Thematic Review Series: Glycerolipids. Phosphatidylcholine and choline homeostasis

Phosphatidylcholine (PC) is made in mammalian cells from choline via the CDP-choline pathway. Animals obtain choline primarily from the diet or from the conversion of phosphatidylethanolamine (PE) to PC followed by catabolism to choline. The main fate of choline is the synthesis of PC. In addition, choline is oxidized to betaine in kidney and liver and converted to acetylcholine in the nervous system. Mice that lack choline kinase (CK) α die during embryogenesis, whereas mice that lack CKβ unexpectedly develop muscular dystrophy. Mice that lack CTP:phosphocholine cytidylyltransferase (CT) α also die during early embryogenesis, whereas mice that lack CTβ exhibit gonadal dysfunction. The cytidylyltransferase β isoform also plays a role in the branching of axons of neurons. An alternative PC biosynthetic pathway in the liver uses phosphatidylethanolamine N-methyltransferase to catalyze the formation of PC from PE. Mice that lack the methyltransferase survive but die from steatohepatitis and liver failure when placed on a choline-deficient diet. Hence, choline is an essential nutrient. PC biosynthesis is required for normal very low density lipoprotein secretion from hepatocytes. Recent studies indicate that choline is recycled in the liver and redistributed from kidney, lung, and intestine to liver and brain when choline supply is attenuated. Phosphatidylcholine (PC) is made in mammalian cells from choline via the CDP-choline pathway. Animals obtain choline primarily from the diet or from the conversion of phosphatidylethanolamine (PE) to PC followed by catabolism to choline. The main fate of choline is the synthesis of PC. In addition, choline is oxidized to betaine in kidney and liver and converted to acetylcholine in the nervous system. Mice that lack choline kinase (CK) α die during embryogenesis, whereas mice that lack CKβ unexpectedly develop muscular dystrophy. Mice that lack CTP:phosphocholine cytidylyltransferase (CT) α also die during early embryogenesis, whereas mice that lack CTβ exhibit gonadal dysfunction. The cytidylyltransferase β isoform also plays a role in the branching of axons of neurons. An alternative PC biosynthetic pathway in the liver uses phosphatidylethanolamine N-methyltransferase to catalyze the formation of PC from PE. Mice that lack the methyltransferase survive but die from steatohepatitis and liver failure when placed on a choline-deficient diet. Hence, choline is an essential nutrient. PC biosynthesis is required for normal very low density lipoprotein secretion from hepatocytes. Recent studies indicate that choline is recycled in the liver and redistributed from kidney, lung, and intestine to liver and brain when choline supply is attenuated. S-adenosylhomocysteine S-adenosylmethionine choline-deficient choline kinase CDP-choline:1,2-diacylglycerol cholinephosphotransferase CTP:phosphocholine cytidylyltransferase endoplasmic reticulum phosphatidylcholine phosphatidylethanolamine phosphatidylethanolamine N-methyltransferase Phosphatidylcholine (PC) is an essential phospholipid in mammalian cells and tissues and is made in all nucleated cells via the choline pathway (Fig. 1). Choline was first identified in ox bile by Strecker (1.Strecker A. Uber eingige neue bestandtheile der schweinegalle.Ann. Chem. Pharmacie. 1862; 183: 964-965Google Scholar) in 1862. The Greek word for bile is chole. After a long interlude, in 1932, Best and Huntsman (2.Best C.H. Huntsman M.E. The effects of the components of lecithine upon deposition of fat in the liver.Journal of Physiology-London. 1932; 75: 405-412Crossref PubMed Scopus (86) Google Scholar) discovered the choline deficiency that results in fatty liver in rodents when insufficient choline is provided in the diet. In animals, choline can be acquired from the diet and via de novo biosynthesis: choline is produced through the methylation of phosphatidylethanolamine (PE) to PC catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) (Fig. 1) (3.Bremer J. Greenberg D.M. Methyltransfering enzyme system of microsomes in the biosynthesis of lecithin (phosphatidylcholine).Biochim. Biophys. Acta. 1961; 46: 205-216Crossref Scopus (358) Google Scholar, 4.Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, New York2008Google Scholar). Choline can then be generated from PC via the action of phospholipases. The PEMT/phospholipase reactions constitute the only known endogenous pathway for choline biosynthesis in animals, whereas in plants and some microbes, choline can be made from the methylation of phosphoethanolamine (5.Witola W.H. Mamoun C.Ben Choline induces transcriptional repression and proteasomal degradation of the malarial phosphoethanolamine methyltransferase.Eukaryot. Cell. 2007; 6: 1618-1624Crossref PubMed Scopus (24) Google Scholar, 6.Weretilnyk E.A. Smith D.D. Wilch G.A. Summers P.S. Enzymes of choline synthesis in spinach (response of phospho-base N-methyltransferase activities to light and salinity).Plant Physiol. 1995; 109: 1085-1091Crossref PubMed Scopus (42) Google Scholar, 7.Pessi G. Kociubinski G. Mamoun C.B. A pathway for phosphatidylcholine biosynthesis in Plasmodium falciparum involving phosphoethanolamine methylation.Proc. Natl. Acad. Sci. USA. 2004; 101: 6206-6211Crossref PubMed Scopus (136) Google Scholar). Thus, choline is made from the methylation of the ethanolamine moiety of phosphoethanolamine or PE. Both exogenous and endogenous choline is converted into PC, which accounts for ∼95% of the total choline pool in most animal tissues. The remaining 5% includes choline, phosphocholine, glycerophosphocholine, CDP-choline, and acetylcholine (8.Zeisel S.H. Blusztajn J.K. Choline and human nutrition.Annu. Rev. Nutr. 1994; 14: 269-296Crossref PubMed Scopus (614) Google Scholar, 9.Zeisel S.H. Mar M.H. Howe J.C. Holden J.M. Concentrations of choline-containing compounds and betaine in common foods.J. Nutr. 2003; 133: 1302-1307Crossref PubMed Scopus (575) Google Scholar). In animals, PEMT is quantitatively significant only in the liver (10.Vance D.E. Ridgway N.D. The methylation of phosphatidylethanolamine.Prog. Lipid Res. 1988; 27: 61-79Crossref PubMed Scopus (201) Google Scholar), and it accounts for ∼30% of hepatic PC biosynthesis in rodents (11.Sundler R. Akesson B. Regulation of phospholipid biosynthesis in isolated rat hepatocytes. Effect of different substrates.J. Biol. Chem. 1975; 250: 3359-3367Abstract Full Text PDF PubMed Google Scholar, 12.DeLong C.J. Shen Y-J. Thomas M.J. Cui Z. Molecular distinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylation pathway.J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 13.Reo N.V. Adinehzadeh M. Foy B.D. Kinetic analyses of liver phosphatidylcholine and phosphatidylethanolamine biosynthesis using (13)C NMR spectroscopy.Biochim. Biophys. Acta. 2002; 1580: 171-188Crossref PubMed Scopus (91) Google Scholar). The other 70% of hepatic PC is made via the choline pathway. Dietary choline is absorbed by the intestine in the form of lysophosphatidylcholine or choline, and uptake of the latter is mediated by choline transporters (14.Tso P. Fujimoto K. The absorption and transport of lipids by the small intestine.Brain Res. Bull. 1991; 27: 477-482Crossref PubMed Scopus (56) Google Scholar). Upon entry into the cell, choline is immediately phosphorylated to phosphocholine or oxidized to betaine in some cell types such as hepatocytes (15.Pritchard P.H. Vance D.E. Choline metabolism and phosphatidylcholine biosynthesis in cultured rat hepatocytes.Biochem. J. 1981; 196: 261-267Crossref PubMed Scopus (86) Google Scholar). The phosphorylation of choline is catalyzed by choline kinase (CK). Two genes encode CK. CKα1 and CKα2 are encoded by Chka on mouse chromosome 19 (16.Aoyama C. Liao H. Ishidate K. Structure and function of choline kinase isoforms in mammalian cells.Prog. Lipid Res. 2004; 43: 266-281Crossref PubMed Scopus (179) Google Scholar). CKβ is encoded by Chkb, which is located on chromosome 15. Mice that do not express either CKα isoform die at an early stage of embryogenesis, around