Mechanisms of Ageing and Development, 64 (1992) 49-59
49
Elsevier ScientificPublishers Ireland Ltd.
EFFECT OF DIETARY RESTRICTION ON LYSOSOMAL BODIES AND TOTAL PROTEIN SYNTHESIS IN HEPATOCYTES OF AGING RATS
GUYLAINE FERLAND, M A U R I C E AUDET and BEATRIZ TUCHWEBER Department of Nutrition, Universit~ de Montreal, Quebec, H3C 3J7 (Canada)
(Received April 23rd, 1991) (Revision receivedNovember 28th, 1991) SUMMARY The accumulation of lysosomal bodies has long been considered to be an important correlate of aging. However it is not well established whether these age related changes interfere with cellular function. In this study, an evaluation of lysosomes by ultrastructural analysis was performed in livers of 4-6 and 20-24-month-old Sprague-Dawley female rats, fed ad libitum (A) or a restricted diet (R). An attempt was made to relate this parameter to hepatic protein synthesis, a liver function known to decrease with age and increase with dietary restriction. Aging was accompanied in both A and R animals with higher number and size of secondary lysosomes (lipofuscin) and by a decrease in total protein synthesis in hepatocytes. When compared to age matched ad libitum fed animals, livers of food restricted rats contained higher number of secondary lysosomes, yet exhibited higher protein synthetic capacity. Thus in hepatocytes, lipofuscin accumulation does not seem to interfere with cellular function.
Key words: Lysosomal bodies; Protein synthesis; Aging; Dietary restriction
INTRODUCTION The accumulation of lysosomal and fluorescence containing bodies (e.g. lipofuscin or age pigment) have long been considered to be an important correlate of aging [1-4]. For instance, a number of authors have reported elevated lysosomal enzyme activities with age [5-8]. In a previous study done with Sprague-Dawley rats [9] we Correspondence to: BeatrizTuchweber,Departmentof Nutrition, Universit~de Montr6al,P.O. Box 6128, Station A, Qu6bec, Canada H3C 3J7.
0047-6374/92/$05.00 © 1992 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
50
showed the activity of four lysosomal enzymes increased with age in parenchymal and non-parenchymal cells of the liver. Likewise, as evidenced by morphologic and chemical analysis lipofuscin pigments have been found to accumulate in many tissues during senescence [3,10,11]. Although many studies have been conducted on the composition and mode of formation of lipofuscin there is still controversy as to the cause of pigment accumulation during cellular aging [12]. Furthermore, it is not clear how the lipofuscin accumulation affects cellular function [3]. It has been claimed that pigment accumulation may in some ways interfere with cellular function. However, few studies have addressed this question directly [3]. In rodents, there is now very good evidence that restriction of calories coupled with the administration of essential nutrients increases both mean and maximum lifespan. Dietary restriction has been shown to retard the age-related decline of many physiological functions and to delay as well as decrease the severity of various agerelated diseases [13,14]. In livers of Sprague-Dawley rats, this dietary regimen has been reported to induce changes in lysosomal enzyme activity, the changes differing with respect to enzyme and cell population [9,15]. In addition, dietary restriction was shown to reduce the levels of fluorescence containing products [13,16], and is thought to protect the organism against free radical-mediated cellular damage [13,16-21]. However, many of these findings have recently been questioned in view of the methodology used to quantify the lipofuscin pigments [12,22]. The measurement of the blue emitting fluorescence in the organic solvent extracts of tissues does not seem to correlate with data obtained by microspectrofluorometry or electron microscopy. Recent work has shown that dietary restriction exerts a beneficial effect on the decline of various liver functions with age [23-27]. In this report, lipofuscin pigments are studied by electron microscopy in hepatocytes during aging in ad libitum fed and food restricted Sprague-Dawley rats. In an attempt to determine whether lipofuscin accumulation correlates with cellular function we evaluated in parallel, total protein synthesis, an important liver function which is known to decrease with age and increase with dietary restriction [23-25,28-30]. MATERIALS AND METHODS
Animals and diets
Female Spragne-Dawley rats, were obtained from the Charles River, St. Constant, Qurbec, Canada at weaning age and were maintained in a barrier facility (22°C, 12/12 h (light/dark) cycle. For the first 4 weeks, animals were fed ad libitum a purified diet (Teklad Test Diets, Madison, WI) consisting of 26% casein, 43% saccharose, 15% cornstarch, 4% corn oil, 7.5% non-nutritive fibre (cellulose), 3.5% mineral mix (William-Briggs modified) and 1% vitamin mix (Teklad). Beginning at 4 weeks of age, the rats designated as food restricted were fed 60% of the food intake of their ad libitum fed litter mates. The composition of the diets and dietary procedures were described previously [26]. For a complete description of the growth and survival characteristics of the colony see Ferland et al. [27].
