Fructose metabolizing enzymes from mouse liver: influence of age and caloric restriction

The influence of caloric restriction (CR) on the activities of liver fructose metabolizing enzymes and metabolite levels were studied in young (3 months) and old (30 months) mice. Fructokinase activity was increased (P<0.05) in both young and old CR mice when compared to controls while triokinase activity was increased (P<0.05) only in old CR versus control mice. Aldolase was not altered by CR in either old or young mice. No age-related differences in activities were observed in controls although a trend towards an increase was observed for triokinase, while significant age-related increases were observed for fructokinase and triokinase, but not aldolase, in CR mice. Both young and old mice on CR showed significant decreases in fructose and fructose-1-phosphate, however, no age-related changes in metabolite levels were observed for either control or CR mice. A fructose-1-phosphate kinase activity was also measured and found to be unchanged in both young and old mice on CR, but the activity was significantly lower in the old mice compared with young. We show here that the enzymes involved in fructose metabolism are influenced by CR and that this could contribute to alterations in gluconeogenesis and glycolysis observed with CR.     

Enzymes of glycerol and glyceraldehyde metabolism in mouse liver: effects of caloric restriction and age on activities

The influence of caloric restriction on hepatic glyceraldehyde- and glycerol-metabolizing enzyme activities of young and old mice were studied. Glycerol kinase and cytoplasmic glycerol-3-phosphate dehydrogenase activities were increased in both young and old CR (calorie-restricted) mice when compared with controls, whereas triokinase increased only in old CR mice. Aldehyde dehydrogenase and aldehyde reductase activities in both young and old CR mice were unchanged by caloric restriction. Mitochondrial glycerol-3-phosphate dehydrogenase showed a trend towards an increased activity in old CR mice, whereas a trend towards a decreased activity in alcohol dehydrogenase was observed in both young and old CR mice. Serum glycerol levels decreased in young and old CR mice. Therefore increases in glycerol kinase and glycerol-3-phosphate dehydrogenase were associated with a decrease in fasting blood glycerol levels in CR animals. A prominent role for triokinase in glyceraldehyde metabolism with CR was also observed. The results indicate that long-term caloric restriction induces sustained increases in the capacity for gluconeogenesis from glycerol.     

Flux of the L-serine metabolism in rat liver. The predominant contribution of serine dehydratase.

L-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by L-serine dehydratase (SDH), another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagon-treated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mM L-serine and 0.25 mM pyruvate) was about (1)/(10) of that through SDH. Flux through GCS accounted for only several percent of the amount of L-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from L-serine were evaluated in vivo based on the principle that 3H at the 3 position of L-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10-20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of L-serine in rat liver.     

Opposing effects on cardiac function by calorie restriction in different‐aged mice

Calorie restriction (CR) increases average and maximum lifespan and exhibits an apparent beneficial impact on age‐related diseases. Several studies have shown that CR initiated either in middle or old age could improve ischemic tolerance and rejuvenate the aging heart; however, the data are not uniform when initiated in young. The accurate time to initiate CR providing maximum benefits for cardiac remodeling and function during aging remains unclear. Thus, whether a similar degree of CR initiated in mice of different ages could exert a similar effect on myocardial protection was investigated in this study. C57BL/6 mice were subjected to a calorically restricted diet (40% less than the ad libitum diet) for 3 months initiated in 3, 12, and 19 months. It was found that CR significantly reversed the aging phenotypes of middle‐aged and old mice including cardiac remodeling (cardiomyocyte hypertrophy and cardiac fibrosis), inflammation, mitochondrial damage, telomere shortening, as well as senescence‐associated markers but accelerated in young mice. Furthermore, whole‐genome microarray demonstrated that the AMP‐activated protein kinase (AMPK)–Forkhead box subgroup ‘O’ (FOXO) pathway might be a major contributor to contrasting regulation by CR initiated in different ages; thus, increased autophagy was seen in middle‐aged and old mice but decreased in young mice. Together, the findings demonstrated promising myocardial protection by 40% CR should be initiated in middle or old age that may have vital implications for the practical nutritional regimen.     

