Stearoyl-CoA Desaturase 1 Deficiency Increases CTP:Choline Cytidylyltransferase Translocation into the Membrane and Enhances Phosphatidylcholine Synthesis in Liver*

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in monounsaturated fatty acid synthesis. Previously, we showed that Scd1 deficiency reduces liver triglyceride accumulation and considerably decreases synthesis of very low density lipoprotein and its secretion in both lean and obese mice. In the present study, we found that Scd1 deficiency significantly modulates hepatic glycerophospholipid profile. The content of phosphatidylcholine (PC) was increased by 40% and the activities of CTP:choline cytidylyltransferase (CCT), the rate-limiting enzyme in de novo PC synthesis, and choline phosphotransferase were increased by 64 and 53%, respectively, in liver of Scd1-/- mice. In contrast, the protein level of phosphatidylethanolamine N-methyltransferase, an enzyme involved in PC synthesis via methylation of phosphatidylethanolamine, was decreased by 80% in the liver of Scd1-/- mice. Membrane translocation of CCT is required for its activation. Immunoblot analyses demonstrated that twice as much CCTα was associated with plasma membrane in livers of Scd1-/- compared with wild type mice, suggesting that Scd1 mutation leads to an increase in CCT membrane affinity. The incorporation of [3H]glycerol into PC was increased by 2.5-fold in Scd1-/- primary hepatocytes compared with those of wild type mice. Furthermore, mitochondrial glycerol-3-phosphate acyltransferase activity was reduced by 42% in liver of Scd1-/- mice; however, the activities of microsomal glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase, and ethanolamine phosphotransferase were not affected by Scd1 mutation. Our study revealed that SCD1 deficiency specifically increases CCT activity by promoting its translocation into membrane and enhances PC biosynthesis in liver.

The regulation of triacylglycerol (TAG) 1 and phospholipid (PL) synthesis plays a critical role in disorders such as obesity, diabetes, and atherosclerosis. TAG is the major energy storage form, as well as a major component of secreted chylomicra and lipoproteins, mainly very low density lipoprotein (VLDL). PLs, the primary lipid component of cellular membranes, are essential for the synthesis and secretion of bile and lipoproteins, as well as providing a reservoir of signaling molecules like lysophosphatidic acid, phosphatidic acid, diacylglycerol, and the arachidonate-and eicosapentanoate-derived eicosanoids (1).
Stearoyl-CoA desaturase 1 (SCD1), an enzyme that catalyzes the conversion of stearoyl-CoA to oleoyl-CoA (or palmitoyl-CoA to palmitoleoyl-CoA), has recently been shown to be an important control point of lipogenesis, leading many to believe that SCD inhibition might play a protective role against obesity and the metabolic syndrome (reviewed in Refs. 2,3). The significance of SCD in TAG synthesis has been confirmed by studies in mouse models that have a disruption in the Scd1 gene. Mice lacking Scd1 have a 50% decrease in epididymal fat pad weights (4), are deficient in hepatic TAG and cholesteryl esters, and have reduced plasma VLDL secretion (5,6). Scd1-deficient animals are resistant to diet-induced weight gain as well as high fat and high carbohydrate diet-induced liver steatosis (4,7) and have increased insulin sensitivity in muscle and brown adipose tissue (8,9). Furthermore, Scd1 has been found to be a major target gene of leptin, and the antisteatotic effects of this hormone in the liver were shown to be, to a large extent, mediated by repression of Scd1 (10). Scd1 deficiency also attenuates liver steatosis in peroxisome proliferator-activated receptor-␣-deficient mice (11). In liver and skeletal muscle, Scd1 deficiency was shown to increase the rate of ␤-oxidation through activation of the AMP-activated protein kinase pathway (12,13) and by up-regulating genes of fatty acid oxidation, e.g. carnitine palmitoyltransferase 1, acyl-CoA oxidase, and very long chain acyl-CoA dehydrogenase (4). SCD1 deficiency also acts to suppress lipid synthesis by reducing the expression of hepatic lipogenic genes, including sterol regulatory elementbinding protein-1c, fatty acid synthase, acetyl-CoA carboxylase, and glycerol-3-phosphate acyltransferase (GPAT) (4,5,7).
