Effect of dietary restriction and N-acetylcysteine supplementation

on intestinal mucosa and liver mitochondrial redox status

and function in aged rats

Ignazio Grattaglianoa,*,1, Piero Portincasaa,1, Tiziana Coccob, Antonio Moschettaa,Marco Di Paolab,c, Vincenzo O. Palmieria, Giuseppe Palascianoa

aSection of Internal Medicine, Department of Internal Medicine and Public Medicine (DIMIMP), University of Bari, P.zza G. Cesare, 11, 70124 Bari, ItalybSection of Medical Biochemistry, Department of Medical Biochemistry and Biology, University of Bari, Bari, Italy

cInstitute of Biomembranes and Bioenergetics (C.N.R.), University of Bari, Bari, Italy

Received 23 February 2004; received in revised form 12 May 2004; accepted 1 June 2004

Available online 25 June 2004

Abstract

The age-related changes of glutathione (GSH) levels and the effect of hypocaloric regimen and N-acetylcysteine (NAC) supplementation

were investigated in intestinal mucosa and liver mitochondria of 28 months rats.

Old rats exhibited lower proteins, GSH and protein sulphydrils (PSH) concentrations, higher GSH-peroxidase (GSH-Px) activity and

protein carbonyl deposit, partial inhibition of succinate stimulated mitochondrial state III respiration and decreased mitochondrial

nitrosothiols (RSNO) concentration. Lower electric potential and current intensity were found in the colonic mucosa.

Old rats undergone hypocaloric regimen showed higher intestinal concentrations of GSH, lower oxidized protein accumulation and GSH-

Px activity and higher mitochondrial RSNO levels. Mitochondrial state III respiration and intestinal transport were improved.

NAC supplementation enhanced GSH and PSH levels in the ileal but not in the colonic mucosa, GSH and RSNO in liver mitochondria,

while GSH-Px and protein carbonyls were decreased everywhere. Mitochondrial respiration ameliorated.

In conclusion, ageing is characterized by a spread decrease of GSH concentrations, increased protein oxidation and decreased

mitochondrial NO content. Hypocaloric diet ameliorated intestinal transport and, as well as NAC, was effective in enhancing GSH levels but

at different extent according to the investigated districts. Both interventions reduced the age-associated increase of GSH-Px and protein

carbonyls and improved mitochondrial respiration.

q 2004 Elsevier Inc. All rights reserved.

Keywords: Glutathione; Glutathione peroxidase; Intestinal transport; Mitochondrial respiration; Nitrosothiols; Protein sulfhydrils; Ussing chamber

1. Introduction

The free radical theory of ageing (Harman, 1992) has

found several confirms in recent years (Miquel, 1992;

Guerrieri et al., 1996). Many cellular and tissue changes

observed in aged animals are currently, at least in part,

attributed to free radical excess and/or to scavenger systems

failure (Stadtman, 1992).

The redox state is therefore one of the major factor

allowing the perpetuation of the functional capacity of

the cell. Deficiency in antioxidant defense is constantly

associated with oxidative product accumulation (Gratta-

gliano et al., 1996; Wieland and Lauterburg, 1995) and

inversely correlates with the concentration of protein

sulphydrils (PSH) (Boscia et al., 2000). PSH oxidation

promotes the mitochondrial permeability transition

(Morin et al., 2001; Chiba et al., 1996), impairs

mitochondrial – nuclear signalling pathway and the

expression of nuclear genes and factors (Ginn-Pease

and Whisler, 1996). By contrast, antioxidant supplemen-

tation has been shown to improve cell response to

physiologic stimuli (Grattagliano et al., 2003) and to

decrease lipid and protein oxidation (Grattagliano et al.,

1999). Glutathione (GSH) and its related enzymes play a

central role in the defense against excess oxygen (Sies,

1999) and nitrogen (Andre and Feley-Bosco, 2003)

0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.exger.2004.06.001

Experimental Gerontology 39 (2004) 1323–1332

www.elsevier.com/locate/expgero

1 These authors contributed equally to this report.

* Corresponding author. Tel.: þ39-80-547-8233; fax: þ39-80-547-8232.

