Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically
2008; Elsevier BV; Volume: 49; Issue: 5 Linguagem: Inglês
10.1194/jlr.m800013-jlr200
ISSN1539-7262
AutoresBoripont Manmontri, Meltem Sarıahmetoğlu, Jimmy Donkor, Maroun Bou Khalil, Meenakshi Sundaram, Zemin Yao, Karen Reue, Richard Lehner, David N. Brindley,
Tópico(s)Adipose Tissue and Metabolism
ResumoGlucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for β-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption. Glucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for β-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption. 8-(4-chlorophenylthio) cyclic AMP diacylglycerol dexamethasone endoplasmic reticulum glucocorticoid lipid phosphate phosphatase phosphatidate phosphatidate phosphatase peroxisome proliferator-activated receptor-coactivator-1α triacylglycerol Mammalian phosphatidate phosphatase (PAP1) activity is Mg2+-dependent and is inhibited by N-ethylmaleimide (1.Jamal Z. Martin A. Gomez-Munoz A. Brindley D.N. Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol.J. Biol. Chem. 1991; 266: 2988-2996Abstract Full Text PDF PubMed Google Scholar). These characteristics distinguish mammalian PAP1 from PAP2 activities that also convert phosphatidate (PA) to diacylglycerol (DAG). PAP2 is now commonly known as a family of lipid phosphate phosphatases (LPPs) that dephosphorylate a variety of lipid phosphate esters. The LPPs are mainly involved in regulating signal transduction (2.Brindley D.N. Lipid phosphate phosphatases and related proteins: signaling functions in development, cell division, and cancer.J. Cell. Biochem. 2004; 92: 900-912Crossref PubMed Scopus (184) Google Scholar). By contrast, PAP1 appears to be specific for PA as a substrate (3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 4.Han G.S. Wu W.I. Carman G.M. The Saccharomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar) and is a required enzyme in the biosynthesis of triacylglycerol (TAG), phosphatidylcholine, and phosphatidylethanolamine (5.Brindley D.N. Phosphatidate Phosphohydrolase: Its Role in Glycerolipid Synthesis. CRC Press, Boca Raton, FL1988: 21-77Google Scholar).Our previous work showed that injecting rats with cortisol or corticotropin produced marked increases in PAP1 activity in the liver (6.Sturton R.G. Butterwith S.C. Burditt S.L. Brindley D.N. Effects of starvation, corticotropin injection and ethanol feeding on the activity and amount of phosphatidate phosphohydrolase in rat liver.FEBS Lett. 1981; 126: 297-300Crossref PubMed Scopus (14) Google Scholar, 7.Glenny H.P. Brindley D.N. The effects of cortisol, corticotropin and thyroxine on the synthesis of glycerolipids and on the phosphatidate phosphohydrolase activity in rat liver.Biochem. J. 1978; 176: 777-784Crossref PubMed Scopus (45) Google Scholar). Subsequent work with rat hepatocytes demonstrated that the glucocorticoid (GC) effect in increasing PAP1 activity was synergized by glucagon and inhibited by insulin (8.Pittner R.A. Fears R. Brindley D.N. Interactions of insulin, glucagon and dexamethasone in controlling the activity of glycerol phosphate acyltransferase and the activity and subcellular distribution of phosphatidate phosphohydrolase in cultured rat hepatocytes.Biochem. J. 1985; 230: 525-534Crossref PubMed Scopus (50) Google Scholar, 9.Pittner R.A. Fears R. Brindley D.N. Effects of cyclic AMP, glucocorticoids and insulin on the activities of phosphatidate phosphohydrolase, tyrosine aminotransferase and glycerol kinase in isolated rat hepatocytes in relation to the control of triacylglycerol synthesis and gluconeogenesis.Biochem. J. 1985; 225: 455-462Crossref PubMed Scopus (61) Google Scholar). We showed that these GC-induced increases in PAP1 activity provide the extra capacity for the liver to sequester excess FAs as TAG when these FAs are not immediately required for β-oxidation (5.Brindley D.N. Phosphatidate