upstream inhibitor and activator of endothelial PPAP2B, respectively. PPAP2B suppression abrogated atheroprotection of unidirectional flow; inhibition of lysophosphatidic acid receptor 1 restored the flow-dependent, anti-inflammatory phenotype in PPAP2B-deficient cells. PPAP2B inhibition resulted in myosin light-chain phosphorylation and intercellular gaps, which were abolished by lysophosphatidic acid receptor 1/2 inhibition. Expression quantitative trait locus mapping demonstrated PPAP2B coronary artery disease risk allele is not linked to PPAP2B expression in various human tissues but significantly associated with reduced PPAP2B in human aortic endothelial cells.ConclusionsAtherorelevant flows dynamically modulate endothelial PPAP2B expression through miR-92a and KLF2. Mechanosensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining vascular integrity of endothelial monolayer under atheroprotective flow.

Genome-wide association studies (GWAS) have identified over 40 loci that affect risk of coronary artery disease (CAD) and the causal mechanisms at the majority of loci are unknown. Recent studies have suggested that many causal GWAS variants influence disease through altered transcriptional regulation in disease-relevant cell types. We explored changes in transcriptional regulation during a key pathophysiological event in CAD, the environmental lipid-induced transformation of macrophages to lipid-laden foam cells. We used a combination of open chromatin mapping with formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and enhancer and transcription factor mapping using chromatin immuno-precipitation (ChIP-seq) in primary human macrophages before and after exposure to atherogenic oxidized low-density lipoprotein (oxLDL), with resultant foam cell formation. OxLDL-induced foam cell formation was associated with changes in a subset of open chromatin and active enhancer sites that strongly correlated with expression changes of nearby genes. OxLDL-regulated enhancers were enriched for several transcription factors including C/EBP-beta, which has no previously documented role in foam cell formation. OxLDL exposure up-regulated C/EBP-beta expression and increased genomic binding events, most prominently around genes involved in inflammatory response pathways. Variants at CAD-associated loci were significantly and specifically enriched in the subset of chromatin sites altered by oxLDL exposure, including rs72664324 in an oxLDL-induced enhancer at the PPAP2B locus. OxLDL increased C/EBP beta binding to this site and C/EBP beta binding and enhancer activity were stronger with the protective A allele of rs72664324. In addition, expression of the PPAP2B protein product LPP3 was present in foam cells in human atherosclerotic plaques and oxLDL exposure up-regulated LPP3 in macrophages resulting in increased degradation of pro-inflammatory mediators. Our results demonstrate a genetic mechanism contributing to CAD risk at the PPAP2B locus and highlight the value of studying epigenetic changes in disease processes involving pathogenic environmental stimuli.

LPIN1 encodes lipin-1, a phosphatidic acid phosphatase (PAP) enzyme that catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerol. Homozygous LPIN1 gene mutations cause severe rhabdomyolysis, and heterozygous LPIN1 missense mutations may promote statin-induced myopathy. We demonstrate that lipin-1-related myopathy in the mouse is associated with a blockade in autophagic flux and accumulation of aberrant mitochondria. Lipin-1 PAP activity is required for maturation of autolysosomes, through its activation of the protein kinase D (PKD)-Vps34 phosphatidylinositol 3-kinase signaling cascade. Statin treatment also reduces PKD activation and autophagic flux, which are compounded by diminished mammalian target of rapamycin (mTOR) abundance in lipin-1-haploinsufficent and -deficient muscle. Lipin-1 restoration in skeletal muscle prevents myonecrosis and statin toxicity in vivo, and activated PKD rescues autophagic flux in lipin-1-deficient cells. Our findings identify lipin-1 PAP activity as a component of the macroautophagy pathway and define the basis for lipin-1-related myopathies.

Group IVA phospholipase A(2) (GIVA PLA(2)) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA(2). We also studied the effect of PAP-1 and PKC on Ca+2 induced and synergy enhanced GIVA PLA(2) activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo(2)-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca+2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA(2) synergistic activation by stimulation with Kdo(2)-Lipid A and ATP.

Cyclooxygenase (COX) has two isoforms, COX-1 and -2, which catalyze the key step in the conversion of cellular arachidonic acid into prostaglandins. In recent years, interest in COX-2 has significantly increased since it has been a target for the development of specific non-steroidal anti-inflammatory drugs. We report that COX-2 expression is up-regulated in phorbol ester (phorbol myristate acetate, PMA)-differentiated human U937 macrophage-like cells stimulated with lipopolysaccharide (LPS), whereas COX-1 is not up-regulated. We show that the LPS-induced up-regulation of COX-2 depends on the activity of the Mg(+2)-dependent phosphatidic acid phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 by bromoenol lactone, propranolol, or ethanol resulted in a decrease in LPS-induced COX-2 mRNA transcript production, COX-2 protein expression, and prostaglandin E(2) release from U937 macrophages. To ensure that these results did not arise because of PMA treatment of the U937 cells, similar experiments were conducted with the P388D(1) cell line, which does not require PMA differentiation. LPS increased the levels of endogenous cellular diacylglycerol (DAG) within 2 min of stimulation. This increase was observed to be sensitive to the PAP-1 inhibitors. Furthermore, phosphatidic acid phosphohydrolase activity assays showed that the bromoenol lactone-sensitive PAP-1 activity was translocated from the cytosolic fraction to the membrane fraction within 2 min of LPS exposure. Finally, DAG add-back experiments demonstrate that LPS-induced COX-2 expression is enhanced by the addition of exogenous DAG.