E-mail address: i.grattagliano@semeiotica.uniba.it (I. Grattagliano).

radicals by forming oxidized glutathione (GSSG) and

nitroso-glutathione (GSNO), respectively (Mathews and

Kerr, 1993). Both these products are highly represented

in aged tissues and are believed to act as bioactive

intermediates (Olafdottir and Reed, 1988; Clancy et al.,

1994; Steffen et al., 2001; Glebska et al., 2003),

especially at mitochondrial level where they modulate

free radical dependent redox cell signalling (Kamata and

Hirata, 1999; Brookes et al., 2002). Therefore, cells

deprived of GSH exhibit defective proliferative response

(Huang et al., 2001; Hamilos et al., 1989; Tanaka et al.,

1998), while the maintenance of GSH levels, especially

in the mitochondrial compartment, prevents the decreased

replication of hepatocytes exposed to xenobiotics or

hormonal dysfunction (Devi et al., 1993; Grattagliano

et al., 2003), increases the tolerance to stressing insults

(Colell et al., 1998) and may extend life-span (Orr and

Sohal, 1994). At this concern, ageing process is

characterized by alterations of both energy production

and GSH metabolism (Guerrieri et al., 1996) and by

defective replication capacity (Li and Holbrook, 2003).

Intestinal mucosa is known to become atrophic (Wang

et al., 2003) and to reduce its function (Schmucker et al.,

2001; Ferraris and Vinnakota, 1993) with years. This might

also result in an impairment of the intestinal epithelial

barrier and ionic transport function.

Despite several investigations have been conducted in

aged animals, poor and controversial data have been

reported on the GSH status and related redox system of

intestines. Indeed, GSH is very important for intestinal

function; GSH depletion is accompanied by colonic cell

degeneration and villi atrophy (Martensson et al., 1990).

Intestinal GSH mostly derives from diet and biliary supply

(Hagen et al., 1990; Ballatori and Truong, 1989): the latter

reflects the hepatic stores (Vendemiale et al., 1994) and is

severely affected by biliary obstruction, the former may be

strongly influenced by food restriction which is believed to

extend life-span and favor the maintenance of organ

function (Weindruch and Sohal, 1997). However, whether

interventions directed to increase intestinal GSH content

does really improve intestinal function in aged individuals is

not known.

Therefore, this study was aimed to investigate the

effect of hypocaloric regimen and GSH precursor

N-acetylcysteine (NAC) supplementation on oxidative

and nitrosative stress markers, intestinal transmucosal

transport and liver mitochondrial respiration in aged rats.

2. Materials

Male Wistar rats, purchased from Harlan Italy, were

housed in a temperature-controlled room with a dark/light

cycle and had free access to food and water for the whole

period of experiment. Rats were killed by decapitation at

age of 2, 5 and 28 months.

Starting at month 12 and continued until sacrifice, some

rats were subjected to a hypocaloric regimen according to

the ‘every-other-day (EOD) feeding’ method, which is

equivalent to a reduction of caloric intake to about 60%

compared to the ‘ad libitum’ fed controls of the same age

(Goodrick et al., 1983). In brief: the food was available all

the time for the ad libitum fed controls, whilst it was given

every second day remaining available for 24 h, and then it

was removed for another 24 h in the diet restricted group.

Last day before killing was always a fasting one (Nagy et al.,

1993). By starting at 12 month and continued until

sacrifice, another group of normally fed rats received

0.3% w/w NAC in addition to the diet as previously reported

(Martinez et al., 2000). No major differences were

registered in rat body weight among groups at time of

sacrifice. The study was conformed to the Guide for the

Care and Use of Laboratory Animals.

3. Methods

3.1. Preparation of ileum and colon

After killing, a middle tract of the small intestine

(approximately 20 cm) and the colon were removed and

immediately rinsed in ice-cold saline. Bowel segments were

opened longitudinally along the mesenteric border and fixed

by small needles on a stiff surface; the mucosa was obtained

by gentle scraping with a spatula.

3.2. Isolation of Liver mitochondria

Livers were homogenized in 10 volumes of a medium

containing 0.25 M sucrose, 10 mM Tris–HCl (pH 7.4),

0.1 mM EGTA, 0.25 mM PMSF. Mitochondria

were isolated by differential centrifugation steps and the

post-mitochondrial fraction was collected during

mitochondria isolation (Cocco et al., 2002).