Phosphatidylcholine (PC) is the primary PL of eukaryotic cellular membranes and has a crucial role in structural maintenance of the lipid bilayer. In mammals, PC is also the predominant phospholipid in plasma lipoproteins and bile and plays a critical role as a second messenger in signal transduction (14). PC is the major component of intracellular transport vesicles, and the synthesis of PC is intimately involved in the regulation of vesicle transport (15). Ongoing PC synthesis is also required for global transcriptional regulation of lipid biosynthesis (reviewed in Ref. 15). In liver, PC is synthesized through two routes, the CDP-choline pathway (the Kennedy pathway) and the methylation of phosphatidylethanolamine (PE) catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). Studies on PemtϪ/Ϫ mice have suggested that PEMT is involved in synthesizing PC that is exported into bile (16) and plays a significant role in synthesis and secretion of VLDL (17,18).
The CDP-choline pathway is a three-step process: phosphorylation of intracellular choline and conversion of phosphocholine to CDP-choline, which reacts with 1,2 diacylglycerol (DAG) to form PC and accounts for ϳ70% of hepatic PC biosynthesis (19). The rate-limiting enzyme of this pathway is CTP:choline cytidylyltransferase (CCT), which catalyzes the conversion of phosphocholine to CDP-choline (19,20). Although hepatic cells express two isoforms of CCT, ␣ and ␤, CCT␣ is the predominant isoform in liver (20,21). One well characterized mode of regulation of CCT activity is through translocation of the protein from an inactive, soluble form to an active, membrane-bound form (20,22). The insertion of CCT into membranes has been shown to increase by the properties of membranes, including loose packing of lipids in the membrane (23), curvature strain (24), and changes in the fatty acid composition of PL (25,26). Because oleic acid is the most abundant monounsaturated fatty acid in mammalian PL, we hypothesized that membrane perturbation that might occur as a consequence of Scd1 deficiency would affect CCT translocation into membranes, and thus SCD might play an important role in regulating PC biosynthesis.
To understand the role of SCD in PL synthesis, we investigated the major steps of glycerophospholipid biosynthesis in liver of Scd1Ϫ/Ϫ mice. We analyzed the activities of key enzymes involved in PL biosynthesis in liver and measured the incorporation of [ 3 H]glycerol into different glycerolipid classes in Scd1Ϫ/Ϫ primary hepatocytes. Our study revealed that Scd1 deficiency specifically increases CCT activity by promoting its translocation into the membrane and thus enhances PC biosynthesis in liver.

EXPERIMENTAL PROCEDURES
Animals-The generation of Scd1Ϫ/Ϫ mice has been previously described (5). Twelve-week-old purebred homozygous (Scd1Ϫ/Ϫ) and wild type male mice on SV-129 background were used. Mice were housed in a pathogen-free barrier facility operating on a 12-h light/12-h dark cycle and were fed a normal nonpurified diet (5008 test diet; PMI Nutrition International Inc., Richmond, IN). The breeding of these animals was in accordance with the protocols approved by the Animal Care Research Committee of the University of Wisconsin-Madison. Mice were sacrificed and livers were isolated, frozen in liquid nitrogen, and stored at Ϫ80°C. Measurement of Lipids-Liver lipids were extracted by the method of Bligh and Dyer (27) and measured as described (28,29). Briefly, the lipids were separated into DAG, TAG, free fatty acids, and PL by thin layer chromatography on silica gel-60 plates (Merck) in heptane/isopropyl ether/glacial acetic acid (60/40/4, v/v/v) with authentic standards. Additionally, PL classes were separated in chloroform/methanol/glacial acetic acid/water (50/37.5/3.5/2, v/v/v/v). The bands corresponding to standards were scraped off the plate and transferred to screw cap glass tubes containing methylpentadecanoic acid as an internal standard. Fatty acids were then transmethylated in the presence of 14% boron trifluoride in methanol. The resulting methyl esters were extracted with hexane and analyzed by gas-liquid chromatography. Total contents were calculated from individual fatty acid content in each fraction.
Western Blot Analysis-For the measurement of total levels of PEMT and CCT␣, liver samples were homogenized in ice-cold 50 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, protease inhibitors, and 10% glycerol and centrifuged at 3,000 ϫ g for 10 min. For measurement of membrane-associated CCT␣, total membranes were isolated by centrifugation at 100,000 ϫ g for 1 h as described (17). Proteins were separated on a 9% SDS-PAGE gel, transferred and immobilized on nitrocellulose membrane, and probed with antibodies against PEMT and CCT␣. The proteins were visualized using ECL (Amersham Biosciences) and quantified by densitometry. Glyceraldehyde-3-phosphate dehydrogenase protein level was used as a loading control.