51
Morphological and morphometric studies The livers of 3 animals per group were taken at each time interval of study (i.e. 4-6 and 20-24 months of age), fixed and processed for electron microscopy as described previously [31]. Lysosomal bodies were studied in hepatocytes of the midzonal area of the hepatic lobule since this area shows a clear cut accumulation of these bodies with age [10,32]. In hepatocytes, the biliary canalicular area was examined because it is well known that lysosomal bodies exhibiting fluorescence accumulate around the bile canaliculi [32-34]. The lysosomal bodies were subdivided into 2 main categories: primary and secondary. The primary lysosomes consisted of regular round vacuoles containing dense homogeneous material while the secondary lysosomes, often multilobular and irregular in shape, contained heterogeneous granules of variable density. We and others [22,34] who have described similar granules have considered them as lipofuscin. These granules have been shown to contain polymerized residues of peroxidised lipids, and proteiris and to arise from autophagocytosis of cellular constituents. Morphometric analysis consisted of counting the number of primary and secondary lysosomes/bile canaliculi and of measurements of volume density for lysosomes according to Weibel et al. [35]. Only canaliculi formed between 2 hepatoytes were considered. Aproximately 10 hepatocytes were randomly photographed from each block at an original magnification of 4 700, centering the bile canaliculi in the field. A total of 20 photographs from canalicular area per animal were made and a total of 80-100 photographs was obtained from 4-5 rats in each group. Fixed areas of 30/zm 2 were used for calculation of number of lysosomes or volume density. Prints were prepared at 16 500 magnification and number of lysosomes were counted in pericanalicular area and expressed/100 bile canaliculi. Volume density for primary and secondary lysosomes were expressed as the ratio of the volume density of a 30-#m 2 area.
Total protein synthesis Total protein synthesis was measured essentially as described by Van Bezooijen et al. [36]. The hepatocytes were isolated by enzymatic digestion [9] and protein synthesis was determined by the incorporation of 14C-leucine into isolated hepatocytes. Between 1.5-2.0 x 106 cells/ml were incubated for 2 h in an enriched Waymouth medium (Gibco, Toronto) containing a mixture of 8 mmol/ml of labelled (New England Nuclear, Montr%al) and unlabelled leucine (ICN Biochemicals Inc, Montrral). The incubation was carried at 37°C under an atmosphere of 95% 02 and 5% CO2 with constant shaking (100 oscillations/min). At the end of the incubation period, the hepatocytes were precipitated with 10% TCA and stored at 0°C for 30 min. The precipitate was then successively washed with 5% TCA, 95% alcohol and ether. The resulting hepatocyte pellet was dissolved in 1 ml NCS (Solubilizer for liquid scintillation, Amersham, Oakville, Ontario). The radioactivity of the samples was counted in 14 ml toluene-based scintillation fluid (composition 40 ml Liquifluor/L toluene, New England Nuclear, Montreal) in a liquid scintillation
Fig. 1. Liver of 24-month-old rat fed ad libitum. Note in the pericanalicular area of hepatocytes primary (thick arrow) and secondary (thin arrow) lysosomes. BC, bile canaliculus; S, sinusoid; MV, multivesicular bodies. × 16 450.
53 counter. Values obtained after incubation were corrected for the amount of radioactivity in a sample withdrawn immediately after the addition of the leucine mixture to all cell suspension. The rate of 14C-leucine incorporation was found to be linear with time and proportional to the number of hepatocytes incubated. Degradation of newly synthesized proteins or reutilization of 14C-leucine was investigated according to Van Bezooijen et al. [36] and was found not to influence the rate of protein synthesis during the incubation period. Values were assessed for significance by two-way analysis of variance (ANOVA), and Newman-Keul's test. A value of P < 0.05 was considered to be significant. RESULTS Figure 1 shows typical primary and secondary lysosomes evaluated in this study: primary lysosomes consisted of round vacuoles containing dense homogeneous material while secondary lysosomes were often irregular in shape and contained heterogeneous granules of variable density. Depicted in Fig. 2 are some of the stages of secondary lysosome formation. They appear to arise from degenerated mitochondria associated with lipid droplets. Lysosomal bodies in liver were evaluated for their number (number of lysosomes/100 canaliculi, Fig. 3) and their size (percent hepatic volume occupied by lysosomes, Fig. 4). Regardless of age and diet, secondary lysosomes were more numerous and larger in size than primary lysosomes (Figs. 3 and 4). Whether expressed per 100 canaliculi or as percent hepatic volume occupied, lysosomes increased with age in both the ad libitum (A) and food restricted (R) animals. The increase was especially marked in secondary lysosomes i.e. the number of lysosomes/100 canaliculi increasing by a factor of 1.7 in A, and by 2 (P < 0.05) in R between 4-6 and 20-24 months. Similarly, secondary lysosomes were affected by diet. When compared to ad libitum fed animals, livers of rats submitted to food restriction contained more lysosomes per 100 canaliculi and the lysosomes occupied a greater percentage of the hepatic volume. However because of the large variance, changes did not prove to be statistically significant. In order to correlate the studies of lysosomal bodies and lipofuscin pigments with hepatic function, total protein synthesis was assessed in isolated hepatocytes (Fig. 5). In both groups of rats, hepatocytes incorporation of 14C-leucine decreased 2-fold between 4-6 and 20-24 months (P < 0.05). Food restriction increased protein synthesis, with the greatest effect being seen at 4-6 months of age (P < 0.05). DISCUSSION Chronic food restriction is now recognized as the most effective means of extending mean and maximum lifespan [13,14]. Studies from this laboratory have focused on liver and have shown that food restriction retards the age related decline of
54
Fig. 2. Possiblemode of formation of secondary lysosomes:(A), lipid containing vacuoleswith markedly electron dense material at their periphery; (B), large vacuole sourrounded by single membrane containing several lipid droplets; (C), two vacuoles containing material of variable electron density probably derived from lipid droplets; (D), the contents of the vacuole are markedly electron dense and probably correspond to degraded lipid, x 32 000.