day 3 (17.Wu G. Aoyama C. Young S.G. Vance D.E. Early embryonic lethality caused by disruption of the gene for choline kinase alpha, the first enzyme in phosphatidylcholine biosynthesis.J. Biol. Chem. 2008; 283: 1456-1462Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Mice that lack CKβ are viable but develop rostrocaudal muscular dystrophy and neonatal bone deformity (18.Sher R.B. Aoyama C. Huebsch K.A. Ji S. Kerner J. Yang Y. Frankel W.N. Hoppel C.L. Wood P.A. Vance D.E. et al.A rostrocaudal muscular dystrophy caused by a defect in choline kinase beta, the first enzyme in phosphatidylcholine biosynthesis.J. Biol. Chem. 2006; 281: 4938-4948Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The connection between PC synthesis and muscular dystrophy in Chkb−/− mice remains unclear. The lack of understanding of how the lack of CKβ could lead to muscular dystrophy reflects the almost complete lack of knowledge about PC biosynthesis and metabolism in muscle. CKα1 contains 435 amino acids, and the splice variant, CKα2, is identical except that an additional 18 amino acids are inserted after methionine-150 in the CKα1 sequence (16.Aoyama C. Liao H. Ishidate K. Structure and function of choline kinase isoforms in mammalian cells.Prog. Lipid Res. 2004; 43: 266-281Crossref PubMed Scopus (179) Google Scholar). CKβ contains 394 amino acids, and the sequence is 60% similar to that of CKα1. The CKs can exist as either homodimers or heterodimers (16.Aoyama C. Liao H. Ishidate K. Structure and function of choline kinase isoforms in mammalian cells.Prog. Lipid Res. 2004; 43: 266-281Crossref PubMed Scopus (179) Google Scholar). Although CK catalyzes the initial and committed step in the conversion of choline to PC, CK is usually not considered to be rate-limiting or to regulate the rate of PC biosynthesis (19.Vance D.E. Phospholipid biosynthesis in eukaryotes.in: Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science, Amsterdam, The Netherlands2002: 205-232Crossref Google Scholar). Rather, the second reaction in the pathway, catalyzed by CTP:phosphocholine cytidylyltransferase (CT), is usually rate-limiting (19.Vance D.E. Phospholipid biosynthesis in eukaryotes.in: Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science, Amsterdam, The Netherlands2002: 205-232Crossref Google Scholar, 20.Vance D.E. Choy P.C. How is phosphatidylcholine biosynthesis regulated?.Trends Biochem. Sci. 1979; 4: 145-148Abstract Full Text PDF Scopus (110) Google Scholar). CT in the mouse is also encoded by two genes (21.Tang W. Keesler G.A. Tabas I. The structure of the gene for murine CTP:phosphocholine cytidylyltransferase, Ctpct.J. Biol. Chem. 1997; 272: 13146-13151Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 22.Lykidis A. Murti K.G. Jackowski S. Cloning and characterization of a second human CTP:phosphocholine cytidylyltransferase.J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 23.Karim M. Jackson P. Jackowski S. Gene structure, expression and identification of a new CTP:phosphocholine cytidylyltransferase beta isoform.Biochim. Biophys. Acta. 2003; 1633: 1-12Crossref PubMed Scopus (78) Google Scholar). Pcyt1a encodes CTα2 and the splice variant CTα3 and is located on chromosome 16 (24.Lykidis A. Baburina I. Jackowski S. Distribution of CTP:phosphocholine cytidylyltransferase (CCT) isoforms: identification of a new CCTβ splice variant.J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Pcyt1b encodes CTβ2 and CTβ3 and is located on the X chromosome (22.Lykidis A. Murti K.G. Jackowski S. Cloning and characterization of a second human CTP:phosphocholine cytidylyltransferase.J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Mice that do not express CTα die during the early stages of embryogenesis (25.Wang L. Magdaleno S. Tabas I. Jackowski S. Early embryonic lethality in mice with targeted deletion of the CTP:phosphocholine cytidylyltransferase alpha gene (Pcyt1a).Mol. Cell. Biol. 2005; 25: 3357-3363Crossref PubMed Scopus (88) Google Scholar). In contrast, mice that do not express CTβ2 survive but