Glycerol-3-phosphate Acyltransferase Activity-GPAT activity was assayed with 300 M [ 3 H]glycerol-3-phosphate and 80 M palmitoyl-CoA according to Muoio et al. (31) in samples containing both microsomes and mitochondria. Microsomal GPAT was estimated by subtracting the N-ethylmaleimide-resistant activity (mitochondrial GPAT) from the total.
Diacylglycerol Acyltransferase (DGAT) Activity and Gene Expression-DGAT activity was determined as described (33). The incorporation of [1-14 C]oleoyl-CoA into triglyceride was measured by adding exogenous diacylglycerol substrate in acetone (4 mM solution). Partially purified membranes (100 g of protein) were used as the enzyme source. Reactions were carried out for 5 min at 37°C, and the products were analyzed as described (34). Total RNA was isolated from liver using TRIzol reagent. Dgat1 and Dgat2 gene expression was analyzed by Northern blotting. 20 g of total RNA were fractionated on 1% agarose-2.2 M formaldehyde gels and transferred to nylon membranes. After UV cross-linking, the membrane was hybridized with cDNA probes labeled with [ 32 P]dCTP by a random primer labeling kit (Promega, Madison, WI). After washing, the membranes were exposed to x-ray film at Ϫ80°C, and signals were quantified by densitometry. The probes for Dgat1 and Dgat2 were obtained from Dr. Robert Farese, Jr. (University of California, San Francisco). pAL15 mRNA was used as an internal control (5).
Protein Content-The protein concentration was determined with Bio-Rad protein assay using bovine serum albumin as a standard.
Statistical Analysis-Results were analyzed using the Student's t test. A difference of p Ͻ0.05 was considered significant. Values are presented as means Ϯ S.D. (n ϭ 6 mice/group).

RESULTS
PC and PE Levels Are Increased in the Liver of Scd1Ϫ/Ϫ Mice-We have previously shown that SCD1 deficiency reduces liver TAG content in both lean (7) and obese (10) mice. Consistently in the present study we found a 48% reduction in TAG content in the liver of Scd1Ϫ/Ϫ compared with wild type mice (Fig. 1A). The total content of liver free fatty acids was decreased by 34% in Scd1Ϫ/Ϫ mice, whereas DAG level was not significantly different between Scd1Ϫ/Ϫ and wild type mice (Fig. 1A). The contents of PC and PE were increased by 40 and 23%, respectively, whereas the contents of phosphatidylinositol (PI) and phosphatidylserine (PS) were decreased by 16 (p ϭ 0.071) and 27%, respectively, in the liver of Scd1Ϫ/Ϫ mice compared with wild type mice (Fig. 1B). Because the increases in PC and PE levels were greater than the decreases in PI and PS, the total content of PLs tends to be higher in the liver of Scd1Ϫ/Ϫ mice (p ϭ 0.27) (Fig. 1A).
SCD1 Deficiency Alters Phospholipid Fatty Acid Composition in the Liver-The FA composition of phospholipids is tightly regulated and believed to stabilize membrane fluidity and functions of intrinsic membrane proteins, including receptors and transporters (35). To determine changes in fatty acid composition of hepatic glycerophospholipids due to Scd1 deficiency, PC, PE, PS, and PI were separated and their fatty acid contents were determined. The relative amounts of palmitoleic (16:1) and oleic (18:1) acids were reduced by 67 and 85% in PC, by 50 and 68% in PE, by 27 and 57% in PS, and by 64 and 65% in PI in the liver of Scd1Ϫ/Ϫ compared with wild type mice. The relative amounts of their saturated precursors, palmitic (16:0) and stearic (18:0) acids, were unchanged or slightly increased due to SCD1 deficiency in most of the PL fractions (Table I). Only in the PS fraction, the 18:0 was increased by 83% and 16:0 was reduced by 20% in liver of Scd1Ϫ/Ϫ as compared with wild type mice (Table I). SCD1 deficiency also affected the relative amounts of polyunsaturated fatty acids in hepatic PLs. Linoleic (18:2) and arachidonic (20:4) acids were increased by 24 and 32%, respectively, in PC and by 17 and 36%, respectively, in PE in the liver of Scd1Ϫ/Ϫ compared with wild type mice (Table I). In the PI fraction, SCD1 deficiency increased 20:4 by 31%, whereas the relative amounts of docosahexanoic acid (22:6) and 18:2 were reduced by 50 and 31%, respectively (Table I). In the PS fraction, 22:6 was increased by 20%, whereas 18:2 was reduced by 23% in liver of Scd1Ϫ/Ϫ mice compared with the controls (Table I). Differences in other major fatty acid species were not significant (data not shown).