Total GSH concentration was measured enzymatically at

412 nm after precipitation of proteins by adding 15%

sulfosalycilic acid (SSA) to intestinal and mitochondria

homogenate. After centrifugation, 20 ml of supernatant

were processed by the glutathione reductase recycling

procedure (Tietze, 1969).

GSSG was measured spectrophotometrically at 412 nm

after incubation of the acidic SSA precipitate with

2-vinylpiridine and triethanolamine (Griffith, 1980).

PSH content was determined with the Elmann’s

procedure, as previously modified (Grattagliano et al.,

1996). In brief, protein pellet, obtained by precipitation with

4% SSA, was resuspended in 800 ml of 6 M guanidine and

successively spectrophotometrically detected at 412 and

530 nm before and after incubation with 50 ml of 10 mM

5,5-dithiobis (2-nitrobenzoic acid).

Protein carbonyls were measured as previously

described (Levine et al., 1990). Briefly, equal aliquots

I. Grattagliano et al. / Experimental Gerontology 39 (2004) 1323–13321324

of proteins were incubated with 2N HCl or 0.2%

dinitro-phenylhydrazine in 2N HCl. Next, proteins were

precipitated by adding 50% TCA and subsequently

washed with 1:1 ethanol/ethylacetate. The final precipi-

tate was dissolved in 6 M guanidine and the spectrum of

absorbance of the hydrazone derivatives was spectro-

photometrically followed. The extinction coefficient for

aliphatic hydrazones (21.5/nM/cm) was used to calculate

carbonyl group concentrations.

Glutathione peroxidase activity was assessed using the

method described by Flohe and Gunzler (1984): one unit of

activity was given by the amount of enzyme that consumes

1.15 mmol NADPH/min at 37 8C (pH 7.0).

RSNO levels were measured following the method

described by Cook et al. (1996), which uses a mixture

of SULF/NEDD (neutral Griess) as reagents. Briefly,

post-mitochondrial fraction and mitochondria, suspended

(1:4) in PBS (pH 7.4), were acidified with 25% SSA

and centrifuged at 10,000g for 10 min. The supernatant

(200 ml) was added to 40 ml of 1% ammonium sulfamate,

200 ml of 0.4N HCl containing 0.3% HgCl2 and 4.6%

sulphanilamide, and 300 ml of 0.4N HCl containing 0.2%

1-naphthyl-ethylendiamide dihydrochloride. After 30 min

of incubation at room temperature, the samples were

spectrophotometrically analyzed at 544 nm. Standards

were prepared by reacting equal molar of GSH and nitrite

in water.

Intestinal electrical activity was evaluated on proximal

colon muscle-stripped mucosa. The mucosa, obtained

after careful dissection and peeling of the mucosa

containing-lamina propria, was mounted in parallel

Ussing chambers (Dipl.-Ing. K.Mussler Scientific Instru-

ments, Aachen, Germany) with a 0.8 cm2 opening surface

area and each mucosal side was bathed in a slightly

modified Krebs-solution (in mM): 107 NaCl, 25

NaHCO3, 1.25 CaCl2, 0.2 NaH2PO4, 1.8 Na2HPO4, 4.5

KCl, 1 MgCl2, 12 glucose, at pH 7.2. (Moschetta et al.,

2003). The solution was continuously oxygenated with

O2/CO2 (95%/5%). Within each chamber, two pairs of

Ag/AgCl electrodes monitored the transmural potential

difference (PD, in mV) under open-circuit conditions or

the short-circuit current (ISC, mA/cm2) with transmural

PD clamped to zero. Changes in ISC depend on net active

ion transport across the epithelium (Greenwood-Van

Meerveld et al., 2000). In this system, the transepithelial

current (ISC) is obtained from measurements of the

transepithelial voltage (Vte) and resistance (Rte) according

to Ohm’s law:

ðISC ¼ Vte=RteÞ:

After an equilibration time of 40–60 min, PD, ISC and

tissue resistance (Rt, in V cm2) were measured in the

basal state every 6 s under voltage-clamped conditions

(Natale et al., 2003).

