Elsevier

Experimental Gerontology

Volume 39, Issue 9, September 2004, Pages 1323-1332
Experimental Gerontology

Effect of dietary restriction and N-acetylcysteine supplementation on intestinal mucosa and liver mitochondrial redox status and function in aged rats

https://doi.org/10.1016/j.exger.2004.06.001Get rights and content

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.

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 (Grattagliano 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 supplementation 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 (Andrè and Feley-Bosco, 2003) 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.

Section snippets

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

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.

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

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

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,

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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