Mechanisms of Ageing and Development

123 (2002) 1183–1189

Age-related changes of cholesterol and dolichol biosynthesisin rat liver

Maria Marino a,*,1, Valentina Pallottini a,2, Cristina D’Eramo a,Gabriella Cavallini b, Ettore Bergamini b, Anna Trentalance a

a Department of Biology, Uni�ersity ‘Roma Tre’, V.le Marconi 446, 00146 Rome, Italyb Department of Experimental Pathology, V. Roma 55, 56126 Pisa, Italy

Received 8 November 2001; received in revised form 10 January 2002; accepted 10 January 2002

Abstract

Ageing has been defined as a gradually decreased ability to maintain homeostatic potential and increased risk todie, associated with a tissue accumulation of altered proteins and lipids. Among other, increased concentration of anisoprenoid compound, dolichol (Dol), in mammalian tissues during ageing has been reported and it has beenconsidered as a new biomarker of ageing. However, the mechanism and the role of this accumulation is still unknown.Aim of this work was to study the mechanism of age-dependent Dol accumulation in the liver analysing the activityof the hepatic rate-limiting enzyme of isoprenoid biosynthesis, the 3-hydroxy 3-methylglutaryl CoA reductase(HMGCoA reductase), the Dol synthesis by mevalonate (MVA), the Dol level in the plasma, and the cholesterol(Chol) synthesis and content of ageing rat fed ad libitum (AL) or subjected to the effect of food restriction. Since thecaloric restrictions are the most reproducible way to slow ageing and to extend life span, animals on these nutritionalregimens were used to study ageing related mechanisms. The data show that during ageing the hepatic Dolaccumulation is associated with an increase of HMGCoA reductase activity, which is affected by diet restriction, andwith an increase of MVA incorporation in Dol and Chol, which is not. In addition, the liver of aged rats maintainsthe capability to regulate its Chol content and to modify Chol delivery into the blood. © 2002 Elsevier ScienceIreland Ltd. All rights reserved.

Keywords: Dolichol; Cholesterol; 3-Hydroxy-3-methyl-glutaryl CoA reductase; Ageing; Caloric restriction; Rat liver

www.elsevier.com/locate/mechagedev

Abbre�iations: AL, ad libitum; Chol, cholesterol; Dol, dolichol; DR, 30–50% food restriction; EOD, every other day ad libitumfeeding; HDL-chol, cholesterol linked to high density lipoprotein; HMGCoA reductase, 3-hydroxy-3-methyl-glutaryl coenzyme Areductase; LDL-chol, cholesterol linked to low density lipoprotein; MVA, mevalonate.

* Corresponding author. Tel.: +39-06-5517-6344; fax: +39-06-5517-6321.E-mail address: m.marino@bio.uniroma3.it (M. Marino).1 This author contributed equally to this work.2 This author contributed equally to this work.

0047-6374/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.

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M. Marino et al. / Mechanisms of Ageing and De�elopment 123 (2002) 1183–11891184

1. Introduction

Dolichol (Dol) is a long chain polyisoprenoidcompound, containing, in dependence on species,from 16 to 23 isoprene units, which may bepresent in free alcohol or phosphorylated or es-terified forms. Although Dol is synthesised by thecommon isoprenoid pathway, the rate of its syn-thesis is slower than that of other final productsof such pathway (i.e. cholesterol (Chol),ubiquinone) (Carroll et al., 1992; Gruenler et al.,1994); Dol, like Chol, is broadly distributed in alltissues and cellular membranes (Chojnacki andDallner, 1988); actually small amounts of Dol,associated with lipoproteins, have been detectedalso in mammalian blood, and its hepatic originhas been postulated (Elmberger et al., 1989). Thiswide Dol presence in organisms may support itsfunctional role, but the knowledge on Dolcatabolism and cell function(s) in the variousanimal tissues is quite limited (Carroll et al.,1992). Dol has been reported to be involved in theorganisation and packing of phospholipids inmodel membranes (Vigo et al., 1984; Valtersson etal., 1985), and its role as the precursor of thephosphorylated form, the dolichyl-phosphate, isdebated (Chojnacki and Dallner, 1988).

In spite of such a poor information on thephysiological role of this isoprenoid compound,the accumulation of Dol has been extensivelyreported in various physiological states (i.e. hep-atic proliferation and early hepatic differentiation;Kin et al., 1983; Marino et al., 1986, 1990) orpathological conditions (i.e. hepatocarcinogenesis,Alzheimer disease, ceroid lipofuscinosis; Ericssonet al., 1993). Increased Dol concentration in mam-malian tissues during ageing has been also re-ported (Rip and Carroll, 1985; Chojnacki andDallner, 1988; Carroll et al., 1992); in particular,we have previously indicated Dol accumulation inliver as a new biomarker of ageing (Marino et al.,1998). However, the mechanism and the role ofthis accumulation is unknown.

