Serine Utilization in Mouse Liver: Inﬂuence of Caloric Restriction and Aging

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FEBS 29411 FEBS Letters 579 (2005) 2009–2013

Serine utilization in mouse liver: Inﬂuence of caloric restriction and aging

Kevork Hagopiana,*, Jon J. Ramseya, Richard Weindruchb,c a Department of Molecular Biosciences, School of Veterinary Medicine, University of California, One Shields Ave, Davis, CA 95616, USA b Department of Medicine, University of Wisconsin Medical School, Madison, WI 53706, USA c Geriatric Research, Educational and Clinical Center, William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terrace, Madison, WI 53705, USA

Received 18 November 2004; revised 11 February 2005; accepted 21 February 2005

Available online 9 March 2005

Edited by Judit Ova´di

about the long-term effects of CR on the major pathways of Abstract The influence of caloric restriction (CR) on the activities of hepatic serine metabolizing enzymes in young (3 months) intermediary metabolism. and old (30 months) mice was studied. Serine dehydratase Serine is synthesized in substantial amounts (370 mg/100 g (SDH) activity increased markedly with age in both diet groups body weight/day in the rat) and, therefore, is classified as non- and in old mice was higher in the CR group. No effects of CR essential amino acid [4]. Serine can be derived from glycine, were observed in the young. Serine:pyruvate transaminase phospholipids, protein degradation and from the glycolytic (SPT) and glycerate kinase activities were unaffected by age metabolite 3-phosphoglycerate. Serine is also important in and diet. However, glycerate dehydrogenase activity was de- the synthesis of the amino acids glycine, cysteine and taurine, creased in old CR mice but not in young CR. The results of this as well as providing the precursors for the synthesis of phos- study show that long-term CR influenced serine utilization only phatidylserine and ceramide [5]. As for utilization, serine is a in the pathway catalyzed by SDH. This suggests that in mouse glucogenic amino acid and in mammals two pathways are liver this pathway is critical for serine utilization in gluconeogenesis, while the SPT pathway plays a minor role. The increase in present for its catabolism. In the first pathway, serine is con- SDH activity with long-term CR is consistent with sustained in- verted directly to pyruvate by the action of serine dehydratase crease in gluconeogenesis. (SDH) (EC 4.3.1.17), while in the second pathway serine is 2005 Federation of European Biochemical Societies. Published converted by the action of SPT (EC 2.6.1.51) to hydroxypyru- by Elsevier B.V. All rights reserved. vate which is then further metabolized by the actions of glycerate dehydrogenase (GlycDH; EC 1.1.1.29) and glycerate kinase Keywords: Caloric restriction; Serine dehydratase; (GlycK; EC 2.7.1.31). Both of these pathways contribute to Serine:pyruvate transaminase; Glycerate dehydrogenase; gluconeogenesis, however, physiological status and species dif- Glycerate kinase ferences play a role in determining which pathway is actually operating [4]. The dehydratase pathway is increased under conditions associated with starvation and diabetes [6], while the transaminase pathway is increased with high-protein feeding [4]. Furthermore, species differences have also been ob- 1. Introduction served, with the transaminase activity being higher in carnivores, such as dogs and cats, while dehydratase being The aging process is associated with a variety of pathophys- higher in non-carnivores, such as mice and rats [7]. iological changes. Caloric restriction (CR), without malnutri- The influence of CR on key hepatic enzymes from several tion, has been shown to be the only environmental metabolic pathways has been reported previously. Those stud- intervention that delays such changes and extends maximum ied reported either increased glycolytic enzyme activities in life span in laboratory rodents and a variety of other species mice with CR [8] or decreased activities in both mice and rats [1,2]. This association between increased longevity and CR [9,10]. Increased gluconeogenic and nitrogen disposal enzyme has been known since the 1930s [3], nevertheless, the mode activities and decreased pyruvate dehydrogenase complex of action of CR is still unknown. The effect of CR on life span (PDH) and acetyl-CoA carboxylase (ACC) activities with is due entirely to a reduction in energy intake rather than a CR were also reported [9,11]. To better understand the effects reduction in a specific nutrient [2]. Despite the central role en- of CR on major metabolic pathways, we began a series of ergy metabolism plays in the actions of CR, little is known experiments investigating hepatic metabolic pathways in mice subjected to CR. The results of these studies on complete pathways have shown that CR leads to decreased glycolysis [12] increased gluconeogenesis and transamination [13], differential *Corresponding author. Fax: +1 530 752 4698. regulation of Krebs cycle [14] and increased activities in fruc- E-mail address: [email protected] (K. Hagopian). tose metabolism enzymes [15]. Since serine is critical in various biochemical events and is a Abbreviations: CR, caloric restriction; SDH, serine dehydratase; SPT, serine:pyruvate transaminase; GlycDH, glycerate dehydrogenase; glucogenic amino acid, and since gluconeogenesis is increased GlycK, glycerate kinase; PDH, pyruvate dehydrogenase complex; by CR, this work was undertaken to see how CR influenced ACC, acetyl-CoA carboxylase serine metabolism.