3.3. Measurement of mitochondrial oxygen

consumption rate

The respiratory activity of freshly prepared mitochondria

was measured polarographically in a Rank Brothers

oxygraph (Bottisham Cambridge, England) at 25 8C.

Mitochondria were suspended at 0.25 mg/ml in a medium

containing 75 mM sucrose, 50 mM KCl, 30 mM Tris–HCl

(pH 7.4), 0.2 mM EDTA, 0.1 mM EGTA, 5 mM KH2PO4.

State 2 respiration started by the addition of glutamate

(5 mM)/malate (2.5 mM) or succinate (10 mM) in the

presence of 1 mg/ml rotenone. State 3 respiration was

induced by addition of 1 mM ADP (Cocco et al., 2002).

Protein concentration was measured by using a Bio-Rad

kit for the assay of proteins (Bio-Rad GmbH, Munich,

Germany).

3.4. Chemicals

All the materials used were purchased from Sigma-

Aldrich Chemical Co. (Milano, Italy) or were of the highest

purity grade commercially available.

3.5. Statistical analysis

For comparison among groups, the one-way analysis of

variance (ANOVA) and the Student’s t-test for unpaired

data were used. P , 0.05 defined significance. All data are

expressed as mean ^ SD.

4. Results

4.1. Age-associated changes in intestinal mucosa

and liver fractions

With the exception of the total protein content and PSH

concentrations (Table 1; Fig. 1), which were higher in the

intestinal mucosa of 5 months old rats, no major differences

were noted with regard to GSH concentration and GSH-Px

activity between young and adult rats at intestinal level.

No differences were also observed with regard to the

intestinal transmembrane transport as well as for liver

mitochondrial respiration in the presence of glutamate plus

malate as substrates.

Compared to young and adult rats, 28 months old rats

exhibited uniformly lower concentrations of GSH and PSH

and higher levels of GSSG in all the investigated districts

(Figs. 1 and 2; Table 2). The total protein content was

decreased in both intestinal segments, while their concen-

trations were unaffected in liver fractions (Table 1). Aged

rats showed a higher GSH-Px activity and accumulation of

protein carbonyls (Table 1) especially in the colonic mucosa

and in liver mitochondria. Old rats exhibited also a partial

inhibition of succinate-supported respiration with no

difference in glutamate/malate-dependent respiration in

I. Grattagliano et al. / Experimental Gerontology 39 (2004) 1323–1332 1325

the presence and in absence of ADP (Fig. 3).

The mitochondrial concentration of RSNO was found to

be lower in old compared to young and adult rats (Table 3).

A lower transmural potential difference was observed in the

colon of old rats together with lower short-circuit current

compared to young rats (Table 4).

4.2. Effect of hypocaloric diet

Compared to normally fed, old rats undergone

hypocaloric regimen showed higher concentrations of

GSH in the small (0.86 ^ 0.17 mmol/g tissue) and large

(0.78 ^ 0.10 mmol/g tissue) intestinal mucosae (Fig. 4)

and lower percentage amount of GSSG (Table 2).

By contrast, the PSH content was not significantly varied.

In the colon, a lower accumulation of oxidized proteins and

a more restricted increase of the GSH-Px activity were

observed (Tables 1 and 3). Diet restriction improved

transmural potential difference and short-circuit current

which were now comparable to those found in young rats

(Table 4).

No significant variation was noted with regard to GSH

(Fig. 4) and PSH concentrations in the liver fractions of old

rats fed with restricted diet. In the mitochondrial

compartment but not in the extra-mitochondrial space,

undernourished rats showed a lower enhancement of

Table 1

Total protein (A) and protein carbonyl (B) concentrations in ileal and colonic mucosae, liver mitochondria and post-mitochondrial fraction of young, adult and

28 months old rats, and effect of hypocaloric regimen and N-acetylcysteine (NAC) supplementation in old rats (see Section 3)

2 months 5 months 28 months Hypocaloric NAC suppl.