The Incorporation of [ 3 H]Glycerol into PC and PE Is
Increased in Scd1Ϫ/Ϫ Mice-To measure the rate of lipid synthesis, we analyzed the incorporation of [ 3 H]glycerol into the various glycerolipids in primary hepatocytes derived from livers of Scd1Ϫ/Ϫ and wild type mice. The incorporation of [ 3 H]glycerol into TAG was decreased by 56%, whereas its incorporation into total PLs was increased by 78% in Scd1Ϫ/Ϫ compared with wild type hepatocytes ( Fig. 2A). [ 3 H]Glycerol incorporation into PC and PE was 2.5-and 1.4-fold higher, respectively, whereas incorporation into PI and PS was reduced by 21 and 38%, respectively, in Scd1Ϫ/Ϫ hepatocytes (Fig. 2B). [ 3 H]Glycerol incorporation into hepatic DAG was not significantly different between wild type and Scd1Ϫ/Ϫ mice ( Fig. 2A).
Mitochondrial GPAT Activity Is Decreased in the Liver of Scd1Ϫ/Ϫ Mice-The initial and committed step in the de novo synthesis of all cellular glycerolipids is the acylation of snglycerol-3-phosphate to form 1-acyl-sn-glycerol-3-phosphate by GPAT. Mammalian tissues contain two GPAT isoforms, one in the outer mitochondrial membrane (mitochondrial GPAT) and the other in the endoplasmic reticulum (microsomal GPAT) (1, 36). Previously, we have shown that GPAT mRNA level is

TABLE I Relative fatty acid composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) in the liver of Scd1Ϫ/Ϫ and wild type mice
The data are expressed as average (n ϭ 6). Bold value denotes statistical significance of p Ͻ0.05 between Scd1Ϫ/Ϫ and wild type mice. significantly reduced in the liver of Scd1Ϫ/Ϫ mice (4). In the present study we found that mitochondrial Gpat activity is 42% lower in the liver of Scd1Ϫ/Ϫ, whereas the activity of microsomal GPAT does not differ between wild type and Scd1Ϫ/Ϫ mice (Fig. 3).

CDP-Choline Pathway of PC Synthesis Is Up-regulated, whereas PEMT Pathway Is Down-regulated in the Liver of Scd1Ϫ/Ϫ Mice-We hypothesized that the increase in PC content and higher incorporation of [ 3 H]glycerol into PC in
Scd1Ϫ/Ϫ liver results from an increased rate of PC biosynthesis. There are two pathways of PC biosynthesis in liver, the CDP-choline pathway and PE methylation by PEMT (19). CCT, the rate-limiting enzyme in the CDP-choline pathway, undergoes post-transcriptional regulation by its translocation from an inactive, soluble form to an active, membrane-bound form (20). To determine whether disruption of the Scd1 gene resulted in increased CCT protein level and/or enhanced its membrane association, immunoblot analysis was performed on liver homogenates and isolated membranes. CCT␣, the predominant isoform of CCT in liver (21), was analyzed. The amount of membrane-associated CCT␣ was increased by 1.6-fold, whereas the total protein level of CCT␣ measured in homogenates was reduced by 20% in the liver of Scd1Ϫ/Ϫ compared with wild type mice (Fig. 4A). Glyceraldehyde-3-phosphate dehydrogenase used as a control was not different in homogenates and membranes. The analysis of CCT subcellular distribution revealed that the liver from Scd1Ϫ/Ϫ mice had a Ͼ2-fold increase in the levels of CCT associated with total membranes than did liver of wild type mice (Fig. 4B). Consequently, the activity of CCT was 62% higher in liver of Scd1Ϫ/Ϫ compared with wild type mice (Fig. 4C).
