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Food restriction in female Wistar rats. III. Thermotropic transition of membrane lipid and 5'-nucleotidase activity in hepatocytes
Arch. Gerontol. Geriatr., 11 (1990) 117-124 Elsevier
117
AGG 00342
Food restriction in female Wistar rats. III. Thermotropic transition of membrane lipid and 5'-nucleotidase activity in hepatocytes C. Pieri, M. Falasca, F. Moroni, F. Marcheselli and R. Recchioni Cytology Center, Gerontological Research Department LN.R.C.A., Via Birarelli 8, 60121 Ancona, Italy
(Received 9 April 1990; revised version received 9 July 1990; accepted 10 July 1990)
Summary The effect of diet restriction was measured on the anisotropy parameter of 1,6-diphenyl-l,3,5hexatriene (DPH) and 5'-nucleotidase enzyme activity in liver plasma membrane preparates. Diet restriction was applied to rats 3.5 months old on an every-other-day schedule (EOD) and the rats were killed at the age of 28-29 months. Six months and 24 month rats, fed ad libitum (AL), were used as controls. The Arrhenius plots of anisotropy parameter of liver membranes from young, old AL and old EOD animals exhibited well defined breakpoints at 16.3° C, 19.5°C and 16.7o C, respectively. The breakpoint temperature of 5'-nucleotidase activity was lower in samples from young rats as compared to those from old AL rats, whereas no difference was observed comparing young and EOD fed rats. Present results support the hypothesis that diet restriction modifies lipid composition of liver plasma membranes in such a way that the appearance of age-dependent alterations is delayed. Undernutrition; Hepatocyte membrane; DPH anisotropy; 5'-Nucleotidase
Introduction Aging of the liver has largely b e e n investigated because its pivotal role i n the metabolism. Liver p l a s m a m e m b r a n e , i n particular, is the p r i m a r y interface i n the homeostatic b a l a n c e b e t w e e n exogenous influence, e.g. diet, a n d e n d o g e n o u s c o n t r o l over the biosynthesis or utilization of various substrates ( C l a n d i n i n et al., 1983). F o r these reasons, a t t e n t i o n has b e e n paid to the a g e - d e p e n d e n t m o d i f i c a t i o n s occurring i n the liver p l a s m a m e m b r a n e s a n d a n increased microviscosity of lipid m a t r i x was d e m o n s t r a t e d i n old a n i m a l s as c o m p a r e d to y o u n g ones (Hegner, 1980; N o k u b o , 1985).
Correspondence to: Dr. Carlo Pieri, Cytology Center, Gerontological Research Department, I.N.R.C.A., Via Birarelli, 8, 60121 Ancona, Italy. 0167-4943/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
118 Recently it has been reported that food restriction reduced the age-related peroxidative membrane deterioration of liver microsomal and mitochondrial fractions (Laganiere and Yu, 1987; 1989; Koizumi et al., 1987). Liver plasma membrane composition can be influenced by the quality of the diet (Clandinin et al., 1983; Brivio-Haughland et al., 1976), but no data are available on the effect of the quantity of food on the chemical composition and biophysical properties of liver plasma membranes. The aim of the present work was to evaluate the effect of undernutrition on the biophysical properties of liver plasma membrane and the activity of 5'-nucleotidase, an enzyme which is regulated mainly by the physicochemical properties of the plasma membrane, the activity of which can be influenced by diet (Gonzales-Calvin et al., 1985).
Material and Methods
Animals Female Wistar rats of our breed were used. The diet restriction applied; the breeding conditions, as well as the survival and body weight curves were reported in our previous paper (Pied et al., 1990). Briefly, the diet restriction was applied to the animals from the age of 3.5 months, feeding them on an every-other-day schedule (EOD) with the same laboratory chow given ad libitum to the control rats (AL). This treatment was able to increase the lifespan of EOD animals when compared to the AL ones (Goodrick et al., 1982; Pieri et al., 1990). The EOD and AL rats were killed at 28-29 and 24 months of age, respectively, together with young control animals 6 months old. In the present experiment six animals of each group were used.
Liver plasma membrane preparation Liver plasma membranes were prepared by differential centrifugation followed by discontinuous gradient centrifugation according to Croze and Morr6 (1984) and washed in 1 mM NaHCO3, pH 7.4. Purity of the membranes was monitored by electron microscopic examination and contamination with mitochondria was assessed by the assay of succinic dehydrogenase (Sottocasa et al., 1967).