various hepatic functions [9,14,26,27,37]. Lysosomes and lipofuscin pigments have long been associated with the aging process, however it is still unclear how these two relate to each other and to cellular function. Although it has been postulated that the accumulation of lipofuscin could be detrimental to the cell [1-3], few studies have addressed this question directly. In this report, hepatic lysosomal bodies were analysed morphometrically and results were correlated to protein synthesis, an important liver function. For the morphometric studies, lysosomal bodies were evaluated from the midzonal area of the hepatic lobule known to accumulate these bodies during senescence [32]. Lysosomes were divided into 2 categories: primary lysosomes consisted of
55
PRIMARY L Y S O S O M E S 200
o
8
150
100
E o o I
01
4-6
20-24
Months
Months
Age SECONDARY LYSOSOMES
m
_o ¢= e.. e= o o o
too
T
100
o
E 0
~ ,.I
so
-t-
4-6 Months
20-24 Months
Age Fig. 3. Number of primary (a) and secondary (b) lysosomes per 100 canaliculi in livers of rats fed ad libitum or food restricted. Values are the means ± S.E.M. of 4 - 6 animals per group. *P < 0.05 when compared to 4 - 6 month old rats fed the same diet. n, rats fed ad libitum; m, rats fed 40% restricted diet.
round vacuoles containing dense homogeneous material while secondary lysosomes were irregular in shape and contained heterogeneous granules of variable density. We and others [38] consider the secondary lysosomes described here to correspond to lipofuscin pigments. The present findings showed an increase in the number and size of the secondary
56
PRIMARY LYSOSOMES 2.2. 2.01 1.8
1.6
E
1.4
>
1.2 1.0~
0.
0.8~ 0.6 0.4~ 0.2OG
20-24 Months
4-6 Months
Age SECONDARY LYSOSOMES 2.22.0~ 1.8" 1.6-' 1.4~ 1.2" 1.00.806-"
-r-
0.4~ 0.2" 0.0' 4-6 Months
20-24 Months
Age Fig. 4. Percent hepatic volume occupied by primary (a) and secondary (b) lysosomes in livers of ad libitum and food restricted fed rats. Values are the means ~ S.E.M. of 4 - 6 animals per group. I"1, rats fed ad libitum; g , rats fed 40% restricted diet.
lysosomes as judged by morphOmetric analysis (Figs. 3 and 4). These results agree with those of Iwasaki et al. [38] who reported, in male Fischer 344 rats, a 2-fold increase in the number of lipofuscin granules between the ages of 6 and 27 months. Furthermore the trends reported in this study during senescence corroborate results from enzymatic and histochemical studies. In a recent article published by this
57 PROTEIN SYNTHESIS *a 15.
m
%
E
v c
~
*b
~
,
o
_c o
0
4-6 Months
20-24 Months
Age
Fig. 5. Total protein synthesis in hepatocytesas measured by incorporation of 14C-leucine(nmol/106 cells per h). Values are the means ± S.E.M. of 5-10 determinationsper group. *a P < 0.05 when compared to rats of the same age fed ad libitum. *b P < 0.05 when compared to 4-6 month old rats fed the same diet. I-I, rats fed ad libitum; B, rats fed 40% restricted diet.