The activity of CPT, an enzyme catalyzing the final step in PC synthesis via the CDP-choline pathway, was increased by 49% in liver of Scd1Ϫ/Ϫ mice (Fig. 5A). Interestingly, the protein level of PEMT, an enzyme involved in PC synthesis via the PE methylation pathway, was reduced by 80% in liver of Scd1Ϫ/Ϫ compared with wild type mice (Fig. 5B). The activity of ethanolamine phosphotransferase, the enzyme involved in de novo synthesis of PE, was not affected by SCD1 deficiency (Fig. 5C). Thus, the increase in PE accumulation in the liver of Scd1Ϫ/Ϫ mice (Fig. 1B) appears to be because of its reduced catabolism rather than increased biosynthesis. DAG, a precursor to PC and PE, is also the substrate for synthesis of TAG, and DAG availability acts as a branchpoint affecting the proportional synthesis of these three lipid fractions (1,36). Therefore, we also analyzed the activity and gene expression of Dgat, an enzyme catalyzing the final, committed step for TAG synthesis. Dgat mRNA level and its activity were not affected by Scd1 deficiency (Fig. 6). DISCUSSION SCD, the rate-limiting enzyme in monounsaturated fatty acid synthesis, has recently been shown to be the critical control point regulating hepatic lipogenesis. Scd1Ϫ/Ϫ mice accu-  3. The activities of microsomal (mcGPAT) and mitochondrial (mtGPAT) glycerol-3-phosphate acyltransferase in the liver of Scd1؊/؊ and wild type mice. GPAT activity was measured using [ 3 H]glycerol-3-phosphate as a substrate, and microsomal GPAT was estimated by subtracting the N-ethylmaleimide-resistant activity (mitochondrial GPAT) from the total as described under "Experimental Procedures." *, p Ͻ0.05 versus wild type mice.
FIG. 4. Membrane-associated CCT␣ protein level and CCT activity are increased in liver of Scd1؊/؊ compared with wild type mice. A, the total CCT␣ protein level measured in liver homogenate and membrane-associated CCT␣ protein measured in membranes isolated from liver of Scd1Ϫ/Ϫ and wild type mice. B, the effect of SCD1 deficiency on distribution of hepatic CCT␣ expressed as the ratio of membrane-associated CCT␣ to total CCT␣ protein content. The protein levels were measured by immunoblotting and quantitated by densitometric scanning, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein content. C, CCT activity analyzed in homogenates as described under "Experimental Procedures." mulate less TAG in liver and other tissues and have reduced plasma VLDL levels (5,6,10). In the present study, we have shown that SCD1 deficiency significantly modulates the hepatic profile of PLs and changes the rate of their biosynthesis.
Analysis of various PL classes revealed that the major PL affected by Scd1 mutation is PC. Increased activities of CCT (Fig. 4C) and CPT (Fig. 5A) and increased [ 3 H]glycerol incorporation into PC (Fig. 2B) indicate increased de novo synthesis of PC in the liver of Scd1Ϫ/Ϫ mice. CCT catalyzes the ratelimiting step of the CDP-choline pathway for PC synthesis (19,20), and translocation of this enzyme into cell membranes has been shown to be critical for its activation (20). The data pre-sented herein showed that SCD1 deficiency increased CCT activity by enhancing its membrane translocation, because twice as much CCT␣ was associated with membrane in livers from Scd1Ϫ/Ϫ compared with wild type mice (Fig. 4B) even though SCD1 deficiency reduced the total content of hepatic CCT␣ (Fig. 4A).
The membrane affinity of CCT is regulated primarily through compositional changes in membrane lipid and through physical properties of the membrane (20,37). Our study has shown that SCD1 deficiency leads to decreased content of monounsaturated FA and increased content of polyunsaturated FA (mainly 20:4) in liver PLs, whereas the relative amount of saturated FA in hepatic PLs was not significantly altered due to Scd1 mutation (Table I). The changes in PL fatty acid composition in liver of Scd1Ϫ/Ϫ mice might be associated with reduced activity of mitochondrial GPAT (Fig. 3), because mitochondrial GPAT plays a key role in establishing initial asymmetric distribution of saturated and unsaturated FA in PL (1,36) and its deficiency was shown to decrease 16:0 and increase 20:4 contents in hepatic PC and PE (38). Given that insertion of CCT into membrane was shown to be enhanced by polyunsaturated fatty acids (39), increased unsaturated PE level (20,37), and by disordered fatty acyl chains of PL (25,26) in vitro, alterations in PL fatty acid composition and its membrane organization due to Scd1 mutation might directly promote CCT membrane binding. Indeed, recent confocal microscopy studies have revealed that Scd expression is a very potent regulator of membrane domain structure (40). Overexpression of Scd in Chinese hamster ovary cells led to an increased content of monounsaturated FA in plasma membrane at the expense of saturated FA and consequently decreased Triton X-100-resistant domains in the plasma membrane (40). Our findings provide the first evidence to suggest that CCT membrane translocation is affected by membrane lipid composition in vivo and might be regulated by changes in SCD activity. It is also possible that the membrane binding of CCT in the Scd1Ϫ/Ϫ mice was caused by changes in the composition of the free fatty acyl-CoA pool due to Scd1 deficiency. The observation that SCD regulates PC biosynthesis and substantially changes membrane organization raises the possibility that many different cellular functions that are attributed to membrane properties, such as activities of nitric-oxide synthase (41) and protein kinase C (42), as well as vesicle transport (15) and functions of intrinsic membrane receptors (43), could be altered by changes in Scd expression.