Fluorescence polarization studies Liver plasma membranes were suspended in phosphate-buffered saline (PBS; 5 mM phosphates, 145 mm NaC1, 4 mM KC1, 1 mM CaC12, pH 7.4) to a final concentration of 75 #g protein/ml. The membranes were loaded with the fluorescent lipid probe 1,6-diphenyl-l,3,5-hexatriene (DPH). Steady state fluorescence polarization was determined by means of SFM 25 spectrofluorimeter (Kontron) and expressed as the fluorescence anisotropy parameter [(ro/r ) - 1 ] -1 where r is the measured fluorescence anisotropy and r o is the limiting anisotropy. The latter is
119 assumed to be 0.362 for DPH (Shinitzky and Barenholz, 1978). The temperature dependency of [ ( r o / r ) - 1] -1 was determined over the range of 4 - 4 0 ° C and the logarithm of the anisotropy parameters was plotted against 1 / o K to detect thermotropic transitions. 5 '-Nucleotidase assay
The assay of 5'-nucleotidase (EC 3.1.3.5) was performed by means of the coupled spectrophotometric method of Dipple and Houslay (1978). Plasma membranes (75-150/~g protein) were suspended in 1 ml of 5 mM Tris-HC1 buffer pH 7.6 (pH adjusted at each temperature) containing 75 /~M adenosine 5'-monophosphate, 50 mM glycerol phosphate, 0.8 units of adenosine deaminase and 0.5 mM MgSO4. The reaction was monitored by the decrease in absorbance at 265 nm in a Beckman DU-7 spectrophotometer equipped with a temperature controlled older sample, in the temperature range 6-40 o C. Protein determination
The protein content of membrane preparations was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. Data analysis
Arrhenius plot parameters, transition temperatures and energies of activities were calculated using nonlinear regression analysis (Duggleby, 1984).
Results
Representative Arrhenius plots of the logarithms of anisotropy parameters of DPH are illustrated in Fig. 1. The values were obtained from liver plasma membrane preparations from the three experimental models: old EOD, old AL and young female Wistar rats. The measured parameters are summarized in Table I. The Arrhenius plot of anisotropy parameters of liver membranes from young, old AL and EOD animals exhibited well defined breakpoints at 16.3, 19.5 and 16.7 o C, respectively. The breakpoint temperature observed in young animals is in close agreement with other reported results obtained by using the same method based on changes in DPH polarization. This method is able to detect only the lower critical temperature of the broad transitions observed with differential scanning calorimetry (Livingstone and Schachter, 1980). Breakpoints in Arrhenius plots are closely associated with lipid phase separations occurring within the membrane and the temperatures at which they occur are closely related to the membrane lipid compositions (Lee et al., 1974; Wunderlich et al., 1975).
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Fig. 1. Representative Arrhenius plots of anisotropy parameter of liver plasma membranes from young (zx), old EOD (0) and old AL (n). The clear increase in break temperature observed in old A L as c o m p a r e d with y o u n g rats indicates changes in m e m b r a n e composition during aging. I m p o r t a n t l y enough, the undernourished rats showed breakpoints very close to those of y o u n g animals, supporting the view that the age-dependent alterations of liver m e m b r a n e composition was prevented by diet restriction. The anisotropy values of liver m e m b r a n e suspension from old A L rats were higher over the entire temperature range of 4 - 4 0 ° C as c o m p a r e d to the E O D animals and the difference was approximately 3 ° C (Fig. 1). As opposed to the pattern of breakpoints, no significant changes of the activation energies of the phase transitions were observed u p o n aging and diet restriction. Typical Arrhenius plots of 5'-nucleotidase activity f r o m isolated hepatic plasma membranes from rats, in the experimental conditions considered, are shown in Fig. 2 and the Arrhenius plot parameters are summarised in Table II.
TABLE I Temperature dependence of DPH fluorescence polarization in liver plasma membrane Models
Young Old AL Old EOD
Temperature induced kinetics Breakpoint ( ° C)
Activation energies (kcal/mol)
16.3 + 0.3 19.5 + 0.6 a 16.7 + 1.4 b
Above b.p. 6.43 + 0.21 5.96 + 1.33 6.39 + 0.21
Below b.p. 7.39 + 0.32 6.93+ 0.23 7.28 + 0.25
Entries are the means+S.E.M, of six separate experiments for each model. Statistical comparisons: a p < 0.01 from young; b p < 0.01 from old AL.
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1 / T 103 (°K) Fig. 2. Arrhenius plots of 5'-nucleotidase activity of liver plasma membranes. Symbols as in Fig. 1.