Prostaglandins are known to play a key role in the initiation of labor in humans, but the mechanisms governing their synthesis in amnion are largely unknown. In this study, we have examined the regulatory pathways for prostaglandin E(2) (PGE(2)) production during protein kinase C-dependent activation of human WISH cells. In these cells, PGE(2) synthesis appears to be limited not by free arachidonic acid availability but by the expression levels of cyclooxygenase-2 (COX-2). Concomitant with the cells being able to synthesize and secrete PGE(2), we detected significant elevations of both COX-2 protein and mRNA levels. Specific inhibition of COX-2 by NS-398 totally ablated PGE(2) synthesis. All of these responses were found to be strikingly dependent on an active phosphatidate phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 activity by three different strategies (i.e. use of bromoenol lactone, propranolol, and ethanol) resulted in inhibition of COX-2 expression and hence of PGE(2) production. These data unveil a novel signaling mechanism for the regulation of PGE(2) production via regulation of COX-2 expression and implicate phosphatidate phosphohydrolase 1 as a key regulatory component of eicosanoid metabolic pathways in the amnion.

Prostaglandins are known to play a central role in the initiation of labor in humans, and amnionic cells constitute a major source of these compounds. Prostaglandin synthesis and release by amnion cells in response to hormones and ligands takes place after a characteristic 4-5 h lag. However, we report herein that free arachidonic acid (AA), the metabolic precursor of prostaglandins, can be induced at much shorter times (1 h) in human amnionic WISH cells by phorbol 12-myristate 13-acetate (PMA) through activation of protein kinase Calpha (PKCalpha). WISH cells were found to possess both cytosolic group IV phospholipase A2 (cPLA2) and Group VI Ca2+-independent phospholipase A2 (iPLA2). Of these, the cPLA2 was found to be the likely mediator of AA mobilization in PMA-activated WISH cells. PMA also activates phospholipase D (PLD) in these cells and ethanol, a compound that inhibits PLD-mediated phosphatidic acid (PA) formation, blocked AA release. Moreover, prevention of PA dephosphorylation by the PA phosphohydrolase inhibitors propranolol and bromoenol lactone, resulted in inhibition of AA release by PMA-treated WISH cells. Collectively, these data suggest that activation of cPLA2 and attendant AA release by phorbol esters in WISH cells requires prior generation of DAG by phosphatidate phosphohydrolase.

Bromoenol lactone (BEL) has previously been identified as a potent, irreversible, mechanism-based phospholipase A2 (PLA2) inhibitor that possesses greater than 1000-fold selectivity for inhibition of Ca2+-independent PLA2 (iPLA2) versus the Ca2+-dependent ones. Thus, this compound has been used as a selective tool for studies aimed at elucidating the role of iPLA2 in certain cellular functions. Herein we report that BEL also inhibits cellular phosphatidic acid phosphohydrolase (PAP) activity in intact P388D1 macrophages with an IC50 of about 8 microM, which is very similar to that previously found for inhibition of iPLA2 under the same experimental conditions. This results in the blockage of the incorporation of exogenous arachidonate and palmitate into diacylglycerol and triacylglycerol. Thus, inhibition of PAP by BEL blocks triacylglycerol biosynthesis in P388D1 cells due to decreased diacylglycerol availability. Because two forms of PAP activity exist in mammalian cells, differential assays were performed to identify which of these forms was inhibited by BEL. The results of these experiments revealed that BEL selectively inhibits the cytosolic, Mg2+-dependent enzyme. No apparent effect of BEL on the membrane-bound Mg2+-independent PAP form could be detected. Collectively, the results reported herein establish that BEL inhibits two cellular phospholipases, namely iPLA2 and Mg2+-dependent PAP, with similar potency. Therefore, the inhibitory effect of BEL on Mg2+-dependent PAP might explain several cellular functions previously attributed to iPLA2.

Many receptors, in response to their specific ligands, trigger activation of phospholipase D (PLD), resulting in the production of phosphatidic acid which, in turn, is acted upon by a specific phosphatase, phosphatidate phosphohydrolase, to produce diacylglycerol. We report here that isolated nuclei from Madin-Darby canine kidneys (MDCK)-D1 cells exhibit a PLD activity that is enhanced by the presence of ATP. PLD activity was measured in the presence of ethanol, by quantitating the production of phosphatidylethanol. Non-phosphorylating ATP analogs were unable to substitute for ATP in activating PLD, indicating that ATP acts as a phosphoryl group donor in a kinase-mediated phosphorylation reaction. The protein kinase C inhibitors chelerythrine and calphostin completely suppressed the ATP-induced nuclear PLD, implicating protein kinase C as the kinase involved in ATP-dependent PLD activity in nuclei from MDCK-D1 cells. In the absence of ethanol, phosphatidic acid was detected in ATP-treated nuclei. Accumulation of phosphatidic acid preceded or closely paralleled that of diacylglycerol, suggesting a precursor-product relationship. Consistent with those results, we detected phosphatidate phosphohydrolase activity in MDCK-D1 cell nuclei. Measurements of phosphatidic acid and diacylglycerol levels at increasing amounts of ethanol demonstrated that PLD and phosphatidate phosphohydrolase are responsible for generating the majority of the diacylglycerol accumulating in MDCK-D1 cell nuclei. The ability of nuclei to generate diacylglycerol from the concerted action of those two enzymes provides a means to regulate nuclear lipid synthesis as well as protein kinase C activity.