show gonadal dysfunction and reduced fertility (26.Jackowski S. Rehg J.E. Zhang Y.M. Wang J. Miller K. Jackson P. Karim M.A. Disruption of CCTbeta2 expression leads to gonadal dysfunction.Mol. Cell. Biol. 2004; 24: 4720-4733Crossref PubMed Scopus (45) Google Scholar). This phenotype reflects the relatively high expression of CTβ2 mRNA in testes and ovary (26.Jackowski S. Rehg J.E. Zhang Y.M. Wang J. Miller K. Jackson P. Karim M.A. Disruption of CCTbeta2 expression leads to gonadal dysfunction.Mol. Cell. Biol. 2004; 24: 4720-4733Crossref PubMed Scopus (45) Google Scholar). The mRNA for CTβ2 is not highly expressed in most tissues but is found in brain, as will be discussed below, and in lung. The regulation of CT activity has been studied intensely. A major mode of regulation involves the reversible movement of CT on and off the endoplasmic reticulum (ER) and/or the nuclear membrane (27.Kent C. Stimulation of phospholipid metabolism in embryonic muscle cells treated with phospholipase C.Proc. Natl. Acad. Sci. USA. 1979; 76: 4474-4478Crossref PubMed Scopus (28) Google Scholar, 28.Vance D.E. Pelech S.L. Enzyme translocation in the regulation of phosphatidylcholine biosynthesis.Trends Biochem. Sci. 1984; 9: 17-20Abstract Full Text PDF Scopus (101) Google Scholar, 29.Cornell R.B. Northwood I.C. Regulation of CTP:phosphocholine cytidylyltransferase by amphitropism and relocalization.Trends Biochem. Sci. 2000; 25: 441-447Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 30.Lykidis A. Jackson P. Jackowski S. Lipid activation of CTP:phosphocholine cytidylyltransferase alpha: characterization and identification of a second activation domain.Biochemistry. 2001; 40: 494-503Crossref PubMed Scopus (43) Google Scholar, 31.Xie M. Smith J.L. Ding Z. Zhang D. Cornell R.B. Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase.J. Biol. Chem. 2004; 279: 28817-28825Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 32.Jackowski S. Fagone P. CTP:phosphocholine cytidylyltransferase: paving the way from gene to membrane.J. Biol. Chem. 2005; 280: 853-856Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 33.Lagace T.A. Ridgway N.D. The rate-limiting enzyme in phosphatidylcholine synthesis regulates proliferation of the nucleoplasmic reticulum.Mol. Biol. Cell. 2005; 16: 1120-1130Crossref PubMed Scopus (69) Google Scholar). When bound to either of these membranes, CT is in its active form. Otherwise, CT is in a soluble form that is inactive. The membrane binding domain of CT is a long amphipathic α helix that enhances the binding of CT to membranes that are deficient in PC (31.Xie M. Smith J.L. Ding Z. Zhang D. Cornell R.B. Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase.J. Biol. Chem. 2004; 279: 28817-28825Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The binding appears to activate CT by relieving an inhibitory restraint in the catalytic domain of the enzyme. Curiously, the soluble form of CTα has been localized to the nucleus in many, but not all, cell types, whereas soluble CTβ, which lacks a nuclear localization signal, is cytoplasmic (24.Lykidis A. Baburina I. Jackowski S. Distribution of CTP:phosphocholine cytidylyltransferase (CCT) isoforms: identification of a new CCTβ splice variant.J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 34.Wang Y. Sweizer T.D. Weinhold P.A. Kent C. Nuclear localization of soluble CTP:phosphocholine cytidylyltransferase.J. Biol. Chem. 1993; 268: 5899-5904Abstract Full Text PDF PubMed Google Scholar, 35.Houweling M. Cui Z. Anfuso C.D. Bussiere M. Chen M.H. Vance D.E. CTP:phosphocholine cytidylyltransferase in both a nuclear and cytoplasmic protein in primary hepatocytes.Eur. J. Cell Biol. 1996; 69: 55-63PubMed Google Scholar). The transcriptional regulation of CTα expression has been investigated in detail and recently reviewed (36.Sugimoto, H., C. Banchio, and D. E. Vance. 