laboratory [9] the activity of 4 lysosomal enzymes (acid phosphatase, betagalactosidase, arylsulfatase B and cathepsin D) was found to increase with age in hepatocytes of female Sprague-Dawley rats. Similarly, in a study by Van Manen et al. [32] various other hepatic lysosomal enzymes were reported to increase during aging in BN/Bi Rij female rats. In this latter study where enzymatic activity was evaluated in 3 intralobular regions of the liver: central, midzonal and peripheral, age-related increase was especially marked in the midzonal area. Interestingly food restricted rats exhibit an increase in the number and size of lysosomes with aging when compared to corresponding ad libitum fed controls (Figs. 3 and 4). The results of food restricted animals disagree with those of Iwasaki et al. [38] who, using electron microscopy, reported a decrease in the number of lipofuscin granules of R rats. However, given the well documented gradient of the hepatic lobule [32] a comparison of the results of the two studies is difficult since Iwasaki et al. [38] give no indication of the lobular location where lipofuscin pigments were evaluated. Thus the divergent trends reported in the two studies could partially be related to iritralobular differences. Other factors such as sex, strain and diet composition may also explain the discrepancy between the two studies. In an attempt to associate hepatic lysosomal accumulation with cellular function, total protein synthesis as determined by the incorporation of ~4C-leucine was evaluated in hepatocytes. Protein synthesis decreased significantly with age in both A and R rats but at a given age, restricted rats exhibited higher protein synthetic
58
capacity than A fed rats, confirming results published by others [24,25]. Interestingly, R rats accumulated significantly more secondary lysosomes. Although the two processes may occur independently our findings suggest that pigment accumulation in hepatocytes may not be detrimental to cell function as measured by protein synthesis. ACKNOWLEDGEMENTS
This work was supported by NSERC (Canada). REFERENCES 1 A. Elens and R. Watiaux, Age-correlated changes in lysosomal enzyme activities: an index of aging? Exp. GerontoL, 4 (1969) 131-135. 2 R. Hochschild, Lysosomes, membranes and aging. Exp. GerontoL, 6 (1971) 153-166. 3 R.S. Sohal (ed.), Age Pigments, Elsevier/North-Holland Biomedical Press, Amsterdam, 1981. 4 K.R. Brizzee, B. Kaack and P. Klara, Lipofuscin: intra- and extraneuronal accumulation and regional distribution. In J.M. Ordy and K.R. Brizzee (eds.), Neurobiology o f Aging, Plenum Press, New York, 1975, pp. 463-484. 5 S. Goto, T. Takano, D. Mizuno, T. Nakano and K. Umaizumi, Aging and location of acid ribonuclease in liver of various animals. J. GerontoL, 24 (1969) 305-308. 6 R. Comoli, Hydrolase activity and intracellular pH in liver, heart and diaphragm of aging rats. Exp. GerontoL, 6 (1971) 219-225. 7 S. Asano, H. Komoriya, E. Hayashi and H. Sawada, Changes in intracellular activities of lysosomal enzymes in tissues of rats during aging. Mech. Ageing Dev., 10 (1979) 81-92. 8 Y. Nakamura, M. Takeda, H. Suzuki, H. Morita, K. Toda, S. Hariguchi and T. Nishimura, Lysosome instability in aged rat brain. Neurosci. Lett., 97 (1989) 215-220. 9 G. Ferland, A. Perea, M. Audet and B. Tuchweber, Characterization of liver lysosomal enzyme activity in hepatocytes, Kupffer and endothelial cells during aging: effect of dietary restriction. Mech. Ageing Dev., 56 (1990) 143-154. 10 H. Tauchi, M. Hananouchi and T. Sato, Accumulation of lipofuscin pigment in human hepatic cells from different races and in different environmental conditions. Mech. Ageing Dev., 12 (1980) 183-195. 11 E.D. Porta, Tissue lipoperoxidation and lipofuscin accumulation as influenced by age, type of dietary fat and levels of vitamin E in rats. In E.A. Tataro, P. Glees and F.A. Pisanti (eds.), Advances in Age Pigments Research, Vol. 64, Pergamon Press, Oxford, New York, 1987, pp. 37-74. 12 E.A. Porta, Advances in age pigment research. Arch. Gerontol. Geriatr., 12 (1991) 303-320. 13 A.M. Holehan and B.J. Merry, The experimental manipulation of ageing by diet. BioL Rev., 61 (1986) 329-368. 14 E.J. Masoro, Food restriction in rodents: an evaluation of its role in the study of aging. J. Gerontol., 43 (1988) B59-64. 15 C. Solomon, B. Tuchweber, U. Srivastava and M. Nadeau, Liver lysosomal enzymes in rats during long-term dietary restriction. 1. Changes during the developmental period of life. Mech. Ageing Dev., 24 (1984) 9-27. 16 H.E. Enesco and P. Kruk, Dietary restriction reduces fluorescent age pigment accumulation in mice. Exp. Gerontol., 16 (1981) 357-361. 17 S. Laganiere and B.P. Yu, Anti-lipoperoxidation action of food restriction. Biochem. Biophys. Res. Commun., 145 (1987) i185-1191. 18 A. Koizumi, R. Weindruch and R. L. Walford, Influences of dietary restriction and age on liver enzyme activities and lipid peroxidation in mice. J. Nutr., 117 (1987) 361-367. 19 S. Laganiere and B.P. Yu, Effect of chronic food restriction in aging rats. I1. Liver cytosolic antioxidant and related enzymes. Mech. Ageing Dev., 48 (1989) 221-230.