TABLE II Arrhenius plot parameters of 5'-nucleotidase activity of liver plasma membrane Temperature induced kinetics Breakpoint ( o C) Young Old AL Old EOD
25.1 +0.6 28.0 + 0.7 a 25.7 5:0.4 b
Activation energies (kcal/mol) Above b.p.
Below b.p.
10.345:0.91 7.37 5:0.43 a 9.99 5:0.73 b
21.2 +0.83 18.77 5:1.1 20.94 + 0.66
Entries are the means+S.E.M, of six separate experiments for each model. Statistical comparisons: a p < 0.02 from young; b p < 0.02 from old AL.
Even in this case the b r e a k p o i n t t e m p e r a t u r e was l o w e r in s a m p l e s f r o m y o u n g a n i m a l s as c o m p a r e d to those of old, whereas n o difference was o b s e r v e d c o m p a r i n g s a m p l e s f r o m y o u n g a n d o l d E O D fed rats. T h e a c t i v a t i o n energies a b o v e the b r e a k p o i n t were higher for the 5 ' - n u c l e o t i d a s e f r o m y o u n g a n d o l d E O D rats as c o m p a r e d those of the o l d A L animals.
Discussion T h e e x p e r i m e n t d e s c r i b e d in the p r e s e n t p a p e r was d e s i g n e d to e x p l o r e the p h y s i c o - c h e m i c a l p r o p e r t i e s of liver p l a s m a m e m b r a n e of rats d u r i n g aging a n d following diet restriction. T h e A r r h e n i u s p l o t s of D P H fluorescence p o l a r i z a t i o n as well as of 5 ' - n u c l e o t i d a s e activity were m e a s u r e d a n d analyzed.
122 In general," breakpoints of Arrhenius plots detected by enzymatic or physical techniques are associated with lipid phase transitions in the membranes and the temperatures at which these phenomena occur are closely related to the chemical composition of the membranes (Lee et al., 1974; Wunderlich et al., 1975). The interpretation of fluorescence polarization of DPH as a measure of membrane microviscosity has been questioned (Kleinfeld et al., 1981; Mc Vey et al., 1981). However, there is agreement that polarization of this type of probe reflects the local properties of its microenvironment (Wunderlich et al., 1975) and here we use the term 'microviscosity' only to indicate changes in the local lipid packing. The results obtained in our experimental models clearly suggest that DPH was partitioned in lipid environment of different composition in the young and old AL liver plasma membranes, their phase separation occurring at 16.3 and 19.5°C, respectively. Moreover, the fact that diet-restricted animals showed about the same breakpoint as the young rats suggests that undernutrition induced changes of liver membrane chemical composition which resulted in a decrease of microviscosity. These conclusions are in agrement with recently reported results on liver mitochondria and microsomes showing that the age-dependent decrease of the unsaturation/saturation ratio of lipids was reversed by diet restriction (Laganiere and Yu, 1987; 1989). Further support to these conclusions comes from the analysis of the data of 5'-nucleotidase activity. This enzyme is a glycoprotein located primarily in the plasma membrane. It is an ectoenzyme, i.e., its active site is exposed at the external surface of the membrane (Evans, 1974), and its activity is regulated by the composition and microviscosity of the external half of the bilayer (Dipple et al., 1982). Even in this case, changes in the Arrhenius plot break temperatures have been related to changes in phospholipid and/or fatty acid composition of the interacting lipids (Widnell, 1974; Merisko et al., 1981). Thus the observation that the Arrhenius break temperature of 5'-nucleotidase is enhanced during aging and that this increase is reversed by underfeeding again supports the hypothesis that diet restriction modifies the liver membrane lipid composition. The way in which undernutrition modifies the physico-chemical properties of liver membranes is not well understood. Laganiere and Yu (1987; 1989) hypothesized that the antiaging mechanism of food restriction was due to its stabilizing and antilipoperoxidative action on cellular membranes. Although we agree with this hypothesis (Pied et al., 1990b), we are of the opinion that another factor of great importance should be taken into account, i.e., the body temperature, which was lower in undernourished animals as compared to the ad libitum fed ones (Nelson and Halberg, 1986; Duffy et al., 1989). Indeed the existence of a relationship between body temperature and lipid composition of cell membranes has been found not only in poikilothermic animals (Hazel, 1979) but also in homeotherms (Houslay and Palmer, 1978). As an example of the latter case, the body temperatures of genetically obese mice were lower than that of their lean littermates (Bray and York, 1979) as a result of defective thermogenesis. However, the body temperature could be normalized by housing the animals at 34 °C or by thyroid hormone treatment (French et al., 1983).