2008. Transcriptional regulation of phosphatidylcholine biosynthesis. Prog. Lipid Res. In press.Google Scholar). CTα expression is largely governed by Sp1, Sp3, Rb, TEF4, Ets-1, and E2F, which enhance the expression of CT, and by Net, a factor that represses CTα expression. Key transcription factors involved in cholesterol or fatty acid metabolism (sterol-regulatory element binding proteins, liver X receptors, peroxisome proliferator-activated receptors) do not appear to play a major role in the transcriptional regulation of CTα. Rather than being linked to cholesterol or energy metabolism, the regulation of CTα at the level of gene expression is linked to the cell cycle, cell growth, and differentiation (36.Sugimoto, H., C. Banchio, and D. E. Vance. 2008. Transcriptional regulation of phosphatidylcholine biosynthesis. Prog. Lipid Res. In press.Google Scholar). The final reaction in the choline pathway for PC biosynthesis is catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT). This enzyme has never been purified from any source, probably because it is an intrinsic membrane protein found primarily on the ER (19.Vance D.E. Phospholipid biosynthesis in eukaryotes.in: Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science, Amsterdam, The Netherlands2002: 205-232Crossref Google Scholar). With new molecular tools, such as the use of a tagged protein in an expression system, CPT purification can now be undertaken with a higher probability of success. Also, the gene(s) in mice that encodes CPT has not been characterized. However, two human CPT cDNAs have been cloned and expressed (37.Henneberry A.L. McMaster C.R. Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidyethanolamine.Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (96) Google Scholar, 38.Henneberry A.L. Wistow G. McMaster C.R. Cloning, genomic organization, and characterization of a human cholinephosphotransferase.J. Biol. Chem. 2000; 275: 29808-29815Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Most studies indicate that there is an excess of CPT activity in cells; hence, the amount of enzyme does not limit the rate of PC biosynthesis. However, in vivo, it seems clear that the CPT reaction is governed by the supply of both CDP-choline and diacylglycerol (19.Vance D.E. Phospholipid biosynthesis in eukaryotes.in: Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science, Amsterdam, The Netherlands2002: 205-232Crossref Google Scholar). The second pathway for PC biosynthesis, catalyzed by the ∼20 kDa protein PEMT, is quantitatively significant only in the liver (10.Vance D.E. Ridgway N.D. The methylation of phosphatidylethanolamine.Prog. Lipid Res. 1988; 27: 61-79Crossref PubMed Scopus (201) Google Scholar). PEMT is localized to the ER and mitochondria-associated membranes (a subfraction of the ER) (39.Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. Cloning and expression of a novel phosphatidylethanolamine N-methyltransferase.J. Biol. Chem. 1993; 268: 16655-16663Abstract Full Text PDF PubMed Google Scholar). PEMT spans the membrane with four transmembrane sequences (40.Shields D.J. Lehner R. Agellon L.B. Vance D.E. Membrane topography of human phosphatidylethanolamine N-methyltransferase.J. Biol. Chem. 2003; 278: 2956-2962Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The methyl donor for the methylation reactions is S-adenosylmethionine (AdoMet), which binds to several residues of PEMT exposed on the cytosolic surface of the ER (41.Shields D.J. Altarejos J.Y. Wang X. Agellon L.B. Vance D.E. Molecular dissection of the AdoMet binding site of phosphatidylethanolamine N-methyltransferase.J. Biol. Chem. 2003; 278: 35826-35836Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The PEMT gene resides on chromosome 11 in the mouse and spans 25 kb (42.Walkey C.J. Cui Z. Agellon L.B. Vance D.E. Characterization of the murine phosphatidylethanolamine N-methyltransferase-2 gene.J. Lipid Res. 1996; 37: 2341-2350Abstract Full Text PDF PubMed Google Scholar). Mice that lack PEMT (Pemt−/−) exhibit no obvious phenotype when fed a chow diet (43.Walkey C.J. Donohue L.R. Bronson R. Agellon L.B. Vance D.E. Disruption of the murine gene encoding phosphatidylethanolamine N-methyltransferase.Proc. Natl. Acad. Sci. USA. 