0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.02.062 2010 K. Hagopian et al. / FEBS Letters 579 (2005) 2009–2013

2. Materials and methods 0.014 * 2.1. Chemicals 0.012 All chemicals were purchased from Sigma Chemical Company (St. Louis, MO) or Roche Diagnostics Corporation (Indianapolis, IN). 0.010 Auxiliary enzymes and coenzymes used in the assays were purchased from Roche. Slide-A-Lyzer mini dialysis units (10,000 MWCO), for 0.008 the removal of ammonium sulfate from the auxiliary enzymes, were from Pierce (Rockford, IL). 0.006 mol/min/mg protein) µ 2.2. Animals 0.004 Male C57BL/6J mice were purchased from Charles River Laborato- ries (Wilmington, MA) at one month of age, housed singly and were 0.002 kept on a 12 h light/12 h dark cycle. All animals were maintained in Activity ( accordance with the Institutional and Federal Guidelines for Ethical 0.000 Animal Experimentation. The mice were fed ad libitum a non-purified 3 Months 30 Months diet, PLI 5001 (Purina Laboratories, St. Louis, MO) for one month and at two months of age they were assigned either to the control or Fig. 1. Activity of serine dehydratase in livers of young and old mice. CR group and fed semi-purified diets, as described elsewhere [16]. Activities were measured from the livers of young control and CR (3 When sacrificed, young animals were three months of age (one month months) and old control and CR (30 months) mice as described in the on either the control or CR diet) and old animals were 30 months of text. All activities were means ± S.E.M of at least six independent age (28 months on either the control or CR diet). Daily caloric intake experiments and expressed as lmol/min/mg protein. Controls, solid per animal was 12 kcal for the controls and 9 kcal for the CR mice. All bars; CR, open bars. \P < 0.02 CR vs old control. feeding procedures were as described previously [14].