A

Ileum 2.0 ^ 0.3 3.7 ^ 0.2a 1.7 ^ 0.2b 1.7 ^ 0.6 2.1 ^ 0.6

Colon 3.0 ^ 0.5 3.4 ^ 0.3 1.0 ^ 0.1b 1.0 ^ 0.5 1.4 ^ 0.2c

L. Mitoc. 18 ^ 5 21 ^ 4 20 ^ 4 17 ^ 5 19 ^ 4

L. Post-mitoc. 12 ^ 4 14 ^ 4 15 ^ 5 13 ^ 5 17 ^ 5

B

Ileum 0.9 ^ 0.2 0.8 ^ 0.2 1.9 ^ 0.3b 1.5 ^ 0.3 1.3 ^ 0.4c

Colon 0.9 ^ 0.2 1.0 ^ 0.1 2.8 ^ 0.5b 2.0 ^ 0.4c 2.5 ^ 0.5

L. Mitoc. 1.1 ^ 0.1 1.2 ^ 0.1 3.9 ^ 0.8b 3.4 ^ 0.3c 3.1 ^ 0.7c

L. Post-mitoc. 1.2 ^ 0.1 1.1 ^ 0.2 2.6 ^ 0.4b 2.2 ^ 0.5 1.9 ^ 0.4c

Total proteins and protein carbonyl concentrations are expressed as mg protein/ml homogenate and nmol/mg protein, respectively.a Significantly different vs. young and old rats.b Significantly different vs. young and adult rats.c Significantly different vs. untreated old rats.

Fig. 1. Protein sulphydril (PSH) concentrations in small intestinal and colonic mucosae, liver mitochondria and post-mitochondrial fractions of young

(2 months), adult (5 months) and old rats (28 months). *Significantly different compared to young and adult rats.

I. Grattagliano et al. / Experimental Gerontology 39 (2004) 1323–13321326

the GSH-Px activity and of GSSG concentration (Tables 2

and 3) and a decreased accumulation of protein carbonyls

(Table 1). RSNO concentration was higher than in control

old rats especially in the mitochondrial compartment

(Table 3). Mitochondrial respiration, in particular in the

presence of ADP (state III), was also improved in underfed

rats with both the substrates (Fig. 3).

4.3. Effect of NAC supplementation

Addition of NAC to the diet significantly increased the

levels of GSH (0.94 ^ 0.11 mmol/g tissue) and PSH

(25.5 ^ 3.4 nmol/mg protein) in the ideal tract but not in

the colon, without major effects on the absolute GSSG

concentrations. Compared to control old rats, the GSH-Px

activity was decreased only in the small intestine (Table 3),

while the protein carbonyl content was significantly lower in

both the intestinal tracts. The colonic tranmucosal electric

activity was unaffected by NAC (Table 4).

NAC supplementation resulted in significantly higher

GSH concentrations in extra- (24.9 ^ 1.3 nmol/mg protein)

and intra-mitochondrial (5.9 ^ 0.8 nmol/mg protein)

compartment (Fig. 4). No such changes were observed for

GSSG and PSH. Mitochondrial RSNO concentrations were

also higher in rats receiving NAC (Table 3) with levels

comparable to those found in adult rats. Rats receiving NAC

showed a lower increase of GSH-Px activity (Table 3) and

protein carbonyl accumulation (Table 1) in both hepatic

fractions, and an improvement of the glutamate plus malate

stimulated mitochondrial state III respiration (Fig. 3).

5. Discussion

Mounting experimental and clinical evidence suggest that

reactive oxygen species play an important role in cellular

senescence and ageing especially in some tissues with high

susceptibility to oxidative alterations (Rebrin et al., 2003).

Little is known, however, on the modifications occurring at

intestinal level and on the effect of diet regimen variation.

Our results show that old rats have decreased GSH and

PSH levels and accumulation of protein carbonyls in the

intestinal mucosa. However, differences were noted

between ileum and colon. In the latter, an increased

GSH-Px activity was associated with higher GSSG content

and with higher extent of protein oxidation, which suggests

that the colonic mucosa is exposed to free radical effect.

Fig. 2. Total glutathione (GSH) concentrations in small intestinal and colonic mucosae, liver mitochondria and post-mitochondrial fractions of young

(2 months), adult (5 months) and old rats (28 months). *Significantly different compared to young and adult rats.