1997; 94: 12880-12885Crossref PubMed Scopus (139) Google Scholar). However, when fed a choline-deficient (CD) diet to restrict the availability of choline for PC synthesis, the mice rapidly develop steatosis, steatohepatitis, and die from liver failure after 3 days (44.Walkey C.J. Yu L. Agellon L.B. Vance D.E. Biochemical and evolutionary significance of phospholipid methylation.J. Biol. Chem. 1998; 273: 27043-27046Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). The liver failure coincides with a 50% decrease in hepatic PC content; thus, it was speculated that this was the reason for the steatohepatitis and liver failure (44.Walkey C.J. Yu L. Agellon L.B. Vance D.E. Biochemical and evolutionary significance of phospholipid methylation.J. Biol. Chem. 1998; 273: 27043-27046Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Because other tissues did not show this dramatic change in PC levels, we hypothesized that the liver failure/decrease in hepatic PC levels might be attributable to the robust secretion of PC into bile [23 mg/day for a 20 g mouse (45.Kuipers F. Oude Elferink R.P. Verkade H.J. Groen A.K. Mechanisms and (patho)physiological significance of biliary cholesterol secretion.Subcell. Biochem. 1997; 28: 295-318Crossref PubMed Scopus (26) Google Scholar), which is equivalent to the entire pool of PC in the liver]. This hypothesis was tested by breeding Pemt−/− mice with mice that lacked MDR2/ABCB4, the protein that transports PC into bile. Mdr2−/− mice secrete no PC and little cholesterol into bile but continue to secrete bile acids (46.Smit J.J. Schinkel A.H. Oude Elferink R.P. Groen A.K. Wagenaar E. Deemter L.van Mol C.A. Ottenhoff R. van der Lugt N.M. van Roon M.A. et al.Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.Cell. 1993; 75: 451-462Abstract Full Text PDF PubMed Scopus (1336) Google Scholar). Remarkably, when fed a CD diet, the Pemt−/−/Mdr2−/− mice survived for >90 days, whereas Pemt−/−/Mdr2+/+ mice experienced end-stage liver failure after 3 days. Unexpectedly, the Pemt−/−/Mdr2−/− mice developed steatosis when fed the CD diet, and the PC levels also decreased by ∼50%. Thus, the rapid liver failure in Pemt−/− mice could not be attributed to the decrease in PC or the accumulation of triacylglycerol (47.Li Z. Agellon L.B. Allen T.M. Umeda M. Jewell L. Mason A. Vance D.E. The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis.Cell Metab. 2006; 3: 321-331Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). The question then became: what difference between Pemt−/− mice and Pemt−/−/Mdr2−/− mice allows the double knockout to survive for 90 days when fed a CD diet, whereas the Pemt−/− mice die after 3 days? One difference that became apparent was that the concentration of hepatic PE decreased in the double knockout mice but not in the Pemt−/− mice (47.Li Z. Agellon L.B. Allen T.M. Umeda M. Jewell L. Mason A. Vance D.E. The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis.Cell Metab. 2006; 3: 321-331Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). Thus, the ratio of PC to PE decreased from ∼1.8 to ∼0.8 in the livers of Pemt−/− mice fed the CD diet for 3 days, whereas the ratio in Pemt−/−/Mdr2−/− mice decreased to only ∼1.4 after being fed the CD diet for 21 days. No changes were observed in hepatic sphingomyelin or cholesterol content in either strain of mouse. PC is a cylindrically shaped molecule, whereas PE usually has an inverted cone shape (48.Dowhan W. Bogdanov M. Functional roles of lipids in membranes.in: Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science, Amsterdam, The Netherlands2002: 1-35Crossref Google Scholar). Because the amount of PC decreased on the plasma membrane of livers from Pemt−/− mice fed a CD diet (47.Li Z. Agellon L.B. Allen T.M. Umeda M. Jewell L. Mason A. Vance D.E. The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis.Cell Metab. 