Ageing has been defined as a gradually de-creased ability to maintain homeostatic potentialand increased risk to die (Masoro, 1995) associ-ated with a tissue accumulation of altered proteins(Vittorini et al., 1999), modified nucleic acids (So-

hal et al., 1994) and lipids (Marino et al., 1998).Restricting the energy intake well below that of adlibitum (AL) feeding slows the rate of ageing anddelays the appearance of many characteristics ofageing (Masoro, 1995). Different types of anti-ageing food restriction (namely, 30–50% foodrestriction (DR) and every other day AL feeding(EOD)) have similar effects on longevity (Roth etal., 1999) and on the abnormal accumulation ofDol (Marino et al., 1998) and altered proteins(Vittorini et al., 1999). Since DR and EOD are themost reproducible way to slow ageing and toextend life span, animal maintained on thesecaloric restrictions can be used to study ageingrelated mechanisms.

Aim of this work was to study the mechanismof the age-dependent Dol accumulation in theliver. The total and the expressed (dephosphory-lated) activity of the hepatic rate-limiting enzymeof isoprenoid biosynthesis, the 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoA reduc-tase), the Dol synthesis by mevalonate (MVA)and the Dol level in the plasma of ageing rat feedAL or subjected to the effects of food restrictionwere investigated. In the same experimental con-ditions Chol synthesis and content have been alsoverified.

The data show that during ageing the hepaticDol accumulation is associated with an increaseof HMGCoA reductase activity, affected by dietrestriction, and with an increase of MVA incorpo-ration in Dol and Chol, which is not. In addition,the liver of aged rats maintains the capability toregulate its Chol content and to modify Choldelivery into the blood.

2. Materials and methods

2.1. Materials

All materials used were of analytical or HPLCgrade.

2.2. Animals

Random groups of 3-month-old Sprague Daw-ley male rats were maintained on standard labora-

M. Marino et al. / Mechanisms of Ageing and De�elopment 123 (2002) 1183–1189 1185

tory food (Harlan autoclavable Teklad diet,Harlan Italy S.r.l., containing: 12.0% water,18.4% crude protein, 5.5% crude fat, 4.4% crudefiber, 5.6% crude ash) and water (AL) or on afood restricted regimen (DR or EOD). Rats hadfree access to water. At given age before thesacrifice, the animals, anaesthetised with the in-traperitoneal injection of pentobarbital (50 mg/kg body weight), were subjected to bloodcollection and right liver lobe excision.

2.3. HMGCoA reductase acti�ity assay

The assay was performed by the radioisotopicmethod, following the production of MVA from3-[14C]-HMGCoA (specific activity 57.0 mCi/mmol. Amersham-Pharmacia, Little Chalfont,UK). Five hundred milligram of liver were ho-mogenised in phosphate buffer containing 0.1 Msucrose, 0.05 M KCl, 0.04 M KH2PO4, 0.03 MEDTA, pH 7.4 in the presence or absence of 50mM NaF (phosphatase inhibitor), to measurethe total (−NaF) and the expressed enzyme ac-tivity (+NaF). Microsomes were prepared bycentrifugation of the homogenates as already re-ported (Bruscalupi et al., 1985). Microsomeswere incubated in the presence of co-factors (20mM glucose-6-phosphate, 205 mM NADPsodium salt, 1 unit of glucose-6-phosphate dehy-drogenase and 5 mM dithiothreitol). Final vol-ume was 200 �l. The assay was started by theaddition of 10 �l (0.088 �Ci/11.7 nmol) of 3-[14C]-HMGCoA. The radioactivity of the synthe-sised MVA, isolated by chromatography onAG1-X8 ion exchange resin (BioRad, Italy), wasmeasured and the recovery calculated on the ba-sis of the internal standard (3-[3H]-MVA, spe-cific activity 24.0 Ci/mmol. Amersham-Pharmacia, Little Chalfont, UK; Bruscalupi etal., 1985).

2.4. 2-[14C]-MVA incorporation andhigh-pressure liquid chromatography assay

About 0.1 g of liver slices (0.5–0.7 mm thick-ness) were incubated, as previously reported(Marino et al., 1986), with 1 �Ci of 2-[14C]-S-MVA (specific activity 58.1 mCi/mmol. Amer-