Reduced food consumption increases water intake and modulates renal aquaporin-1 and -2 expression in autoimmune prone mice

Aquaporin-1(AQP1) and AQP2 are members of the aquaporin family of cell membrane water channel transport proteins and have been implicated in the regulation of renal water excretion. We have previously shown that calorie restriction (CR) relative to ad libitum (AL) feeding extends lifespan and delays the onset of autoimmune kidney disease in lupus-prone (NZBxNZW)F1 (B/W) mice. To determine if AQP1 and/or AQP2 expression is influenced by CR, mice were fed an AL or CR (40% less food) diet until 4 (young) or 9 (old) months of age when mice were sacrificed. Kidneys were removed and the expression of AQP1 and AQP2 was determined at the protein and mRNA levels using western blotting and RT-PCR respectively. While age did not significantly increase AQP1 expression in the AL groups, CR did increase both the protein (1.4-fold) and mRNA (2.4-fold) levels. In old mice, AQP1 expression was higher (1.8-fold) in CR compared to the AL group while CR had no effect in young mice. In contrast, AQP2 showed an age related decrease (55%) in the AL groups and an increase in the protein (8.4-fold) and mRNA (1.7-fold) levels in the CR groups. Relative to AL, CR decreased AQP2 expression at the protein (90%) and mRNA (50%) levels in the young mice while an increase at the protein (2.9-fold) and mRNA (1.9-fold) levels was evident in the old mice. Interestingly, a significant increase in water intake per gram body weight was found in both young and old CR fed mice when compared to their AL counterparts which may contribute to the prevention of autoimmune disease with age and differences in longevity. These data show, for the first time, significant age and diet influences in renal AQP1 and AQP2 expression at both protein and mRNA levels in lupus-prone mice.     

Possibility of mitochondrial-cytosolic cooperation in gluconeogenesis from serine via hydroxypyruvate

Intracellular localization of D-glycerate dehydrogenase (D-glycerate : NAD⁺ oxidoreductase, EC 1.1.1.29), one of the enzymes of the pathway for gluconeogenesis from serine via hydroxypyruvate, was studied by differential centrifugation. Almost all enzyme activity was found in cytosol. Since the major activities of two other enzymes, serine : pyruvate aminotransferase (EC 2.6.1.51) and glycerate kinase (ATP : D-glycerate 2-phosphotransferase, EC 2.7.1.31), of the pathway via hydroxypyruvate are localized in mitochondrial inner membrane and/or matrix, the possible localization of D-glyceratedehydrogenase in mitochondria was examined. Detailed analysis of mitochondrial fraction prepared by differential centrifugation indicated that rat liver mitochondria do not contain any D-glycerate dehydrogenase activity. Based on these results, a cooperative connection between mitochondria and cytosol in gluconeogenesis from serine via hydroxypyruvate is proposed. Possible mechanisms for transport of intermediates of the pathway via hydroxypyruvate across the mitochondrial membranes are also discussed.     

Influence of aging and caloric restriction on activation of Ras/MAPK, calcineurin, and CaMK-IV activities in rat T cells

The signaling cascade mediated by Ras (p21ras) and MAPK (mitogen-activated protein kinase) and calcium/calmodulin regulating enzymes, calcineurin (CaN) and CaMK-IV, are considered to be essential for T-cell growth and function. In the present study, the effect of aging and caloric restriction (CR) on the induction of Ras and MAPK activation by concanavalin A (ConA) was studied. Splenic T cells were isolated from young (4-6 months) and old (22-24 months) rats that had free access to food (control group), and from caloric restricted old (22-24 months) rats that beginning at 6 weeks of age were fed 60%(40% caloric restriction) of the diet consumed by the control rats. We found that the induction of Ras activity in T cells isolated from control old rats was lower (P<0.001) than that in control young rats. However, the levels of Ras activity in T cells isolated from CR old rats were similar to the levels in the age-matched control rats. The induction of MAPK activity in T cells isolated from control old rats and CR old rats was significantly less than in T cells isolated from control young rats, and caloric restriction significantly (P<0.05) reduced the age-related decline in MAPK activation. We also measured the induction of CaN and CaMK-IV activities by ConA in T cells from control young and old and CR old rats. The induction of both CaN and CaMK-IV activity decreased with age. Caloric restriction significantly (P<0.05) reduced the age-related decline in CaN activity, but had no significant effect on CaMK-IV activity. The changes in Ras/MAPK activation and in CaN and CaMK-IV activity with age or with CR were not associated with alterations in their corresponding protein levels. Thus, caloric restriction has a differential effect on the activation of the upstream signaling molecules that are altered with age.     

Quantification of 3-ketodihydrosphingosine using HPLC-ESI-MS/MS to study SPT activity in yeast Saccharomyces cerevisiae

Serine palmitoyltransferase (SPT) catalyzes the rate-limiting step of condensation of L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (3KDS). Here, we report a HPLC-ESI-MS/MS method to directly quantify 3KDS generated by SPT. With this technique, we were able to detect 3KDS at a level comparable to that of dihydrosphingosine in yeast Saccharomyces cerevisiae. An in vitro SPT assay measuring the incorporation of deuterated serine into deuterated 3KDS was developed. The results show that SPT kinetics in response to palmitoyl-CoA fit into an allosteric sigmoidal model, suggesting the existence of more than one palmitoyl-CoA binding site on yeast SPT and positive cooperativity between them. Myriocin inhibition of yeast SPT activity was also investigated and we report here, for the first time, an estimated myriocin K_i for yeast SPT of approximately 10 nM. Lastly, we investigated the fate of serine α-proton during SPT reaction. We provide additional evidence to support the proposed mechanism of SPT catalytic activity in regard to proton exchange between the intermediate NH₃⁺ base formed on the active Lys residue with surrounding water. These findings establish the current method as a powerful tool with significant resolution and quantitative power to study SPT activity.     