CPT activity is mainly regulated by availability of its substrates, DAG and CDP-choline (19). Because CCT catalyzes CDP-choline synthesis, increased CCT activity due to Scd1 mutation leads to activation of CPT in liver of Scd1Ϫ/Ϫ mice (Fig. 5A). Because DGAT and CPT draw from the same pool of DAG for synthesis of TAG and PC, respectively, CPT activity might indirectly determine the rate of TAG synthesis as well (1,44). Although DAG content (Fig. 1A) and [ 3 H]glycerol incorporation into DAG ( Fig. 2A) were not affected by SCD1 deficiency, the rate of TAG synthesis was decreased by 56% ( Fig. 2A) despite normal DGAT activity (Fig. 6B) in the liver of Scd1Ϫ/Ϫ mice. The lower incorporation of glycerol into TAG in Scd1deficient hepatocytes was accompanied by a 2.5-fold increase in glycerol incorporation into PC (Fig. 2B). Given that the affinity of CPT for DAG is severalfold higher than that of DGAT (1,44), increased CPT activity in the liver of Scd1Ϫ/Ϫ mice might result in suppression of DAG conversion to TAG. Increased rate of PC synthesis has previously been shown to inhibit TAG synthesis through diversion of DAG in rat hepatocytes (44) and yeast (45). Taken together, these data suggest that increased PC biosynthesis via the CDP-choline pathway might be par- tially responsible for the decreased rate of TAG synthesis in liver of Scd1Ϫ/Ϫ mice. PE methylation by PEMT, an additional pathway of hepatic PC synthesis in liver, is down-regulated in Scd1Ϫ/Ϫ mice (Fig.  5B), supporting the idea that the two major pathways of PC biosynthesis (CDP-choline pathway and PEMT pathway) are reciprocally regulated (19). Experiments with hepatocytes as well as in vivo studies have demonstrated a specific role for PEMT in VLDL secretion (17,18). In male PemtϪ/Ϫ mice fed a high fat/high cholesterol diet, plasma TAG and PC levels were reduced by 50 and 20%, respectively, compared with wild type mice (18). In hepatocytes from male PemtϪ/Ϫ compared with wild type mice, apoB100 secretion was reduced by 60% and TAG secretion was reduced by 75% (17). Therefore, it is possible that alterations in plasma lipoprotein metabolism and reduced VLDL secretion observed in Scd1Ϫ/Ϫ mice (5) might result from reduction in PEMT.
In summary, the data presented herein for the first time demonstrate the importance of SCD1 in synthesis of PL. The total levels and fatty acid composition of several PL species were significantly changed, with PC demonstrating the greatest alteration. As shown schematically in Fig. 7, disruption of the Scd1 gene increases activities of CCT and CPT and leads to an increase in de novo PC synthesis and its accumulation. Our study revealed that Scd1 deficiency activates CCT by promoting its membrane binding and suggests that alteration in PL fatty acid composition due to Scd1 mutation leads to an increase in membrane affinity of CCT. Because mitochondrial GPAT is responsible for asymmetric proportion of PL fatty acids (36), a decrease in mitochondrial GPAT activity might be partially accountable for membrane fatty acid modification in the liver of Scd1Ϫ/Ϫ mice. PC synthesis via PE meth-ylation pathway is down-regulated in Scd1Ϫ/Ϫ mice, leading to accumulation of PE. Increased PC synthesis via the CDPcholine pathway in the liver of Scd1Ϫ/Ϫ mice was coupled with decreased synthesis of TAG, even though DGAT gene expression and activity were not significantly different. Because DGAT and CPT draw from the same pool of DAG, the data suggest that, in addition to increased ␤-oxidation (4,12) and decreased lipogenesis (2), increased PC biosynthesis could be responsible for reduced TAG synthesis in Scd1Ϫ/Ϫ mice.