sham-Pharmacia, Little Chalfont, UK) and 3�M mevinoline (HMGCoA reductase inhibitor,Sigma Chemical Co., St. Louis, MO, USA), toprevent the isotopic dilution by endogenousMVA, in 3 ml 0.1 M KH2PO4 oxygenatedbuffer (pH 7.2) containing: 0.4 mM MgCl2, 0.03M nicotinamide and 0.125 M sucrose. After 3 hat 37 °C, the incubation was stopped by placingthe samples in ice. Liver slices were added to0.5 ml 0.25% pyrogallol in methanol (w/v) and0.5 ml 60% KOH and heated at 100 °C for 30min, and the saponifiable lipids were extractedtwice with diethyl ether/petroleum ether (1/1, v/v), then washed with methanol/water (1/1, v/v)and dried under nitrogen atmosphere. Reversed-phase HPLC was carried out by a Perkin–Elmer Apparatus (Norwalk, CT, USA) equippedwith an ODS-C18 column (5 �m, 4.6×150 mm)and precolumn (5 �m, 4.6×50 mm) wavelengthof 210 nm. Dol and Chol were eluted at 1 ml/min with a linear gradient of 20% (v/v) isopro-pyl-alcohol in methanol over 20 min, followedby isocratic elution with 80% isopropyl-alcoholin methanol for 15 min. Dol was eluted as afamily of peaks corresponding to Dol-17, -18,-19, -20, and -21. The radioactivity of each classof compound was measured in a liquid scintilla-tion beta counter (Camberra Pakard srl, Italy).The sensitivity of the method allowed us tomeasure at least 1.7 ng of total Dol and Chol.Recovery was 95% as determined by internalstandard (Dol-23) addition to samples (Eggenset al., 1983). A calibration curve of total peakheight against quantity injected (ng) was builtby using Dol standard (Sigma Chemical Co., St.Louis, MO, USA) and was linear in the 1.7–1000 ng range.

2.5. Plasma Dol and Chol content assays

Blood was collected on EDTA (0.4 mg/mlblood). Plasma was separated by centrifugation.

Saponification and analysis of Dol were per-formed on 1 ml of plasma as described for theliver. The analysis of total and lipoprotein-linked plasma Chol was performed using the di-agnostics kits for Total-Chol, HDL-Chol andLDL-Chol (Sigma Chemical Co.).

M. Marino et al. / Mechanisms of Ageing and De�elopment 123 (2002) 1183–11891186

2.6. Statistical analysis

The analysis of variance (ANOVA) test wasused to evaluate difference among multiple condi-tions. If positive, the Bonferroni test was em-ployed to evaluate the statistical significance.Values of P�0.05 were considered to besignificant.

3. Results

In the liver tissue of older AL rats, the levels ofDol increased while the levels of Chol did not,and the increase in Dol content was prevented bycaloric restriction (data not shown) confirmingour previous observation (Marino et al., 1998).

In order to study the mechanisms underlyingthe accumulation and the effects of food restric-tions, we measured HMGCoA reductase activityin the liver. The HMGCoA reductase activity isregulated by phosphorylation–dephosphorylationmechanisms, the unphosphorylated form (10–15% of total enzyme) representing the active en-zyme and the phosphorylated form the inactive.The activation of the enzyme by lisosomal phos-phatases during the preparative procedures can beavoided in presence of NaF (Bruscalupi et al.,1985). Therefore, the expressed and total enzymeactivities were measured in microsomes preparedin presence and in absence of NaF, respectively.

The expressed (+NaF) activity increased in theliver of 24-months-aged AL fed rats and caloricrestriction prevented the increase (Fig. 1). Totalenzyme activity (−NaF) was unaltered at anyconsidered age and dietetic regimen. The +NaF/−NaF ratio, which reflects the activation state ofthe enzyme in vivo, increased during ageing indi-cating that enzyme regulation may be impaired inAL (but not in the longer-lived food-restricted)rats.

The incorporation of MVA into Dol duringageing was studied in presence of mevinoline toavoid isotopic dilution and compared with theMVA incorporation into Chol. The resultsshowed that incorporation in either Dol or Cholincreased during ageing, and that diet restrictionsdid not significantly affect the change (Fig. 2).

Fig. 1. HMGCoA reductase activity in liver of 3- and 24-month-aged AL or DR or EOD fed rats. Microsomes wereprepared in presence or in absence of NaF (50 mM). The datarepresent the mean�S.D. of at least four different experi-ments. r represents the ratio between HMGCoA reductaseactivity in presence or in absence of NaF. P�0.001 performedby Bonferroni’s t-test: (a) significantly different from 3months, (b) significantly different from AL 24 months.

During ageing, as already observed in the Dolaccumulation (Marino et al., 1998), the newlyformed radioactive Dol maintained the chainlength distribution observable in the adult (datanot shown).

Fig. 2. Incorporation of [14C]MVA in liver Chol and Dol of 6-,12-, and 24-month-aged AL or DR or EOD fed rats. Data areexpressed as percentage change with respect to the control rats(3-month-aged). The data represent the mean�S.D. of atleast four different experiments. P�0.001 performed by Bon-ferroni’s t-test: (a) significantly different from 3 months, (b)significantly different from 6 months, (c) significantly differentfrom 12 months.