Table 2

Oxidized glutathione (GSSG) content in ileal and colonic mucosae, liver

mitochondria and post-mitochondrial fraction of young and 28 months old

rats, and effect of hypocaloric regimen and N-acetylcysteine (NAC)

supplementation in old rats

2 months 28 months Hypocaloric NAC suppl.

Ileum 1.6 ^ 0.3 3.2 ^ 0.6a 2.3 ^ 0.5b 2.8 ^ 0.9

Colon 1.4 ^ 0.5 4.0 ^ 0.7a 2.6 ^ 0.6b 3.4 ^ 0.8

L. Mitoc. 1.8 ^ 0.5 3.0 ^ 0.5a 1.7 ^ 0.5b 2.3 ^ 0.7

L. Post-mitoc. 1.2 ^ 0.4 2.7 ^ 0.5a 2.3 ^ 0.5 2.6 ^ 0.6

GSSG is expressed as percentage of total glutathione.a Significantly different vs. young rats.b Significantly different vs. untreated old rats.

I. Grattagliano et al. / Experimental Gerontology 39 (2004) 1323–1332 1327

An increased GSH oxidative consumption more than a

decreased ex-novo synthesis may be the plausible

mechanism explaining the lower intestinal GSH content in

old rats.

A decreased GSH availability renders intestinal cells

more exposed to toxic insults and injury (Altomare et al.,

1998; Victor et al., 1991), and because cell replication in

aged animals is defective (Iakova et al., 2003), not repairable

damages may result in tissue dysfunction. As a

consequence, the lower PSH content may depend on a

reduced GSH availability (Augusteyn, 1979) or on

a decreased synthesis, even if increased oxidation or

diminished reduction capacity (Nordberg and Arner, 2001)

may not be excluded. The decreased content of PSH in

the intestinal mucosa of aged rats may affect transport and

absorption capacity (Mirabelli et al., 1988).

However, although a lower GSH content in the intestinal

mucosa of old rats would point to a less active filter by

epithelial barrier against luminal toxins, GSH-Px activity

was higher, indicating a persistently active antioxidant

barrier.

In the present study, there was an impairment of the

intestinal transmucosal ionic transport in old rats. The lower

short current intensity observed in colonic mucosae of old

rats compared to those of young rats mounted in Ussing

chambers, points to a decrease of net active ion transport

across the epithelium (Greenwood-Van Meerveld et al.,

2000). The effect of aging on active ion transport and

epithelial cell morphology were studied in the rabbit colon

where it was shown that cAMP-dependent secretion of

Fig. 3. Succinate-stimulated (left panels) and glutamate/malate-stimulated (right panels) respiration in liver mitochondria of young and 28 months old rats and

effect of hypocaloric regimen and N-acetylcysteine (NAC) supplementation in old rats. *Significantly different compared to young and adult rats;oSignificantly different compared to untreated old rats.

Table 3

Glutathione peroxidase (GSH-Px) activity (A) in ileal and colonic mucosae, liver mitochondria and post-mitochondrial fractions, and nitroso-thiols (RSNO)

concentrations (B) in liver mitochondria and post-mitochondrial fractions of young, adult and 28 months old rats, and effect of hypocaloric regimen and

N-acetylcysteine (NAC) supplementation in old rats (see Section 3)

2 months 5 months 28 months Hypocaloric NAC suppl.

A

Ileum 876 ^ 112 1002 ^ 322 1122 ^ 153 1067 ^ 158 726 ^ 144a

Colon 410 ^ 151 494 ^ 182 941 ^ 195b 717 ^ 177 855 ^ 99

L. Mitoc. 2029 ^ 95 1924 ^ 198 3107 ^ 179b 2712 ^ 140a 2609 ^ 198a

L. Post-mitoc. 1717 ^ 104 1882 ^ 172 2397 ^ 154b 2011 ^ 171 1952 ^ 134a

B

L. Mitoch. 0.54 ^ 0.11 0.51 ^ 0.12 0.34 ^ 0.11b 0.44 ^ 0.13a 0.58 ^ 0.08a

L. Post-mitoc. 0.43 ^ 0.21 0.50 ^ 0.22 0.29 ^ 0.16b 0.35 ^ 0.12 0.54 ^ 0.16a

GSH-Px activity and RSNO concentrations are expressed as nmol/mg protein/min and nmol/mg protein, respectively.a Significant difference compared to untreated old rats.b Significant difference compared to young and adult.