Phosphohydrolase: Its Role in Glycerolipid Synthesis. CRC Press, Boca Raton, FL1988: 21-77Google Scholar). The interaction of GC with insulin explains the diurnal rhythm of PAP1 activity in rat livers (10.Knox A.M. Sturton R.G. Cooling J. Brindley D.N. Control of hepatic triacylglycerol synthesis. Diurnal variations in hepatic phosphatidate phosphohydrolase activity and in the concentrations of circulating insulin and corticosterone in rats.Biochem. J. 1979; 180: 441-443Crossref PubMed Scopus (22) Google Scholar). The GC effect is also consistent with increases in hepatic PAP1 seen after sham operations or in liver remnants after partial hepatectomy (11.Mangiapane E.H. Lloyd-Davies K.A. Brindley D.N. A study of some enzymes of glycerolipid biosynthesis in rat liver after subtotal hepatectomy.Biochem. J. 1973; 134: 103-112Crossref PubMed Scopus (56) Google Scholar), in starvation (6.Sturton R.G. Butterwith S.C. Burditt S.L. Brindley D.N. Effects of starvation, corticotropin injection and ethanol feeding on the activity and amount of phosphatidate phosphohydrolase in rat liver.FEBS Lett. 1981; 126: 297-300Crossref PubMed Scopus (14) Google Scholar), diabetes (12.Whiting P.H. Bowley M. Sturton R.G. Pritchard P.H. Brindley D.N. Hawthorne J.N. The effect of chronic diabetes, induced by streptozotocin, on the activities of some enzymes of glycerolipid synthesis in rat liver.Biochem. J. 1977; 168: 147-153Crossref PubMed Google Scholar), insulin resistance (13.Jamal Z. Martin A. Gomez-Munoz A. Hales P. Chang E. Russell J.C. Brindley D.N. Phosphatidate phosphohydrolases in liver, heart and adipose tissue of the JCR:LA corpulent rat and the lean genotypes: implications for glycerolipid synthesis and signal transduction.Int. J. Obes. Relat. Metab. Disord. 1992; 16: 789-799PubMed Google Scholar), and hypoxia (14.Kinnula V.L. Hassinen I. Metabolic adaptation to hypoxia. Redox state of the cellular free NAD pools, phosphorylation state of the adenylate system and the (Na+-K+)-stimulated ATP-ase in rat liver.Acta Physiol. Scand. 1978; 104: 109-116Crossref PubMed Scopus (10) Google Scholar), and in toxic conditions (5.Brindley D.N. Phosphatidate Phosphohydrolase: Its Role in Glycerolipid Synthesis. CRC Press, Boca Raton, FL1988: 21-77Google Scholar). Increases in hepatic PAP1 also occur in response to dietary modification in rodents, for instance, when glucose or starch is replaced by fructose, sorbitol, glycerol, or ethanol (15.Sturton R.G. Pritchard P.H. Han L.Y. Brindley D.N. The involvement of phosphatidate phosphohydrolase and phospholipase A activities in the control of hepatic glycerolipid synthesis. Effects of acute feeding with glucose, fructose, sorbitol, glycerol and ethanol.Biochem. J. 1978; 174: 667-670Crossref PubMed Scopus (31) Google Scholar), and these effects are exaggerated by high-fat feeding (16.Brindley D.N. Cooling J. Glenny H.P. Burditt S.L. McKechnie I.S. Effects of chronic modification of dietary fat and carbohydrate on the insulin, corticosterone and metabolic responses of rats fed acutely with glucose, fructose or ethanol.Biochem. J. 1981; 200: 275-283Crossref PubMed Scopus (41) Google Scholar). These changes in PAP1 are also associated with increased GC concentrations relative to insulin. PAP1 activity is also increased in the livers of baboons (17.Savolainen M.J. Baraona E. Pikkarainen P. Lieber C.S. Hepatic triacylglycerol synthesizing activity during progression of alcoholic liver injury in the baboon.J. Lipid Res. 1984; 25: 813-820Abstract Full Text PDF PubMed Google Scholar) and human alcoholics (18.Simpson K.J. Venkatesan S. Martin A. Brindley D.N. Peters T.J. Activity and subcellular distribution of phosphatidate phosphohydrolase (EC 3.1.3.4) in alcoholic liver disease.Alcohol Alcohol. 