Serine palmitoyltransferase (SPT) deficient mice absorb less cholesterol

Serine palmitoyltransferase (SPT) is the key enzyme for the biosynthesis of sphingolipids. It has been reported that oral administration of myriocin (an SPT inhibitor) decreases plasma sphingomyelin (SM) and cholesterol levels, and reduces atherosclerosis in apoE knockout (KO) mice. We studied cholesterol absorption in myriocin-treated WT or apoE KO animals and found that, after myriocin treatment, the mice absorbed significantly less cholesterol than controls, with no observable pathological changes in the small intestine. More importantly, we found that heterozygous Sptlc1 (a subunit of SPT) KO mice also absorbed significantly less cholesterol than controls. To understand the mechanism, we measured protein levels of Niemann-Pick C1-like 1 (NPC1L1), ABCG5, and ABCA1, three key factors involved in intestinal cholesterol absorption. We found that NPC1L1 and ABCA1 were decreased, whereas ABCG5 was increased in the SPT deficient small intestine. SM levels on the apical membrane were also measured and they were significantly decreased in SPT deficient mice, compared with controls. In conclusion, SPT deficiency might reduce intestinal cholesterol absorption by altering NPC1L1 and ABCG5 protein levels in the apical membranes of enterocytes through lowering apical membrane SM levels. This may be also true for ABCA1 which locates on basal membrane of enterocytes. Manipulation of SPT activity could thus provide a novel alternative treatment for dyslipidemia.     

Glycerate 2-kinase of Thermotoga maritima and genomic reconstruction of related metabolic pathways

Members of a novel glycerate-2-kinase (GK-II) family were tentatively identified in a broad range of species, including eukaryotes and archaea and many bacteria that lack a canonical enzyme of the GarK (GK-I) family. The recently reported three-dimensional structure of GK-II from Thermotoga maritima (TM1585; PDB code 2b8n) revealed a new fold distinct from other known kinase families. Here, we verified the enzymatic activity of TM1585, assessed its kinetic characteristics, and used directed mutagenesis to confirm the essential role of the two active-site residues Lys-47 and Arg-325. The main objective of this study was to apply comparative genomics for the reconstruction of metabolic pathways associated with GK-II in all bacteria and, in particular, in T. maritima. Comparative analyses of ~400 bacterial genomes revealed a remarkable variety of pathways that lead to GK-II-driven utilization of glycerate via a glycolysis/gluconeogenesis route. In the case of T. maritima, a three-step serine degradation pathway was inferred based on the tentative identification of two additional enzymes, serine-pyruvate aminotransferase and hydroxypyruvate reductase (TM1400 and TM1401, respectively), that convert serine to glycerate via hydroxypyruvate. Both enzymatic activities were experimentally verified, and the entire pathway was validated by its in vitro reconstitution.     

Caloric Restriction: Conservation of in Vivo Cellular Replicative Capacity Accompanies Life-Span Extension in Mice

In male mice of a long-lived hybrid strain (B6D2F1), long-term 40% caloric restriction (CR) extended both mean and maximum life spans by 36 and 20%, respectively, over that of ad libitum fed (AL) controls. Measurements of entry into S-phase were made in vivo of six different cell types in five different organs using 2-week exposures to BrdU. The labeling index (L.I.) in all organs studied was lower in young CR mice than in young AL fed mice. In most cases, the L.I. in AL mice fell to the levels of that in the CR mice by 13 months of age, and the two groups then remained so through old age. However, when the L.I. was measured in old CR mice which had been placed on the AL diet for a period of 4 weeks (this was termed refeeding (RF)), it was found to be above that of similar age AL or CR mice and almost at the level of young AL mice. This was still true, but to a lesser degree, in a repeat study using an 8-week period of RF. In a separate but parallel in vitro study (companion paper, this volume), the superiority of CR over AL for retention of cellular replication capacity was confirmed by clone size distribution measurements made in several cell types in mice of several age groups. These results indicate that: (1) the rate of cell replication in AL diet mice diminishes greatly by early middle age in all organ sites studied and then plateaus or declines much more slowly; (2) CR broadly preserves in vivo cellular replicative capacity but often requires the energy levels provided by a switch to AL feeding to demonstrate this late in life; (3) accordingly, the replicative deficit in AL fed mice appears to be cumulative and is significant only in old age. The mechanism(s) involved is yet to be discovered but may be related to, or even the same as, that which extends life spans in CR animals. Correspondingly, and with corroborative data from our in vitro companion study, (W. R. Pendergrass et al., 1995. Exp. Cell. Res. 217, 309-316), we suggest that cell populations sustain an accrual of biochemical damage or physiological alterations which increasingly limit their replicative capacity as the animal ages, and that CR reduces the accrual of this damage.     