59 20 21 22 23
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
B.P. Yu, Food restriction research: past and present status. Rev. Biol. Res. Aging., 4 (1990) 349-371. S. Chipalkatti, A.K. De and A.S. Aijar, Effect of diet restriction on some biochemical parameters related to aging in mice. J. Nutr., 113 (1983) 944-950. R.S. Sohal and U.T. Brunk, Lipofuscin as an indicator of oxidative stress and aging. In: E.A. Porta (ed.), Lipofuscin and Ceroid Pigments, Plenum Press, New York, 1989, pp. 17-29. B.J. Merry, A.M. Holehan, S.E.M. Lewis and D.F. Goldspink, The effect of ageing and chronic dietary restriction on in vivo hepatic protein synthesis in the rat. Mech. Ageing Dev., 39 (1987) 189-199. M.C. Birchenall-Sparks, M.S. Roberts, J. Staecker, J.P. Hardwick and A. Richardson, Effect of dietary restriction on liver protein synthesis in rats. J. Nutr., 115 (1985) 944-950. W.F. Ward, Enhancement by food restriction of liver protein synthesis in the aging Fischer 344 rats. J. Gerontol. 43 (1988) B50-53. B. Tuchweber, A. Perea, G. Ferland, and I.M. Yousef, Dietary restriction influences bile formation in aging rats. Life Sci., 41 (1987) 2091-2099. G. Ferland, B. Tuchweber, A. Perea and I.M. Yousef, Effect of aging and dietary restriction on bile acid metabolism in rats. Lipids, 24 (1989) 842-848. G.A. Ricea, D.S.H. Liu, J.J. Coniglio and A. Richardson, Rates of protein synthesis by hepatocytes isolated from rats of various ages. J. Cell Physiol., 97 (1978) 137-146. J.J. Coniglio, D.S.H. Liu and A. Richardson, A comparison of protein synthesis by liver parenchymal cells isolated from Fischer F344 rats of various ages. Mech. Ageing Dev., 11 (1979) 77-90. A. Richardson, The relationship between aging and protein synthesis in: J.R. Florini (ed), CRC Handbook Series in Biochemistry in Aging, CRC Press, Boca Raton, FL, 1981, pp. 79-101. M. Salas, B. Tuchweber and P. Kourounakis, Liver ultrastructure during acute stress. Pathol. Res. Pract., 167 (1980) 217-233. R. Van Manen, W. De Priester and D.L. Knook, Lysosomal activity in aging rat liver: I. Variation in enzyme activity within the liver Iobule. Mech. Ageing Dev., 22 (1983) 159-165. W. De Priester, R. Van Manen and D.L. Knook, Lysosomal activity in the aging rat liver: II. Morphometry of acid phosphatase positive dense bodies. Mech. Ageing Dev., 26 (1984) 205-216. H. Ikeda, H. Tauchi and T. Sato, Fine structural analysis of lipofuscin in various tissues of rats of different ages. Mech. Ageing Dev., 33 (1985) 77-93. E.R. Weibel, W. Staubli, H.R. Gmago and F.A. Hess, Correlated morphometric and biochemical studies on the liver cell. J., Cell Biol., 42 (1969) 68-91. C.F.A. Van Bezooijen, T. Grell and D.L. Knook, The effect of age on protein synthesis by isolated liver parenchymal cells. Mech. Ageing Dev., 6 (1977) 293-304. G. Ferland, B. Tuchweber, P.V. Bhat and A. Lacroix, Effect of dietary restriction on hepatic vitamin A content in aging rats. J. Gerontol., (in press). K. Iwasaki, H. Maeda, I. Shimokawa, M. Hayashida, B.P. Yu, E.J. Masoro and T. Ikeda, An electron microscopic examination of age-related changes in the rat liver. Acta Pathol. Jpn., 38 (1988) 1119-1130.
49
Elsevier ScientificPublishers Ireland Ltd.
EFFECT OF DIETARY RESTRICTION ON LYSOSOMAL BODIES AND TOTAL PROTEIN SYNTHESIS IN HEPATOCYTES OF AGING RATS
GUYLAINE FERLAND, M A U R I C E AUDET and BEATRIZ TUCHWEBER Department of Nutrition, Universit~ de Montreal, Quebec, H3C 3J7 (Canada)
(Received April 23rd, 1991) (Revision receivedNovember 28th, 1991) SUMMARY The accumulation of lysosomal bodies has long been considered to be an important correlate of aging. However it is not well established whether these age related changes interfere with cellular function. In this study, an evaluation of lysosomes by ultrastructural analysis was performed in livers of 4-6 and 20-24-month-old Sprague-Dawley female rats, fed ad libitum (A) or a restricted diet (R). An attempt was made to relate this parameter to hepatic protein synthesis, a liver function known to decrease with age and increase with dietary restriction. Aging was accompanied in both A and R animals with higher number and size of secondary lysosomes (lipofuscin) and by a decrease in total protein synthesis in hepatocytes. When compared to age matched ad libitum fed animals, livers of food restricted rats contained higher number of secondary lysosomes, yet exhibited higher protein synthetic capacity. Thus in hepatocytes, lipofuscin accumulation does not seem to interfere with cellular function.