2.3. Tissue harvesting and preparation 3.2. Serine:pyruvate transaminase pathway After overnight fast, all mice were sacrificed and their livers har- The activity of SPT was uninfluenced by either age or diet vested between 9 and 10 AM, as described previously [14]. Livers were (Fig. 2A). GlycDH activity (Fig. 2B) was unaffected by diet rapidly freeze-clamped in situ and placed immediately in liquid nitro- in young CR mice. In old mice, GlycDH activity was decreased gen. Livers were powdered in a mortar and pestle, under liquid nitro- (13%) in old CR mice (P < 0.04). While no differences were ob- gen, and the powders stored in liquid nitrogen for future use. served in GlycDH activity between young and old controls, old CR mice showed 19% decrease in activity (P < 0.025) when 2.4. Measurement of enzyme activities compared with young CR. In the case of GlycK (Fig. 2C), Liquid nitrogen-stored powders were removed as rapidly as possible no differences were observed between control and CR mice and placed in an ice-cold glass homogenizer, weighed and homoge- nized at a 1:10 ratio (w/v) and the supernatants saved for assays. in both young and old groups. No differences were observed SDH [17], serine:pyruvate transaminase (SPT) [18], D-GlycDH [19] between young and old controls but activity in old CR was and GlycK [19] were assayed according to the indicated methods. 16% lower (P < 0.03) than in the young CR. All assays were performed at 25 C, except for SPT which was at 30 C, using a Perkin–Elmer Lambda 25 UV/VIS spectrophotometer set at 340 nm and equipped with a Peltier heating control and nine-cell changer. Enzyme activities were expressed as lmol/min/mg protein 4. Discussion using an extinction coefficient of 6.22 mMÀ1 cmÀ1. In mammals, there are two distinct routes for serineÕs metab- 2.5. Other methods olism and utilization for gluconeogenesis (Fig. 3), which are Protein concentrations were measured with a Bio-Rad protein assay catalyzed by SDH or SPT [5,6]. We have previously shown kit, using BSA as the standard. The auxiliary enzymes, supplied in that CR resulted in increases in the activities of gluconeogenic 3.2 M ammonium sulfate suspensions, were first centrifuged at enzymes and transaminases of several amino acids [13]. There- 10 000 · g in a microfuge at 4 C, the supernatant discarded and the pellets re-suspended in a volume of dialysis buffer equal to the dis- fore, it was of interest to investigate the influence of CR on ser- carded supernatants. This was then dialyzed at 4 C, using the Slide- ine metabolism and to see whether this influence was exerted A-Lyzer mini dialysis units, either overnight or for 6 h with three via SDH or SPT pathways. changes of buffer. Statistical comparisons between diet and age groups The results show that serine metabolism in mouse liver pro- were performed using StudentÕs t test where values of P < 0.05 were ta- ceeds mainly via the direct SDH-catalyzed conversion of serine ken as statistically significant. to pyruvate, in agreement with previous reports [6,7]. The activity of SDH (Fig. 1) was increased markedly by age and further by CR in old mice (P < 0.02). Rat liver SDH activity 3. Results has been reported to be induced under gluconeogenic conditions [6], such as 24 h starvation or glucagon treatment of rats 3.1. Dehydratase pathway [20]. Since CR is accompanied by increases in gluconeogenesis The activity of SDH, which is involved in the direct conver- [13] and higher glucagon levels [21], it is likely that mouse liver sion of serine to pyruvate, was determined in both young and SDH behaves in a manner similar to that of rat liver. In rat li- old mice fed on control or CR diets (Fig. 1). In young CR ver, changes of activity with age have been reported [6,22,23], mice, no differences in activity were observed between control with adults showing higher SDH activity than juvenile and CR groups. However, old CR mice showed a 20% increase animals. in activity when compared with controls (P < 0.02). Moreover, SPT activity was, on the other hand, not influenced by age or in old mice, both control and CR were 6-fold higher in their diet (Fig. 2A), suggesting that the pathway involving serine activity (P < 0.0001) than young control and CR mice. conversion by SPT is not the critical one in serine metabolism. K. Hagopian et al. / FEBS Letters 579 (2005) 2009–2013 2011

0.0015 SPT Protein Catabolism

0.0012 SPT SHMT Hydroxypyruvate SERINE Glycine Fructose 0.0009 GlycDH F-1-P SDH Nucleic Acids GlyAld + DHAP 0.0006 AldDH mol/min/mg protein) TK TIM µ D-Glycerate Phospholipids 0.0003 GAP Pyruvate GlycK PYR-OXA-PEP Steps of Gluconeogenesis

Activity ( 0.0000 2-Phosphoglycerate Bypassing PYR-OXA-PEP steps of Gluconeogenesis A 3 Months 30 Months Gluconeogenesis

0.030 GlycDH Glucose

0.025 * Fig. 3. Serine utilization pathways in liver. Serine utilization pathways ‡ are indicated, with the two pathways studied here shown in solid 0.020 arrows, while the third pathway is shown in dotted arrows. The SDH catalyzed pathway, which is increased in activity, is indicated in bold 0.015 arrows. Serine utilizing enzymes are indicated by gray shading. SHMT, serine hydroxymethyltransferase; AldDH, aldehyde dehydrogenase; TK, triokinase; TIM, triosphosphate isomerase; PYR, pyruvate; OXA, mol/min/mg protein) 0.010 µ oxaloacetate; PEP, phosphoenolpyruvate; F-1-P, fructose-1-phosphate; GlyAld, glyceraldehyde; DHAP, dihydroxyacetone phosphate; 0.005 GAP, glyceraldehydes-3-phosphate.