1995; 30: 31-36PubMed Google Scholar). The involvement of GC in ethanol-induced increases in PAP1 activity is confirmed because this is attenuated in adrenalectomized rats (19.Brindley D.N. Cooling J. Burditt S.L. Pritchard P.H. Pawson S. Sturton R.G. The involvement of glucocorticoids in regulating the activity of phosphatidate phosphohydrolase and the synthesis of triacylglycerols in the liver. Effects of feeding rats with glucose, sorbitol, fructose, glycerol and ethanol.Biochem. J. 1979; 180: 195-199Crossref PubMed Scopus (45) Google Scholar).The physiological expression of PAP1 activity involves a FA-induced translocation of the reservoir of cytosolic PAP1 to become functional on membranes of the endoplasmic reticulum (ER), where PA is synthesized (20.Cascales C. Mangiapane E.H. Brindley D.N. Oleic acid promotes the activation and translocation of phosphatidate phosphohydrolase from the cytosol to particulate fractions of isolated rat hepatocytes.Biochem. J. 1984; 219: 911-916Crossref PubMed Scopus (109) Google Scholar, 21.Martin A. Hopewell R. Martin-Sanz P. Morgan J.E. Brindley D.N. Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes.Biochim. Biophys. Acta. 1986; 876: 581-591Crossref PubMed Scopus (44) Google Scholar). The activity of the membrane-bound PAP1 correlates closely with the conversion of PA to DAG and the synthesis of TAG and phosphatidylcholine in intact rat hepatocytes (21.Martin A. Hopewell R. Martin-Sanz P. Morgan J.E. Brindley D.N. Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes.Biochim. Biophys. Acta. 1986; 876: 581-591Crossref PubMed Scopus (44) Google Scholar).Further work in this area was severely hampered because of the inability of any group to purify or identify the structure of PAP1. This situation changed with a publication by Han, Wu, and Carman (4.Han G.S. Wu W.I. Carman G.M. The Saccharomyces cerevisiae lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme.J. Biol. Chem. 2006; 281: 9210-9218Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar), who identified the yeast PAP1 (PAH1; previously known as SMP2) as an ortholog of mammalian lipin. They also showed that recombinant mammalian lipin-1 had PAP1 activity. Mammals express a family of lipins consisting of lipin-1A and its splice variant lipin-1B, plus lipin-2 and lipin-3 (22.Peterfy M. Phan J. Xu P. Reue K. Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin.Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (463) Google Scholar). In mature adipocytes, lipin-1A is preferentially located in the nucleus, whereas most of the lipin-1B is found in the cytosol (23.Peterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis.J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Our recent studies demonstrated that all of these lipins possess Mg2+-dependent PAP1 activity and that they are expressed in a tissue-specific manner (3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). For example, lipin-1 provides the majority, if not all, of the PAP1 activity in white and brown adipose tissue, skeletal muscle, and heart, whereas liver expresses lipin-1, -2, and -3 (3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 24.Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr, J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). This observation explains why the fld mouse, which has a null mutation in the Lpin1 gene, exhibits lipodystrophy, because lipin-1 is required for the development of mature adipocytes by regulating the expression of peroxisome proliferator-activated receptor γ (PPARγ) and TAG synthesis (25.Phan J. Peterfy M. Reue K. Lipin expression preceding peroxisome proliferator-activated receptor-gamma is critical for adipogenesis in vivo and in vitro.J. Biol. Chem. 2004; 279: 29558-29564Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The fld mouse also develops a fatty liver and hypertriglyceridemia in the preweaning period, which indicates an ability of the liver to synthesize and secrete TAG (26.Langner C.A. Birkenmeier E.H. Ben-Zeev O. Schotz M.C. Sweet H.O. Davisson M.T. Gordon J.I. The fatty liver dystrophy (fld) mutation. A new mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue-specific defects in lipoprotein lipase and hepatic lipase activities.J. Biol. Chem. 1989; 264: 7994-8003Abstract Full Text PDF PubMed Google Scholar). This capacity in the fld mouse is explained by the expression of PAP1 activity through lipin-2 and -3 (3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 24.Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr, J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). In fact, livers from fld mice show normal PAP1 activity and increased lipin-3 mRNA levels, presumably as an adaptive response to the lack of lipin-1 (3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). In addition to controlling TAG synthesis, lipin-1 increases the capacity of the liver for β-oxidation in fasting by facilitating transcriptional regulation by peroxisome proliferator-activated receptor-coactivator-1α (PGC-1α) and PPARα (27.Finck B.N. Gropler M.C. Chen Z. Leone T.C. Croce M.A. Harris T.E. Lawrence Jr., J.C. Kelly D.P. Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway.Cell Metab. 2006; 4: 199-210Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar).The discovery that the liver expresses lipin-1A, -1B, -2, and -3 (3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar) provokes the question of which lipins respond to hormonal regulation to explain the observed physiological changes in the composite PAP1 activity. Answering this question and describing the mechanisms that control the expression of the different lipins are essential to establishing their functions in hepatic metabolism and for understanding the hormonal regulation of their expression. To investigate this, we compared the responses of primary cultures of rat and mouse hepatocytes over a time course after treatment with hormones and 8-(4-chlorophenylthio) cyclic AMP (CPTcAMP). The results show that the members of the lipin family were differentially regulated by dexamethasone (dex), glucagon, and insulin. Dex with glucagon or CPTcAMP markedly increased total PAP1 activity, and this effect was accounted for by the increased synthesis of lipin-1. Insulin attenuated the dex- + CPTcAMP-induced increases in lipin-1 synthesis. These results provide the first evidence for the differential regulation of the activity of different lipins in the liver. They help to explain how the composite changes in PAP1 (lipin) activity may coordinate increased TAG synthesis, β-oxidation, and VLDL secretion in conditions of starvation, metabolic stress, insulin resistance, and diabetes.METHODSMaterialsDex, CPTcAMP, insulin, and glucagon was purchased from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal antibodies against lipin-1 were prepared in accordance with University of Ottawa Heart Institute regulations relating to Animal Care Procedures using the peptide SKTDSPSRKKDKRSRHLGADG essentially as described previously (28.Huffman T.A. Mothe-Satney I. Lawrence Jr, J.C. Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin.Proc. Natl. Acad. Sci. USA. 2002; 99: 1047-1052Crossref PubMed Scopus (185) Google Scholar). The antibody was used at a dilution of 1:500. Mouse monoclonal antibody for the V5 tag and GAPDH were purchased from Invitrogen and Sigma and used at dilutions of 1:1,000 and 1:5,000, respectively. Secondary antibodies were IRDye 800 goat anti-rabbit IgG (Rockland Immunochemicals, Gilbertsville, PA) and goat anti-mouse IgG conjugated to Alexa Fluor 680 (Molecular Probes, Eugene, OR).Preparation and culture of hepatocytesHepatocytes were prepared from male Sprague-Dawley rats (200–540 g) or C57BL/6J mice (22.5–32 g) as described previously (29.Sahoo D. Trischuk T.C. Chan T. Drover V.A. Ho S. Chimini G. Agellon L.B. Agnihotri R. Francis G.A. Lehner R. ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from murine hepatocytes.J. Lipid Res. 2004; 45: 1122-1131Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). They were plated onto collagen-coated dishes in DMEM containing 15% serum in an atmosphere of 95% air and 5% CO2 for 45–90 min to allow attachment. The medium was changed to remove nonviable cells, and the hepatocytes were incubated for a further 4 h to allow them to spread. The medium was then changed, and the hepatocytes were incubated for different times in serum-free medium containing 0.1% BSA with the addition of hormones or agonists as indicated. All incubations contained 0.5% DMSO, which was used as a vehicle for dex.Gene expression analysis in fasting/refeeding conditionsLivers were harvested from 16 week old female C57BL/6J mice after fasting for 16 h (fasted samples) or fasting for 16 h followed by refeeding for 4 h (refed samples). Total RNA was isolated with TRIzol (Invitrogen, Carlsbad, CA) and cDNA synthesized from 2 μg of RNA using the Omniscript reverse transcriptase kit (Qiagen, Valencia, CA). Real-time RT-PCR was performed with the iCycler (Bio-Rad, Hercules, CA) using SYBR Green PCR reagents (Qiagen) as described previously (25.Phan J. Peterfy M. Reue K. Lipin expression preceding peroxisome proliferator-activated receptor-gamma is critical for adipogenesis in vivo and in vitro.J. Biol. Chem. 2004; 279: 29558-29564Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Gene expression was normalized to levels of β2-microglobin and 18S rRNA. Primers used for this fasting study are listed in Table 1 .TABLE 1.Oligonucleotide primers used for real-time RT-PCR in fasting experiments with miceProteinForward PrimerReverse PrimerReferenceMouse 18s rRNAaccgcagctaggaataatggagcctcagttccgaaaaccaMouse β2-microglobingtctttcagcaaggactggtccaaatgcggcatcttcaaacc48.Xu J. Lee W.N. Phan J. Saad M.F. Reue K. Kurland I.J. Lipin deficiency impairs diurnal metabolic fuel switching.Diabetes. 2006; 55: 3429-3438Crossref PubMed Scopus (33) Google ScholarMouse lipin-1Aggtcccccagccccagtccttgcagcctgtggcaattca23.Peterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis.J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (173) Google ScholarMouse lipin-1Bcagcctggtagattgccagagcagcctgtggcaattca23.Peterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis.J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (173) Google ScholarMouse lipin-2agttgaccccatcaccgtagcccaaagcatcagacttggt3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google ScholarMouse lipin-3tggaattgggatgacaaggtcactgcaagtaccccttggt3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google ScholarMouse PGC-1αctcacagagacactggacagttgtagctgagctgagtgttggMouse PPARαaatgcaattcgctttggaagggccttgaccttgttcatgt48.Xu J. Lee W.N. Phan J. Saad M.F. Reue K. Kurland I.J. Lipin deficiency impairs diurnal metabolic fuel switching.Diabetes. 2006; 55: 3429-3438Crossref PubMed Scopus (33) Google ScholarPGC-1α, peroxisome proliferator-activated receptor-coactivator-1α; PPARα, peroxisome proliferator-activated receptor α. Where not referenced, primers were designed by Primer3 or Primer Express version 2.0 software using default parameters. Open table in a new tab RNA quantitation for hepatocyte samples by real-time RT-PCRRNA was collected using the RNAqueous Kit (Ambion, Inc., Austin, TX) according to the manufacturer's directions. Reverse transcription was performed using SuperScript II, random primers, and RNaseOUT according to instructions from the supplier (Invitrogen). PCR was performed on an iCycler (Bio-Rad) using SYBR Green PCR reagents (Applied Biosystems, Foster City, CA). Primer sequences for PCR are listed in Table 2 . Gene expression was normalized to the housekeeping genes cyclophilin A and GAPDH. In initial experiments, the relative changes in mRNA expression for the lipins were essentially the same when expressed relative to both reference mRNAs; therefore, we routinely expressed results relative to cyclophilin A mRNA.TABLE 2.Oligonucleotide primers used for