Key words: Lysosomal bodies; Protein synthesis; Aging; Dietary restriction
INTRODUCTION The accumulation of lysosomal and fluorescence containing bodies (e.g. lipofuscin or age pigment) have long been considered to be an important correlate of aging [1-4]. For instance, a number of authors have reported elevated lysosomal enzyme activities with age [5-8]. In a previous study done with Sprague-Dawley rats [9] we Correspondence to: BeatrizTuchweber,Departmentof Nutrition, Universit~de Montr6al,P.O. Box 6128, Station A, Qu6bec, Canada H3C 3J7.
0047-6374/92/$05.00 © 1992 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
50
showed the activity of four lysosomal enzymes increased with age in parenchymal and non-parenchymal cells of the liver. Likewise, as evidenced by morphologic and chemical analysis lipofuscin pigments have been found to accumulate in many tissues during senescence [3,10,11]. Although many studies have been conducted on the composition and mode of formation of lipofuscin there is still controversy as to the cause of pigment accumulation during cellular aging [12]. Furthermore, it is not clear how the lipofuscin accumulation affects cellular function [3]. It has been claimed that pigment accumulation may in some ways interfere with cellular function. However, few studies have addressed this question directly [3]. In rodents, there is now very good evidence that restriction of calories coupled with the administration of essential nutrients increases both mean and maximum lifespan. Dietary restriction has been shown to retard the age-related decline of many physiological functions and to delay as well as decrease the severity of various agerelated diseases [13,14]. In livers of Sprague-Dawley rats, this dietary regimen has been reported to induce changes in lysosomal enzyme activity, the changes differing with respect to enzyme and cell population [9,15]. In addition, dietary restriction was shown to reduce the levels of fluorescence containing products [13,16], and is thought to protect the organism against free radical-mediated cellular damage [13,16-21]. However, many of these findings have recently been questioned in view of the methodology used to quantify the lipofuscin pigments [12,22]. The measurement of the blue emitting fluorescence in the organic solvent extracts of tissues does not seem to correlate with data obtained by microspectrofluorometry or electron microscopy. Recent work has shown that dietary restriction exerts a beneficial effect on the decline of various liver functions with age [23-27]. In this report, lipofuscin pigments are studied by electron microscopy in hepatocytes during aging in ad libitum fed and food restricted Sprague-Dawley rats. In an attempt to determine whether lipofuscin accumulation correlates with cellular function we evaluated in parallel, total protein synthesis, an important liver function which is known to decrease with age and increase with dietary restriction [23-25,28-30]. MATERIALS AND METHODS
Animals and diets
Female Spragne-Dawley rats, were obtained from the Charles River, St. Constant, Qurbec, Canada at weaning age and were maintained in a barrier facility (22°C, 12/12 h (light/dark) cycle. For the first 4 weeks, animals were fed ad libitum a purified diet (Teklad Test Diets, Madison, WI) consisting of 26% casein, 43% saccharose, 15% cornstarch, 4% corn oil, 7.5% non-nutritive fibre (cellulose), 3.5% mineral mix (William-Briggs modified) and 1% vitamin mix (Teklad). Beginning at 4 weeks of age, the rats designated as food restricted were fed 60% of the food intake of their ad libitum fed litter mates. The composition of the diets and dietary procedures were described previously [26]. For a complete description of the growth and survival characteristics of the colony see Ferland et al. [27].
51
Morphological and morphometric studies The livers of 3 animals per group were taken at each time interval of study (i.e. 4-6 and 20-24 months of age), fixed and processed for electron microscopy as described previously [31]. Lysosomal bodies were studied in hepatocytes of the midzonal area of the hepatic lobule since this area shows a clear cut accumulation of these bodies with age [10,32]. In hepatocytes, the biliary canalicular area was examined because it is well known that lysosomal bodies exhibiting fluorescence accumulate around the bile canaliculi [32-34]. The lysosomal bodies were subdivided into 2 main categories: primary and secondary. The primary lysosomes consisted of regular round vacuoles containing dense homogeneous material while the secondary lysosomes, often multilobular and irregular in shape, contained heterogeneous granules of variable density. We and others [22,34] who have described similar granules have considered them as lipofuscin. These granules have been shown to contain polymerized residues of peroxidised lipids, and proteiris and to arise from autophagocytosis of cellular constituents. Morphometric analysis consisted of counting the number of primary and secondary lysosomes/bile canaliculi and of measurements of volume density for lysosomes according to Weibel et al. [35]. Only canaliculi formed between 2 hepatoytes were considered. Aproximately 10 hepatocytes were randomly photographed from each block at an original magnification of 4 700, centering the bile canaliculi in the field. A total of 20 photographs from canalicular area per animal were made and a total of 80-100 photographs was obtained from 4-5 rats in each group. Fixed areas of 30/zm 2 were used for calculation of number of lysosomes or volume density. Prints were prepared at 16 500 magnification and number of lysosomes were counted in pericanalicular area and expressed/100 bile canaliculi. Volume density for primary and secondary lysosomes were expressed as the ratio of the volume density of a 30-#m 2 area.