菠菜叶片乙醇酸氧化酶的氧化甘油酸活性

首先从菠菜叶片中纯化了乙醇酸氧化酶（GO）。通过鉴定反应中氧的消耗以及反应产物H2O2的生成,证实菠菜GO具有氧化光呼吸途径中间代谢物甘油酸的活性。该氧化活性依赖于辅因子FMN和FAD,而不依赖核黄素和光黄素;其最适反应pH值为8．0,Km（甘油酸）值为7．14mmol／L,kcat值为1．04s^-1,活化能为17．29kJ／mol;草酸和丙酮酸对该氧化活性有明显的抑制作用,其中前者为典型的竞争性抑制。进一步通过两底物竞争作图表明：菠菜叶片GO氧化甘油酸反应和氧化乙醇酸反应为同一活性中心所催化。     

Identification and mutation of a gene required for glycerate kinase activity from a facultative methylotroph, Methylobacterium extorquens AM1.

A gene (gckA) responsible for the activity of glycerate kinase has been identified within a chromosomal fragment of the serine cycle methylotroph Methylobacterium extorquens AM1. A mutation in gckA leads to a specific C1-negative phenotype. The polypeptide sequence derived from gckA showed high similarity to a product of ttuD essential for tartrate metabolism in Agrobacterium vitis. Our data suggest that gckA and ttuD might be structural genes for glycerate kinase and that the serine cycle and the tartrate utilization pathway share a series of reactions.     

Only plant-type (GLYK) glycerate kinases produce d-glycerate 3-phosphate

d-Glycerate kinases (GK) occur in three phylogenetically distinct classes. Class II GKs produce glycerate 2-phosphate, while both class I GK and class III GK (GLYK) are thought to produce glycerate 3-phosphate. We report on the identification of a bacterial-type class I GK in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 and of a plant-type GLYK in the filamentous cyanobacterium Nostoc sp. strain PCC 7120. The comparison with other prokaryotic and eukaryotic GKs of both classes shows that glycerate 3-phosphate is produced only by the GLYKs, but, in contrast to current thinking, not by any of the examined class I enzymes.     

Enzymes of serine metabolism in normal and neoplastic rat tissues

Enzymes involved in the pathway of de novo serine biosynthesis (L-phosphoserine aminotransferase) and in alternative pathways of serine utilization (L-serine hydroxymethyltransferase, L-serine dehydratase and L-serine aminotransferase) were assayed in normal adult and fetal rat tissues and in a range of transplantable sat tumors. Serine dehydratase and serine aminotransferase activities were essentially confined to normal adult liver and kidney, whereas phosphoserine aminotransferase and serine hydroxymethyltransferase activities showed a more ubiquitous tissue distribution. In particular, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were appreciable in neoplastic tissues, in the absence of the other enzymes of serine utilization. The pattern of enzyme distribution suggests that the synthesis of serine de novo is metabolically coupled to its utilization for nucleotide biosynthesis in tumors of differing tissue origins.     

Enzymes of serine metabolism in normal and neoplastic rat tissues

Enzymes involved in the pathway of de novo serine biosynthesis (L-phosphoserine aminotransferase) and in alternative pathways of serine utilization (L-serine hydroxymethyltransferase, L-serine dehydratase and L-serine aminotransferase) were assayed in normal adult and fetal rat tissues and in a range of transplantable rat tumors. Serine dehydratase and serine aminotransferase activities were essentially confined to normal adult liver and kidney, whereas phosphoserine aminotransferase and serine hydroxymethyltransferase activities showed a more ubiquitous tissue distribution. In particular, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were appreciable in neoplastic tissues, in the absence of the other enzymes of serine utilization. The pattern of enzyme distribution suggests that the synthesis of serine de novo is metabolically coupled to its utilization for nucleotide biosynthesis in tumors of differing tissue origins.     

Peroxisomal and mitochondrial targeting of serine: Pyruvate/alanine: Glyoxylate aminotransferase in rat liver

Serine: pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, STPp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.