I. Grattagliano et al. / Experimental Gerontology 39 (2004) 1323–13321328

the Chloride ion (but not Na þ absorption), was

significantly decreased in mature compared with young

animals (Braaten et al., 1988). Morphologically, there was

decreased density in nongoblet cells in colonic crypts which

was paralleled by decreased stool water content in mature

animals. Also, our findings seem to parallel those obtained

in rat colonic mucosae upon incubation with prosecretory

and cytotoxic (Moschetta et al., 2003) as well as

proinflammatory stimuli (Natale et al., 2003) and represent

the functional impairment of the intestinal epithelial

barrier. Taken together, such electrophysiological and

morphological studies point to an age-dependent decrease

in ion transports (Veeze et al., 1994) which parallel other

associated events, namely changes in water permeability

(Marin and Aperia, 1984), colonic motor function

(McDougal et al., 1984), and endocrine cell population

(Sandstrom et al., 1998). Such events may partly explain the

increased prevalence of delayed colonic transit and

constipations in adults.

The liver, chosen as a referent organ for intestinal

investigations, showed alterations similar to those observed

in the intestinal mucosa with comparable decline in GSH

and PSH both in the mitochondrial as well as in the

extra-mitochondrial space. The higher GSH-Px activity and

the wider protein carbonyl deposit found in mitochondria of

old rats together with the low GSH level are expressions of a

high susceptibility of these organelles to oxidative damages,

and renders this cellular compartment particularly prone to

the action of pro-apoptotic molecules such as nitroxides

(Glebska et al., 2003). The differences in the redox balance

between intra- and extra-mitochondrial compartments also

suggest that mitochondria are elective targets of age induced

damages and that they may play, in turn, a central role

in ageing process by initiating and promoting senescence-

associated intracellular alterations (Nicholls, 2002).

In line with other authors (Goodell and Cortopassi,

1998), no relevant age-related difference in respiratory

fluxes were observed in liver mitochondria, with the

exception of a significant decrease of succinate-stimulated

state III respiration (if compared with adult rats).

Differently, other investigators (Ventura et al., 2002)

found a deficit of glutamate–malate stimulated respiration.

Whereas mitochondrial respiratory dysfunction and

Fig. 4. Effect of hypocaloric regimen (Hyp.) and N-acetylcysteine (NAC) supplementation on total glutathione (GSH) concentrations in small intestinal and

colonic mucosae, liver mitochondria and post-mitochondrial fractions of old rats (28 mnths). oSignificantly different compared to untreated old rats.

Table 4

Transmucosal electrical parameters in muscle-stripped proximal colonic

mucosa of young, and 28 months old rats, and effects of hypocaloric

regimen and N-acetylcysteine (NAC) supplementation in old rats

(see Section 3)

2 months 28 months Hypocaloric NAC suppl.

PD (mV) 3.3 ^ 1.9 0.9 ^ 0.1a 3.7 ^ 0.3 1.2 ^ 0.4a

ISC (mA/cm2) 48 ^ 47 13 ^ 1a 41 ^ 7 18 ^ 0.3a

Rt (V cm2) 153 ^ 170 81 ^ 9a 84 ^ 6a 88 ^ 14a

PD, transmural potential difference; ISC, short-circuit current; Rt, tissue

resistance.a Significantly different compared to young rats.

I. Grattagliano et al. / Experimental Gerontology 39 (2004) 1323–1332 1329

imbalance of antioxidant defense existed, significant

changes of hepatic morphology in old rats were absent.

Another interesting result of this study is the lower

mitochondrial concentration of RSNO in aged rats,

observation which opens to a wide range of interpretations.