Total protein synthesis Total protein synthesis was measured essentially as described by Van Bezooijen et al. [36]. The hepatocytes were isolated by enzymatic digestion [9] and protein synthesis was determined by the incorporation of 14C-leucine into isolated hepatocytes. Between 1.5-2.0 x 106 cells/ml were incubated for 2 h in an enriched Waymouth medium (Gibco, Toronto) containing a mixture of 8 mmol/ml of labelled (New England Nuclear, Montr%al) and unlabelled leucine (ICN Biochemicals Inc, Montrral). The incubation was carried at 37°C under an atmosphere of 95% 02 and 5% CO2 with constant shaking (100 oscillations/min). At the end of the incubation period, the hepatocytes were precipitated with 10% TCA and stored at 0°C for 30 min. The precipitate was then successively washed with 5% TCA, 95% alcohol and ether. The resulting hepatocyte pellet was dissolved in 1 ml NCS (Solubilizer for liquid scintillation, Amersham, Oakville, Ontario). The radioactivity of the samples was counted in 14 ml toluene-based scintillation fluid (composition 40 ml Liquifluor/L toluene, New England Nuclear, Montreal) in a liquid scintillation
Fig. 1. Liver of 24-month-old rat fed ad libitum. Note in the pericanalicular area of hepatocytes primary (thick arrow) and secondary (thin arrow) lysosomes. BC, bile canaliculus; S, sinusoid; MV, multivesicular bodies. × 16 450.
53 counter. Values obtained after incubation were corrected for the amount of radioactivity in a sample withdrawn immediately after the addition of the leucine mixture to all cell suspension. The rate of 14C-leucine incorporation was found to be linear with time and proportional to the number of hepatocytes incubated. Degradation of newly synthesized proteins or reutilization of 14C-leucine was investigated according to Van Bezooijen et al. [36] and was found not to influence the rate of protein synthesis during the incubation period. Values were assessed for significance by two-way analysis of variance (ANOVA), and Newman-Keul's test. A value of P < 0.05 was considered to be significant. RESULTS Figure 1 shows typical primary and secondary lysosomes evaluated in this study: primary lysosomes consisted of round vacuoles containing dense homogeneous material while secondary lysosomes were often irregular in shape and contained heterogeneous granules of variable density. Depicted in Fig. 2 are some of the stages of secondary lysosome formation. They appear to arise from degenerated mitochondria associated with lipid droplets. Lysosomal bodies in liver were evaluated for their number (number of lysosomes/100 canaliculi, Fig. 3) and their size (percent hepatic volume occupied by lysosomes, Fig. 4). Regardless of age and diet, secondary lysosomes were more numerous and larger in size than primary lysosomes (Figs. 3 and 4). Whether expressed per 100 canaliculi or as percent hepatic volume occupied, lysosomes increased with age in both the ad libitum (A) and food restricted (R) animals. The increase was especially marked in secondary lysosomes i.e. the number of lysosomes/100 canaliculi increasing by a factor of 1.7 in A, and by 2 (P < 0.05) in R between 4-6 and 20-24 months. Similarly, secondary lysosomes were affected by diet. When compared to ad libitum fed animals, livers of rats submitted to food restriction contained more lysosomes per 100 canaliculi and the lysosomes occupied a greater percentage of the hepatic volume. However because of the large variance, changes did not prove to be statistically significant. In order to correlate the studies of lysosomal bodies and lipofuscin pigments with hepatic function, total protein synthesis was assessed in isolated hepatocytes (Fig. 5). In both groups of rats, hepatocytes incorporation of 14C-leucine decreased 2-fold between 4-6 and 20-24 months (P < 0.05). Food restriction increased protein synthesis, with the greatest effect being seen at 4-6 months of age (P < 0.05). DISCUSSION Chronic food restriction is now recognized as the most effective means of extending mean and maximum lifespan [13,14]. Studies from this laboratory have focused on liver and have shown that food restriction retards the age related decline of
54
Fig. 2. Possiblemode of formation of secondary lysosomes:(A), lipid containing vacuoleswith markedly electron dense material at their periphery; (B), large vacuole sourrounded by single membrane containing several lipid droplets; (C), two vacuoles containing material of variable electron density probably derived from lipid droplets; (D), the contents of the vacuole are markedly electron dense and probably correspond to degraded lipid, x 32 000.
