Phospholipid homeostasis in phosphatidylserine synthase-2-deficient mice

Phosphatidylserine (PS) is synthesized in mammalian cells by two distinct serine-exchange enzymes, phosphatidylserine synthase-1 and -2. We recently demonstrated that mice lacking PS synthase-2 develop normally and exhibit no overt abnormalities [Bergo et al., (2002) J. Biol. Chem. 277:47701-47708]. We now show that PS synthase-2 mRNA levels are up to 80-fold higher in livers of embryos than in adults. Despite reduced serine-exchange activity in several tissues of PS synthase-2 deficient mice, the phospholipid composition of mitochondria and microsomes from these tissues is normal. Although PS synthase-2 is highly expressed in neurons, axon extension of cultured sympathetic neurons is not impaired by PS synthase-2 deficiency. We hypothesized that mice compensate for PS synthase-2 deficiency by modifying their phospholipid metabolism. Our data show that the rate of PS synthesis in hepatocytes is not reduced by PS synthase-2 deficiency but PS synthase-1 activity is increased. Moreover, PS degradation is decreased by PS synthase-2 deficiency, probably as a result of decreased PS degradation via phospholipases rather than decreased PS decarboxylation. These experiments underscore the idea that cellular phospholipid composition is tightly controlled and show that PS synthase-2-deficient hepatocytes modify phospholipid metabolism by several compensatory mechanisms to maintain phospholipid homeostasis.     

Defining the importance of phosphatidylserine synthase 2 in mice

Phosphatidylserine synthase 1 (Pss1) and phosphatidylserine synthase 2 (Pss2) produce phosphatidylserine by exchanging serine for the head groups of other phospholipids. Pss1 and Pss2 are structurally similar (approximately 32% amino acid identity) but differ in their substrate specificities, with Pss1 using phosphatidylcholine for the serine exchange reaction and Pss2 using phosphatidylethanolamine. Whether Pss1 and Pss2 are both required for mammalian growth and development is not known, and no data exist on the relative contributions of the two enzymes to serine exchange activities in different tissues. To address those issues and also to define the cell type-specific expression of Pss2, we generated Pss2-deficient mice in which a beta-galactosidase marker is expressed from Pss2 regulatory sequences. Histologic studies of Pss2-deficient mice revealed very high levels of beta-galactosidase expression in Sertoli cells of the testis and high levels of expression in brown fat, neurons, and myometrium. The ability of testis extracts from Pss2-deficient mice to catalyze serine exchange was reduced by more than 95%; reductions of approximately 90% were noted in the brain and liver. However, we found no perturbations in the phospholipid content of any of these tissues. As judged by Northern blots, the expression of Pss1 was not up-regulated in Pss2-deficient cells and tissues. Testis weight was reduced in Pss2-deficient mice, and some of the male mice were infertile. We conclude that Pss2 is responsible for the majority of serine exchange activity in in vitro assays, but a deficiency in this enzyme does not cause perturbations in phospholipid content or severe developmental abnormalities.     

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. III. Genetic evidence for utilization of phosphatidylcholine and phosphatidylethanolamine as precursors

In the preceding paper, we reported that Chinese hamster ovary (CHO) cells contain two different serine-exchange enzymes (I and II) which catalyze the base-exchange reaction of phospholipid(s) with serine and that a phosphatidylserine-requiring mutant (strain PSA-3) of CHO cells is defective in serine-exchange enzyme I and lacks the ability to synthesize phosphatidylserine (Kuge, O., Nishijima, M., and Akamatsu, Y. (1986) J. Biol. Chem. 261, 5790-5794). In this study, we examined precursor phospholipids for phosphatidylserine biosynthesis in CHO cells. When mutant PSA-3 and parent (CHO-K1) cells were cultured with [32P]phosphatidylcholine, phosphatidylserine in the parent accumulated radioactivity while that in the mutant was not labeled significantly. On the contrary, when cultured with [32P]phosphatidylethanolamine, the mutant incorporated the label into phosphatidylserine more efficiently than the parent. Furthermore, we found that mutant PSA-3 grew normally in growth medium supplemented with 30 microM phosphatidylethanolamine as well as phosphatidylserine and that the biosynthesis of phosphatidylserine in the mutant was biosynthesis of phosphatidylserine in the mutant was normal when cells were cultured in the presence of exogenous phosphatidylethanolamine. The simplest interpretation of these findings is that phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions: phosphatidylcholine----phosphatidylserine----phosphatidylethanolamine--- - phosphatidylserine. The three reactions are catalyzed by serine-exchange enzyme I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively.     