Activity ( 0.000 B 3 Months 30 Months over, this high activity of SDH in mouse is in agreement with 0.012 GlycK previous ﬁndings where the activity of SDH is inversely related 0.010 to body size in higher animals, while SPT is higher in carnivores presumably because of the high-protein content of their † 0.008 diets [7]. Unlike SDH, no age-related changes in SPT were observed between young and old mice, which is similar to previ- 0.006 ously reported results from rat liver [6,26]. It is interesting to note that in rat liver, prolonged starvation mol/min/mg protein) 0.004 µ caused SDH activity to increase at a greater rate than SPT, suggesting that the potential for gluconeogenesis from pyru- 0.002 vate was increased greatly relative to that from hydroxypyruvate [27]. SPT activity has been reported not to increase with

Activity ( 0.000 starvation [24,28,29], and this has been attributed to the sup- C 3 Months 30 Months pressive eﬀects of cortisone [22]. Also, it has been observed that Fig. 2. Activities of serine:pyruvate transaminase, glycerate dehydro- in starved rats, alanine was the main amino acid used in gluco- genase and glycerate kinase activities in livers of young and old mice. neogenesis [30,31] and that alanine transaminase activity was Activities were measured from the livers of young control and CR (3 six to eight times higher than that of SDH [20]. We have also months) and old control and CR (30 months) mice as described in the text. All activities were means ± S.E.M of at least six independent reported high alanine transaminase activities from mouse liv- experiments and expressed as lmol/min/mg protein. Controls, solid ers [13], which are much higher than the SDH activities re- bars; CR, open bars. \P < 0.04 old control vs old CR; àP < 0.025 old ported here. Although alanine is a more dominant amino

CR vs young CR; P < 0.03, old CR vs young CR. acid in gluconeogenesis, nevertheless, under CR conditions serine is also utilized in this process predominantly by the action of SDH. While CR is not starvation, it seems that there are This is in agreement with previous findings in rat liver where certain similarities in the way these two amino acids and their SPT contributed to gluconeogenesis at an almost insignificant transaminases are influenced by these two dietary states. rate [24] and SPT contributed only 10–20%, even after As in SDH and SPT, no information is available concerning enhancement of activity by glucagon, suggesting that in rat li- the effects of CR on GlycDH and GlycK. These two enzymes ver SDH played a more important role than SPT [20,25].Itis sequentially catalyze the two reactions following SPT. also interesting to note that the SPT activity in this work rep- GlycDH activity was not altered by dietary treatment in young resents only 10% of the SDH activity in the old control and CR mice, however a 13% decrease (P < 0.04) was observed in old mice while in young controls and CR mice it is about 60% of CR mice when compared with old controls (Fig. 2B). The en- the SDH activity. This change could indicate that the pattern zyme shows an activity pattern similar to that of SPT, as re- of activity development is altered as animals get older, where ported previously in rat liver [6,32]. It has also been reported SDH increases and SPT decreases in older animals, which is that GlycDH activity was inhibited by several glycolytic in agreement with previous findings in rodents [6,23]. More- metabolites, such as 2- or 3-phosphoglycerate and fructose- 2012 K. Hagopian et al. / FEBS Letters 579 (2005) 2009–2013

1,6-bisphosphate [6]. Interestingly, this decreased activity in [5] De Koning, T.J., Snell, K., Duran, M., Berger, R., Poll-The, B.-T. old CR mice is paralleled by increased levels of 3-phosphoglyc- and Surtees, R. (2003) L-Serine in disease and development. erate and fructose-1,6-bisphosphate [12]. The unchanged Biochem. J. 371, 653–661. [6] Snell, K. (1984) Enzymes of serine metabolism in normal, GlycDH activity in the young control and CR mice were also developing and neoplastic rat tissues. Adv. Enzyme Regul. 22, matched by the unchanged levels of 2- or 3-phosphoglycerate 325–400. and fructose-1,6-bisphosphate levels reported previously [12]. [7] Rowsell, E.V., Carnie, J.A., Wahbi, S.D. and Al-Tai, A.H. (1979) This inhibitory influence of the glycolytic metabolites on L-serine dehydratase and L-serine-pyruvate aminotransferase activities in different animal species. Comp. Biochem. Physiol. 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