various hepatic functions [9,14,26,27,37]. Lysosomes and lipofuscin pigments have long been associated with the aging process, however it is still unclear how these two relate to each other and to cellular function. Although it has been postulated that the accumulation of lipofuscin could be detrimental to the cell [1-3], few studies have addressed this question directly. In this report, hepatic lysosomal bodies were analysed morphometrically and results were correlated to protein synthesis, an important liver function. For the morphometric studies, lysosomal bodies were evaluated from the midzonal area of the hepatic lobule known to accumulate these bodies during senescence [32]. Lysosomes were divided into 2 categories: primary lysosomes consisted of
55
PRIMARY L Y S O S O M E S 200
o
8
150
100
E o o I
01
4-6
20-24
Months
Months
Age SECONDARY LYSOSOMES
m
_o ¢= e.. e= o o o
too
T
100
o
E 0
~ ,.I
so
-t-
4-6 Months
20-24 Months
Age Fig. 3. Number of primary (a) and secondary (b) lysosomes per 100 canaliculi in livers of rats fed ad libitum or food restricted. Values are the means ± S.E.M. of 4 - 6 animals per group. *P < 0.05 when compared to 4 - 6 month old rats fed the same diet. n, rats fed ad libitum; m, rats fed 40% restricted diet.
round vacuoles containing dense homogeneous material while secondary lysosomes were irregular in shape and contained heterogeneous granules of variable density. We and others [38] consider the secondary lysosomes described here to correspond to lipofuscin pigments. The present findings showed an increase in the number and size of the secondary
56
PRIMARY LYSOSOMES 2.2. 2.01 1.8
1.6
E
1.4
>
1.2 1.0~
0.
0.8~ 0.6 0.4~ 0.2OG
20-24 Months
4-6 Months
Age SECONDARY LYSOSOMES 2.22.0~ 1.8" 1.6-' 1.4~ 1.2" 1.00.806-"
-r-
0.4~ 0.2" 0.0' 4-6 Months
20-24 Months
Age Fig. 4. Percent hepatic volume occupied by primary (a) and secondary (b) lysosomes in livers of ad libitum and food restricted fed rats. Values are the means ~ S.E.M. of 4 - 6 animals per group. I"1, rats fed ad libitum; g , rats fed 40% restricted diet.
lysosomes as judged by morphOmetric analysis (Figs. 3 and 4). These results agree with those of Iwasaki et al. [38] who reported, in male Fischer 344 rats, a 2-fold increase in the number of lipofuscin granules between the ages of 6 and 27 months. Furthermore the trends reported in this study during senescence corroborate results from enzymatic and histochemical studies. In a recent article published by this
57 PROTEIN SYNTHESIS *a 15.
m
%
E
v c
~
*b
~
,
o
_c o
0
4-6 Months
20-24 Months
Age
Fig. 5. Total protein synthesis in hepatocytesas measured by incorporation of 14C-leucine(nmol/106 cells per h). Values are the means ± S.E.M. of 5-10 determinationsper group. *a P < 0.05 when compared to rats of the same age fed ad libitum. *b P < 0.05 when compared to 4-6 month old rats fed the same diet. I-I, rats fed ad libitum; B, rats fed 40% restricted diet.
laboratory [9] the activity of 4 lysosomal enzymes (acid phosphatase, betagalactosidase, arylsulfatase B and cathepsin D) was found to increase with age in hepatocytes of female Sprague-Dawley rats. Similarly, in a study by Van Manen et al. [32] various other hepatic lysosomal enzymes were reported to increase during aging in BN/Bi Rij female rats. In this latter study where enzymatic activity was evaluated in 3 intralobular regions of the liver: central, midzonal and peripheral, age-related increase was especially marked in the midzonal area. Interestingly food restricted rats exhibit an increase in the number and size of lysosomes with aging when compared to corresponding ad libitum fed controls (Figs. 3 and 4). The results of food restricted animals disagree with those of Iwasaki et al. [38] who, using electron microscopy, reported a decrease in the number of lipofuscin granules of R rats. However, given the well documented gradient of the hepatic lobule [32] a comparison of the results of the two studies is difficult since Iwasaki et al. [38] give no indication of the lobular location where lipofuscin pigments were evaluated. Thus the divergent trends reported in the two studies could partially be related to iritralobular differences. Other factors such as sex, strain and diet composition may also explain the discrepancy between the two studies. In an attempt to associate hepatic lysosomal accumulation with cellular function, total protein synthesis as determined by the incorporation of ~4C-leucine was evaluated in hepatocytes. Protein synthesis decreased significantly with age in both A and R rats but at a given age, restricted rats exhibited higher protein synthetic
58
capacity than A fed rats, confirming results published by others [24,25]. Interestingly, R rats accumulated significantly more secondary lysosomes. Although the two processes may occur independently our findings suggest that pigment accumulation in hepatocytes may not be detrimental to cell function as measured by protein synthesis. ACKNOWLEDGEMENTS
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