Phosphatidylserine synthase-1 and -2 are localized to mitochondria-associated membranes

We report the subcellular localization of enzymes involved in phosphatidylserine biosynthesis in mammalian cells. Several lines of evidence suggest that phosphatidylserine synthase-1 (PSS1) is highly enriched in mitochondria-associated membranes (MAM) and is largely excluded from the bulk of the endoplasmic reticulum (ER). Taking advantage of the substrate specificity of PSS1, we showed that (i) MAM contain choline exchange activity, whereas this activity is very low in the bulk of the ER, (ii) serine exchange activity is inhibited by choline to a much greater extent in MAM than in ER, and (iii) MAM use phosphatidylcholine and phosphatidylethanolamine as substrates for phosphatidylserine biosynthesis, whereas the ER utilizes only phosphatidylethanolamine. According to immunoblotting of proteins from both CHO-K1 cells and murine liver, PSS1 is localized to MAM, and in hepatoma cells stably expressing PSS1 this protein is highly enriched in MAM. Since the ER contains serine and ethanolamine exchange activities, we had predicted that PSS2 would account for the serine exchange activity in the ER. Unexpectedly, using immunoblotting experiments, we found that (i) PSS2 of CHO-K1 cells is present only in MAM and (ii) PSS2 is restricted to MAM of McArdle cells expressing recombinant PSS2. These data leave open the question of which enzyme imparts PSS activity to the ER and suggest that a third isoform of PSS might be located in the ER.     

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.     

Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells.

Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-overproducing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO-K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a approximately 4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA-directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer-mediated inhibition of PSS II.     

Sequential synthesis and methylation of phosphatidylethanolamine promote lipid droplet biosynthesis and stability in tissue culture and in vivo

Triacylglycerols are stored in eukaryotic cells within lipid droplets (LD). The LD core is enwrapped by a phospholipid monolayer with phosphatidylcholine (PC), the major phospholipid, and phosphatidylethanolamine (PE), a minor component. We demonstrate that the onset of LD formation is characterized by a change in cellular PC, PE, and phosphatidylserine (PS). With induction of differentiation of 3T3-L1 fibroblasts into adipocytes, the cellular PC/PE ratio decreased concomitant with LD formation, with the most pronounced decline between confluency and day 5. The mRNA for PS synthase-1 (forms PS from PC) and PS decarboxylase (forms PE from PS) increased after day 5. Activity and protein of PE N-methyltransferase (PEMT), which produces PC by methylation of PE, are absent in 3T3-L1 fibroblasts but were induced at day 5. High fat challenge induced PEMT expression in mouse adipose tissue. PE, produced via PS decarboxylase, was the preferred substrate for methylation to PC. A PEMT-GFP fusion protein decorated the periphery of LD. PEMT knockdown in 3T3-L1 adipocytes correlated with increased basal triacylglycerol hydrolysis. Pemt(-/-) mice developed desensitization against adenosine-mediated inhibition of basal hydrolysis in adipose tissue, and adipocyte hypotrophy was observed in Pemt(-/-) animals on a high fat diet. Knock-out of PEMT in adipose tissue down-regulated PS synthase-1 mRNA, suggesting coordination between PE supply and converting pathways during LD biosynthesis. We conclude that two consecutive processes not previously related to LD biogenesis, (i) PE production via PS and (ii) PE conversion via PEMT, are implicated in LD formation and stability.     