Mitochondrial NO synthase activity is considered the major

source of NO within cells (Brookes et al., 2002; Ghafourifar

and Richter, 1997; Lacza et al., 2003). Mitochondrial NO is

supposed to act as a physiological regulator of cell

respiration and ATP production (Giulivi et al., 1998);

physiological levels of NO seems to act also as anti-

apoptotic factor (Kim et al., 1997). The assessment of

RSNO represents a useful method to quantify the NO

content of a system (Mathews and Kerr, 1993), since it

represents a storage and a transport system (Steffen et al.,

2001), and a metabolic pathway to scavenge excess NO

radicals (Mathews and Kerr, 1993; Steffen et al., 2001). It is

ascribed to NO also a role in intracellular signalling

pathway. In fact, S-nitrosation/denitrosation reactions are

claimed as modulator of apoptosis (Mannick et al., 1999;

Steffen et al., 2001) through concentration-dependent

effects on the mitochondrial permeability transition,

cytochrome c release (Brookes et al., 2000) and S-nitrosa-

tion of PSH (Ji et al., 1999). In our setting, the lower RSNO

concentration may plausibly reflect an increased S-nitrosa-

tion of mitochondrial proteins including respiratory

complexes (Nishikawa et al., 1998) or might be the result

of a reduced mitochondrial NO synthase activity whose

inducible isoform seems to be regulated by GSH (Chen et al.,

2000). A lower mitochondrial NO concentration may be

associated with a reduced anti-apoptotic protection, which is

likely to occur in aged cells.

Caloric restriction has been proposed to extend life-

span and reduce the risk of cancer (Weindruch and

Walford, 1982) through an improvement of the cell redox

balance (Rebrin et al., 2003). In our study, when

compared to normally fed, old rats under hypocaloric

diet showed higher GSH concentrations in both the

intestinal segments and an improved transmembrane

electric activity. This finding suggests that diet restriction

improves oxidative balance, which is mainly based on the

GSH/GSSG ratio (Schafer and Buettner, 2003), and the

transport function.

It is of interest that hypocaloric diet is able to restore the

age-induced changes in transmucosal intestinal ionic

transport in old rats. It is unlikely a GSH-dependent effect,

since NAC treatment did not induce the same effect of diet

restriction, despite improving mitochondrial redox status by

increasing the levels of GSH and PSH. The intra- and/or

extra-cellular signaling mechanism responsible for the

direct role of hypocaloric diet on preventing intestinal

transport impairment should be addressed in the near future

by more specific studies.

Concerning liver fractions, the most evident effects were

noted at mitochondrial level where the reduction in GSH-Px

activity and protein carbonyl deposit and the increase in

RSNO levels clearly indicate that a restricted diet is

associated with an increased capacity to quench free

radicals (Youngman et al., 1992) and with an improvement

of mitochondrial respiration with both the substrates used,

i.e. glutamate/malate and succinate.

Prolonged supplementation of NAC to the diet was

likewise effective in improving the redox state of intestinal

and hepatic cells. In particular, the ileal tract was more

advantaged as showed by the increased levels of GSH and

PSH, revealing a likely utilization of luminal NAC. In the

liver, NAC improved mitochondrial redox status by

increasing the concentrations of GSH and RSNO and

by reducing the accumulation of protein carbonyls. It was as

effective as the hypocaloric diet in improving mitochondrial

respiration, whose inhibition is known to be reversed by

low-molecular weight thiols (Clementi et al., 1998).

Since the effects of NAC were better expressed in the

mitochondrial compartment, it could be supposed that NAC

is used with priority by mitochondria in order to improve

their antioxidant status and to contrast the pro-apoptotic

attitude of hepatocytes in old animals.

In conclusion, this study shows that ageing is associated

with a spread decrease of GSH and with alterations of

protein content and redox status in intestinal mucosa and

liver subcellular fractions. Intestinal transport and

mitochondrial respiration were also altered. Hypocaloric

diet and NAC administration were both effective in

enhancing GSH levels but with different effects according

to the investigated districts. Both interventions reduced the

ageing-associated increase of GSH-Px activity and protein

carbonyl accumulation, improved liver mitochondrial

respiration and NO metabolism, while the transmucosal

intestinal ionic transport was ameliorated only by diet

restriction.

Acknowledgements

This study was financially supported in part by the grant

within the National Research Project (PRIN) for ‘Brain

Aging in animal models’ of MURST, Italy.

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