M. Marino et al. / Mechanisms of Ageing and De�elopment 123 (2002) 1183–1189 1187

Fig. 3. Dol levels in plasma of 3- and 24-month-aged AL orDR or EOD fed rats. The data represent the mean�S.D. ofat least four different experiments. P�0.001 performed byBonferroni’s t-test, (a) significantly different from 3 months.

4. Discussion

An accumulation of Dol during ageing hadbeen reported in many tissues and in differentanimal species (Rip and Carroll, 1985; Chojnackiand Dallner, 1988; Carroll et al., 1992; Marino etal., 1998). It represents a common feature of theageing process but the mechanism by which Dolaccumulates during ageing is still unclear.

Aim of this study was to investigate the mecha-nisms underlying the Dol accumulation duringageing and the effect exerted by caloric restriction.The obtained results show that the Dol synthesisis increased always during ageing, but that accu-mulation of Dol in the ageing liver occurs onlywhen HMGCoA reductase too is increased (bothaccumulation of Dol and alteration of HMGCoAreductase are prevented by an anti-ageing caloricrestriction). Levels of plasma Dol indicate thatbesides the increase in the rate of Dol synthesis anincrease of Dol release may occur and that thesize of these increments could be, at least par-tially, connected with the accumulation observed.In addition, other processes as well as a modifiedrate of Dol disappearance in the liver cannot beexcluded, even if conflicting data are available onthe Dol degradation. In fact, while Rip and Car-roll (1985), Carroll et al. (1992) were unsuccessfulto demonstrate catabolism of Dol, Adair andKeller (1982) sustained the existence of suchcatabolic pathway measuring in vivo a Dol halflife ranging between 80 and 118 h in the liver.Moreover, recently Nanni et al. (2000) demon-strated the susceptibility of the Dol molecule tooxidative degradation. At the present, such degra-dative processes require further studies to definethe not yet described enzymatic steps.

Our data indicate that HMGCoA reductasemay undergo a severe age-related impairment ofregulation without any significant change in liverChol. In particular, the high expressed activity ofHMGCoA reductase seems related to a loss ofregulation by phosphorylation–dephosphoryla-tion mechanisms, prevented by anti-ageing diets.Thus, the aged rat might keep the hepatic Cholhomeostasis balancing the increased synthesiswith an increased release in the blood; the final

At any considered age and dietary regimen, theincrease in Dol synthesis was associated with anincrease in plasma Dol content (Fig. 3). Con-versely, the ageing-related increase in Chol syn-thesis was associated with an unchanged Cholcontent in the liver and with an increase in all thelipoprotein fractions considered, both in AL andin EOD or DR fed rats (Fig. 4), suggesting thatthe ageing liver maintains Chol homeostasis bymodifying the release of the molecule in theblood.

Fig. 4. Total Chol, HDL-Chol and LDL-Chol in plasma of 3-and 24-month-aged AL or DR or EOD fed rats. The datarepresent the mean�S.D. of at least four different experi-ments. P�0.001 performed by Bonferroni’s t-test, (a) signifi-cantly different from 3 months, (b) significantly different fromAL 24 months.

M. Marino et al. / Mechanisms of Ageing and De�elopment 123 (2002) 1183–11891188

result is an ipercholesterolemic status, more seri-ous in the AL-fed-animal than in the DR orEOD-fed ones. The ageing-related hypercholes-terolemia is not prevented fully by caloric restric-tion even if under such diet regimen the activity ofHMGCoA reductase is as low as in adult age.Other Authors reported age-related hypercholes-terolemia associated with low activity of prenyltransferases (Stahlberg et al., 1991; Thelin et al.,1994). Our data, showing an enhanced MVA con-version into isoprenoid compounds, could be onlyapparently conflicting with those observationssince, beside the HMGCoA reductase, other en-zymes of the MVA pathway (i.e. MVA kinase)could be altered by ageing. The alteration of thesebranch-point enzymes of the MVA pathway(Gruenler et al., 1994) could be also responsible ofthe different behaviour of Dol and Chol levelduring ageing.

In conclusion, the presented data show that thehepatic accumulation of Dol is related with theloss of enzymatic regulation characteristic of age-ing (Conconi et al., 1996). In fact, a higher MVAavailability deriving from an increased expressedactivity of HMGCoA reductase could cause anincreased production of Dol not balanced by aproportional enhancement of its release in theblood.

Acknowledgements

The generous gift of Dolicol 23 from Dr EvaSwiezewska (Institute of Biochemistry and Bio-physics, Polish Academy of Science, Warszawa,Poland) is gratefully acknowledged. This workwas supported by grant ‘ex 60%’ 2000 to M.Marino from University of ‘Roma Tre’.

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