Trafficking of cholesterol from cell bodies to distal axons in Niemann Pick C1-deficient neurons

Niemann Pick type C (NPC) disease is a progressive neurodegenerative disorder. In cells lacking functional NPC1 protein, endocytosed cholesterol accumulates in late endosomes/lysosomes. We utilized primary neuronal cultures in which cell bodies and distal axons reside in separate compartments to investigate the requirement of NPC1 protein for transport of cholesterol from cell bodies to distal axons. We have recently observed that in NPC1-deficient neurons compared with wild-type neurons, cholesterol accumulates in cell bodies but is reduced in distal axons (Karten, B., Vance, D. E., Campenot, R. B., and Vance, J. E. (2002) J. Neurochem. 83, 1154-1163). We now show that NPC1 protein is expressed in both cell bodies and distal axons. In NPC1-deficient neurons, cholesterol delivered to cell bodies from low density lipoproteins (LDLs), high density lipoproteins, or cyclodextrin complexes was transported into axons in normal amounts, whereas transport of endogenously synthesized cholesterol was impaired. Inhibition of cholesterol synthesis with pravastatin in wild-type and NPC1-deficient neurons reduced axonal growth. However, LDLs restored a normal rate of growth to wild-type but not NPC1-deficient neurons treated with pravastatin. Thus, although LDL cholesterol is transported into axons of NPC1-deficient neurons, this source of cholesterol does not sustain normal axonal growth. Over the lifespan of NPC1-deficient neurons, these defects in cholesterol transport might be responsible for the observed altered distribution of cholesterol between cell bodies and axons and, consequently, might contribute to the neurological dysfunction in NPC disease.     

Externalization of phosphatidylserine during apoptosis does not specifically require either isoform of phosphatidylserine synthase

Phosphatidylserine (PtdSer) is made in mammalian cells by two PtdSer synthases, PSS1 and PSS2. In the plasma membrane PtdSer is normally localized on the inner leaflet but undergoes transbilayer movement during apoptosis and becomes exposed on the cell surface. We induced apoptosis with staurosporine in four Chinese hamster ovary (CHO) cell lines that are deficient in PSS1 and/or PSS2 to determine if PtdSer generated by either of these enzymes is required for externalization on the cell surface during apoptosis. The onset of apoptosis was confirmed by the appearance of morphological changes and DNA fragmentation while the plasma membrane remained largely intact. In all cell lines, regardless of their content of PSS1 and/or PSS2, apoptosis occurred to approximately the same extent, and within approximately the same time frame, as in parental CHO-K1 cells. The exposure of PtdSer on the cell surface was assessed by annexin V labeling and flow cytometry. Cells that were deficient in either PSS1 or PSS2, as well as cells that were deficient in both PSS1 and PSS2, externalized normal amounts of PtdSer. Our study demonstrates, that reduction of in vitro serine-exchange activity, even by 97%, does not restrict the externalization of PtdSer during apoptosis. Moreover, a normal level of expression of PSS1 and/or PSS2 is not required for generating the pool of PtdSer externalized during apoptosis.     

N-myristoyltransferase 1 is essential in early mouse development

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a beta-galactosidase marker gene. beta-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1+/-) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1+/- mice yielded no viable homozygotes (Nmt1-/-), and heterozygotes were born at a less than predicted frequency. Nmt1-/- embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1-/- embryos. To explore this concept, we generated Nmt1-/- embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1-/- ES cells, but the total NMT activity levels were reduced by approximately 95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1-/- ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis.     

Effects of genetic background on thermoregulation and fatty acid-induced uncoupling of mitochondria in UCP1-deficient mice

An interaction between free fatty acids and UCP1 (uncoupling protein-1) leading to de-energization of mitochondria was assumed to be a key event for triggering heat production in brown fat. Recently, Matthias et al., finding indistinguishable de-energization of isolated brown fat mitochondria by fatty acids in UCP1-deficient mice and control mice, challenged this assumption (Matthias, A., Jacobsson, A., Cannon, B., and Nedergaard, J. (1999) J. Biol. Chem. 274, 28150-28160). Since their results were obtained using UCP1-deficient and control mice on an undefined genetic background, we wanted to determine unambiguously the phenotype of UCP1 deficiency with the targeted Ucp1 allele on congenic C57BL/6J and 129/SvImJ backgrounds. UCP1-deficient congenic mice have a very pronounced cold-sensitive phenotype; however, deficient mice on the F1 hybrid background were resistant to cold. We propose that heterosis provides a mechanism to compensate for UCP1 deficiency. Contrary to the results of Matthias et al., we found a significant loss of fatty acid-induced de-energization, as reflected by membrane potential and oxygen consumption, in brown fat mitochondria from UCP1-deficient mice. Unlike cold sensitivity, fatty acid-induced uncoupling of mitochondria was independent of the genetic background of UCP1-deficient mice. We propose that intracellular free fatty acids directly regulate uncoupling activity of UCP1 in a manner consistent with models described in the literature.     

Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice

Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E(2) synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE(2) synthase-1 (mPGES-1) isomerizes COX-2-derived PGH(2) to PGE(2). Here, we evaluated the effect of mPGES-1-deficiency on the nociceptive behavior in various models of nociception that depend on PGE(2) synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE(2) synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE(2) synthesis to PGD(2), PGF(2alpha) and 6-keto-PGF(1alpha) (stable metabolite of PGI(2)). Since the latter prostaglandins serve also as mediators of nociception they may compensate the loss of PGE(2) synthesis in mPGES-1-deficient mice.     

An alternative splicing form of phosphatidylserine-specific phospholipase A1 that exhibits lysophosphatidylserine-specific lysophospholipase activity in humans

Phosphatidylserine-specific phospholipase A1 (PS-PLA1), which acts specifically on phosphatidylserine (PS) and 1-acyl-2-lysophosphatidylserine (lyso-PS) to hydrolyze fatty acids at the sn-1 position of these phospholipids, was first identified in rat platelets (Sato, T., Aoki, J., Nagai, Y., Dohmae, N., Takio, K., Doi, T., Arai, H., and Inoue, K. (1997) J. Biol. Chem. 272, 2192-2198). In this study we isolated and sequenced cDNA clones encoding human PS-PLA1, which showed 80% homology with rat PS-PLA1 at the amino acid level. In addition to an mRNA encoding a 456-amino acid product (PS-PLA1), an mRNA with four extra bases inserted at the boundary of the exon-intron junction was detected in human tissues and various human cell lines. This mRNA is most probably produced via an alternative use of the 5'-splicing site (two consensus sequences for RNA splicing occur at the boundary of the exon-intron junction) and encodes a 376-amino acid product (PS-PLA1DeltaC) that lacks two-thirds of the C-terminal domain of PS-PLA1. Unlike PS-PLA1, PS-PLA1DeltaC hydrolyzed exclusively lyso-PS but not PS appreciably. Any other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and their lyso derivatives were not hydrolyzed at all. These data demonstrated that PS-PLA1DeltaC exhibits lyso-PS-specific lysophospholipase activity and that the C-terminal domain of PS-PLA1 is responsible for recognizing diacylphospholipids. In addition, human PS-PLA1 gene was mapped to chromosome 3q13.13-13.2 and was unexpectedly identical to the nmd gene, which is highly expressed in nonmetastatic melanoma cell lines but poorly expressed in metastatic cell lines (van Groningen, J. J., Bloemers, H. P., and Swart, G. W. (1995) Cancer Res. 55, 6237-6243).     

Incorporation of serine into the phospholipids of phosphatidylethanolamine-depleted Tetrahymena

Phosphatidylserine formation and decarboxylation are decreased in Tetrahymena in which phosphatidylethanolamine has been replaced by its isosteric analog 3-aminopropylphosphonolipid (1,2-diacylglyceryl-3-O-(3-aminopropylphosphonate). The combined activity of the phosphatidylethanolamine: serine phosphatidyltransferase/ phosphatidylserine decarboxylase complex in isolated mitochondria from lipid-altered cells [J. D. Smith and D. A. Giegel (1981) Arch. Biochem, Biophys. 206, 420-423] is about 20% of the activity in mitochondria from control cells. The enzyme activity in the lipid-altered mitochondria is stimulated by the addition of exogenous phosphatidylethanolamine to the assay system while the enzymes of the control mitochondria are not. In vivo the lipid-altered cells are able to incorporate radioactivity from [3-14C]- or [3-3H]serine into phosphatidylserine and phosphatidylcholine in amounts comparable to normal cells. Thus, under conditions of "stress" (e.g., the depletion of phosphatidylethanolamine), the phosphatidyltransferase is apparantly capable of utilizing other phospholipids besides its normal substrate phosphatidylethanolamine.     