2006; 3: 321-331Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar), we considered that PC might be replaced by PE. In such a circumstance, the packing of the bilayer might become permeable, leading to steatohepatitis and liver failure. Subsequent studies showed that, indeed, there was increased PE on the hepatic cell surface of Pemt−/− mice fed a CD diet as well as increased plasma alanine aminotransferase (normally only found in the liver), indicating membrane permeability (47.Li Z. Agellon L.B. Allen T.M. Umeda M. Jewell L. Mason A. Vance D.E. The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis.Cell Metab. 2006; 3: 321-331Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). Moreover, the leakage of alanine aminotransferases was attenuated by inhibiting the biosynthesis of PE, thereby increasing the ratio of PC to PE in the livers or hepatocytes from Pemt−/− mice fed the CD diet (47.Li Z. Agellon L.B. Allen T.M. Umeda M. Jewell L. Mason A. Vance D.E. The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis.Cell Metab. 2006; 3: 321-331Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). These results provided strong support for the hypothesis that Pemt−/− mice fed the CD diet develop steatohepatitis as a result of increased permeability of the hepatic plasma membrane caused by a decreased ratio of PC to PE. In a preliminary study on liver biopsies from humans with nonalcoholic steatohepatitis, the PC-to-PE ratio was reduced markedly (47.Li Z. Agellon L.B. Allen T.M. Umeda M. Jewell L. Mason A. Vance D.E. The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis.Cell Metab. 2006; 3: 321-331Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). Thus, when the dietary supply of choline limits PC biosynthesis, PEMT plays an important role by providing PC/choline for normal liver function. PC biosynthesis is required for the normal secretion of VLDL by hepatocytes. Elimination of choline and methionine (two precursors of PC synthesis) from hepatocyte culture medium reduced VLDL secretion (49.Yao Z. Vance D.E. The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes.J. Biol. Chem. 1988; 263: 2998-3004Abstract Full Text PDF PubMed Google Scholar). The requirement for choline was specific, and choline could not be replaced by dimethylethanolamine, monomethylethanolamine, or ethanolamine (50.Yao Z. Vance D.E. Head group specificity in the requirement of phosphatidylcholine biosynthesis for very low density lipoprotein secretion from cultured hepatocytes.J. Biol. Chem. 1989; 264: 11373-11380Abstract Full Text PDF PubMed Google Scholar). In rodents, a CD diet markedly reduced plasma levels of apolipoprotein B, a major component of VLDL (51.Yao Z. Vance D.E. Reduction in VLDL, but not HDL in plasma of rats deficient in choline.Biochem. Cell Biol. 1989; 68: 552-558Crossref Scopus (99) Google Scholar). If, however, only choline were removed from hepatocyte culture medium, VLDL secretion was not impaired because PC synthesis was not reduced (52.Kulinski A. Vance D.E. Vance J.E. A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion.J. Biol. Chem. 2004; 279: 23916-23924Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Consequently, these studies did not establish whether or not PC synthesis from the choline pathway is required for VLDL secretion. In subsequent studies, the choline pathway for PC biosynthesis was shown to be required for normal VLDL secretion in mice that lacked CTα specifically in hepatocytes (53.Jacobs R.L. Devlin C. Tabas I. Vance D.E. Targeted deletion of hepatic CTP:phosphocholine cytidylyltransferase A in mice decreases plasma high density and very low density lipoproteins.J. Biol. Chem. 2004; 279: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). In CTα-deficient hepatocytes, there was an almost 2-fold increase in PEMT activity. However, this increased capacity for the generation of PC did not substitute for the attenuation of the CDP-choline pathway in CTα-deficient hepatocytes. The generation of Pemt−/− mice allowed the role of PEMT in VLDL secretion to be examined. When these mice were fed a chow diet, the concentration of hepatic PC was the same as in Pemt+/+ mice. Unexpectedly, the secretion of triacylglycerol from Pemt−/− hep