real-time RT-PCR in hepatocyte experimentsProteinForward PrimerReverse PrimerReferenceCyclophilin Acaccgtgttcttcgacatcacccagtgctcagagctcgaaag49.Morris K.E. Schang L.M. Brindley D.N. Lipid phosphate phosphatase-2 activity regulates S-phase entry of the cell cycle in Rat2 fibroblasts.J. Biol. Chem. 2006; 281: 9297-9306Abstract Full Text Full Text PDF PubMed Scopus (28) Google ScholarMouse GAPDHtgtgtccgtcgtggatctgacctgcttcaccaccttcttgaMouse lipin-1Agcctgctcgtgaatcctctcgatgcatcccgacagcgt23.Peterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis.J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (173) Google ScholarMouse lipin-1Bcagcctggtagattgccagagcagcctgtggcaattca23.Peterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis.J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (173) Google ScholarRat lipin-1tcactacccagtaccagggctgagtccaatcctttcccagRat lipin-1BagcagcctggtagattgtcataaggggctggagtctttcatRat and mouse lipin-2tagatgcagaccctgttcccctggtgctggcttcttttgtRat and mouse lipin-3aaagactggacacaccagggtgctggatatcactcaggcaMouse PGC-1αggcacgcagccctattcacgacacggagagttaaaggaagaMouse PPARαactacggagttcacgcatgtgttgtcgtacaccagcttcagc50.Yang J. Sambandam N. Han X. Gross R.W. Courtois M. Kovacs A. Febbraio M. Finck B.N. Kelly D.P. CD36 deficiency rescues lipotoxic cardiomyopathy.Circ. Res. 2007; 100: 1208-1217Crossref PubMed Scopus (192) Google ScholarRat PGC-1αcacaacgcggacagaactgaccgcagatttacggtgcattRat PPARαtggagtccacgcatgtgaagcgccagctttagccgaatagWhere not referenced, primers were designed by Primer3 or Primer Express version 2.0 software using default parameters. Open table in a new tab Measurement of PAP1 activityHepatocytes were lysed in 0.25 M sucrose containing 2 mM dithiothreitol, 0.15% Tween 20, and a protease inhibitor cocktail (Sigma). We developed the following assay specifically to give accurate measurements of relative PAP1 activity in tissue and cell homogenates, which are able to metabolize PA by several different routes (3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 30.Martin A. Gomez-Munoz A. Jamal Z. Brindley D.N. Characterization and assay of phosphatidate phosphatase.Methods Enzymol. 1991; 197: 553-563Crossref PubMed Scopus (49) Google Scholar). We chose to measure the formation of DAG from PA labeled with [3H]palmitate (3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 30.Martin A. Gomez-Munoz A. Jamal Z. Brindley D.N. Characterization and assay of phosphatidate phosphatase.Methods Enzymol. 1991; 197: 553-563Crossref PubMed Scopus (49) Google Scholar) in preference to the release of water-soluble 32P from [32P]PA. In liver or hepatocyte homogenates, glycerol-32P can be produced by phospholipase A action, and this product is further converted to inorganic 32P (31.Sturton R.G. Brindley D.N. Problems encountered in measuring the activity of phosphatidate phosphohydrolase.Biochem. J. 1978; 171: 263-266Crossref PubMed Scopus (39) Google Scholar). Thus, this latter assay with crude enzyme preparations has to be used with care to ensure that the measured 32P is only produced by PAP activity (32.Jasinska R. Zhang Q.X. Pilquil C. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. et al.Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters.Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (129) Google Scholar). We also mixed the PA in the molar ratio of 3:2 with nonradioactive PC, because this form of the substrate maximizes PAP1 activity relative to that of PAP2 (1.Jamal Z. Martin A. Gomez-Munoz A. Brindley D.N. Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol.J. Biol. Chem. 1991; 266: 2988-2996Abstract Full Text PDF PubMed Google Scholar). In our assays, ∼90% of the PAP activity is from PAP1 (3.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Had we used Triton X-100 to solubilize the PA, this would extract lipids, hydrophobic proteins, and amphiphilic proteins
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