Presphenoidal synchondrosis fusion in DBA/2J mice

Cranial base growth plates are important centers of longitudinal growth in the skull and are responsible for the proper anterior placement of the face and the stimulation of normal cranial vault development. We report that the presphenoidal synchondrosis (PSS), a midline growth plate of the cranial base, closes in the DBA/2J mouse strain but not in other common inbred strains. We investigated the genetics of PSS closure in DBA/2J mice by evaluating F1, F1 backcross, and/or F1 intercross offspring from matings with C57BL/6J and DBA/1J mice, whose PSS remain open. We observed that PSS closure is genetically determined, but not inherited as a simple Mendelian trait. Employing a genome-wide SNP array, we identified a region on chromosome 11 in the C57BL/6J strain that affected the frequency of PSS closure in F1 backcross and F1 intercross offspring. The equivalent region in the DBA/1J strain did not affect PSS closure in F1 intercross offspring. We conclude that PSS closure in the DBA/2J strain is complex and modified by different loci when outcrossed with C57BL/6J and DBA/1J mice.     

In vivo role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the regulation of intracellular redox state and accumulation of abdominal adipose tissue

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that utilizes NAD(P)H as an electron donor, catalyzing the two-electron reduction and detoxification of quinones and their derivatives. NQO1-/- mice deficient in NQO1 activity and protein were generated in our laboratory (Rajendirane, V., Joseph, P., Lee, Y. H., Kimura, S., Klein-Szanto, A. J. P., Gonzalez, F. J., and Jaiswal, A. K. (1998) J. Biol. Chem. 273, 7382-7389). Mice lacking a functional NQO1 gene (NQO1-/-) were born normal and reproduced adeptly as the wild-type NQO1+/+ mice. In the present report, we show that NQO1-/- mice exhibit significantly lower levels of abdominal adipose tissue as compared with the wild-type mice. The NQO1-/- mice showed lower blood levels of glucose, no change in insulin, and higher levels of triglycerides, beta-hydroxy butyrate, pyruvate, lactate, and glucagon as compared with wild-type mice. Insulin tolerance test demonstrated that the NQO1-/- mice are insulin resistant. The NQO1-/- mice livers also showed significantly higher levels of triglycerides, lactate, pyruvate, and glucose. The liver glycogen reserve was found decreased in NQO1-/- mice as compared with wild-type mice. The livers and kidneys from NQO1-/- mice also showed significantly lower levels of pyridine nucleotides but an increase in the reduced/oxidized NAD(P)H:NAD(P) ratio. These results suggested that loss of NQO1 activity alters the intracellular redox status by increasing the concentration of NAD(P)H. This leads to a reduction in pyridine nucleotide synthesis and reduced glucose and fatty acid metabolism. The alterations in metabolism due to redox changes result in a significant reduction in the amount of abdominal adipose tissue.     

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway

The complement system plays an important role in innate immunity. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme. It has been suggested that MASP-2 is responsible for the activation of C4. Other serine proteases (MASP-1 and MASP-3) are also associated with MBL or ficolins; however, their functions are still controversial. In this study, a MASP-1- and MASP-3-deficient mouse model (MASP1/3(-/-)) was generated by a gene targeting strategy to investigate the roles of MASP-1 and MASP-3 in the lectin pathway. Serum derived from MASP1/3(-/-) mice showed significantly lower activity of both C4 and C3 deposition on mannan-agarose, and this low activity was restored by the addition of recombinant MASP-1. MASP-1/3-deficient serum showed a significant delay for activation of MASP-2 compared with normal serum. Reconstitution of recombinant MASP-1 in MASP-1/3-deficient serum was able to promote the activation of MASP-2. From these results, we propose that MASP-1 contributes to the activation of the lectin pathway, probably through the activation of MASP-2.