123 A c o m p l e t e l y different p i c t u r e e m e r g e d f r o m the n o r m a l i z a t i o n of b o d y t e m p e r a ture of these mice c o n c e r n i n g b o t h liver m e m b r a n e c o m p o s i t i o n a n d b r e a k p o i n t s o f A r r h e n i u s plots o f b o t h D P H p o l a r i z a t i o n a n d 5 ' - n u c l e o t i d a s e activity ( F r e n c h et al., 1983). T h u s we can h y p o t h e s i z e that u n d e r n u t r i t i o n m o d i f i e s m e m b r a n e l i p i d c o m p o s i t i o n s t i m u l a t i n g the cells to react a g a i n s t the l o w e r i n g of b o d y t e m p e r a t u r e to m a i n t a i n the cellular m e m b r a n e in a p r o p e r f u n c t i o n a l state. T o g e t h e r with this, the m a i n t e n a n c e of the activity o f e n z y m e s which p r o t e c t against p e r o x i d a t i o n is the o t h e r key event which prevents the a g e - d e p e n d e n t d e t e r i o r a t i o n of cell m e m b r a n e s . T h e c o n t r i b u t i o n o f these two events m a y e x p l a i n w h y m e m b r a n e m i c r o v i s c o s i t y of o l d E O D a n i m a l s was lower t h a n t h a t of A L l i t t e r m a t e s even if one assumes a d e c r e a s e of b o d y t e m p e r a t u r e o f 2 ° C ( T a b l e I).
References Bray, G.A. and York, D.A. (1979): Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiol. Rev., 59, 719-809. Brivio-Haughland, R.P., Louis, S.L., Musch, K., Waldeck, N. and Williams, M.A. (1976): Liver plasma membranes from essentially fatty acid deficient rats, isolation, fatty acid composition and activities of 5'-nucleotidase, ATPase and adenylate cyclase. Biochim. Biophys. Acta, 433, 150-163. Clandinin, M.T., Foot, M. and Robson, L. (1983): Plasma membrane: can its structure and function be modulated by dietary fat? Comp. Biochem. Physiol., 76B, 335-339. Croze, E.M. and Morrr, J. (1984): Isolation of plasma membrane, Golgi apparatus and endoplasmic reticulum fractions from single homogenates of mouse liver. J, Cell Physiol., 119, 46-57. Dipple, I. and Houslay, M.D. (1978): The activity of glucagon-stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its hpid environment. Biochem. J., 174, 179-190. Dipple, I., Gordon, L.M. and Houslay, M.D. (1982): The activity of 5'-nucleotidase in liver plasma membranes is affected by the increase in bilayer fluidity achieved by anionic drugs but not by cationic drugs. J. Biol. Chem., 257, 1811-1815. Duffy, P.H., Feuers, R.J., Leakey, J.A., Nakamura, K.D., Turturro, A. and Hart, R.W. (1989): Effect of chronic caloric restriction o n physiological variables related to energy metabolism in male Fischer-344 rats. Mech. Ageing Dev., 48, 117-133. Duggleby, R.G. (1984): Regression analysis of nonlinear Arrhenius plots: an empirical model and a computer program. Comput. Biol. Med., 14, 447-455. Evans, W.H. (1974): Nucleotide pyrophosphatase: a sialoglycoprotein located on the hepatocyte surface. Nature, 250, 391-394. French, R.R., York, D.A., Portman, J.M. and Isaac, K. (1983): Hepatic plasma membranes from genetically obese (ob/ob) mice: studies on fluorescence polarization, phospholipid composition and 5'-nucleotidase activity. Comp. Biochem. Physiol., 76B, 309-319. Gonzales-Calvin, J., Saunders, J.B., Crossley, I.R., Dickerson, C.J., Smith, H.M., Tredger, J.M., and Williams, R. (1985): Effect of ethanol administration on rat hver plasma membrane-bound enzymes. Biochem. Pharmacol., 34, 2685-2689. Goodrick, C.L., Ingrain, D.K., Reynolds, M.A., Freeman, J.R. and Cider, N.L. (1982): Effect of intermittent feeding upon growth and life span of rats. Gerontology, 28, 233-241. Hazel, J. (1979): Influence of thermal acclimatation on membrane hpid composition of rainbow trout liver. Am. J. Physiol., 236, R91-101. Hegner, D. (1980): Age-dependence of molecular and functional change in biological membrane properties. Mech. Ageing Dev., 14, 101-108. Houslay, M.D. and Palmer, R.W. (1978): Changes in the form of Arrhenius plots of the activity of glucagon stimulated adenylate cyclase and other hamster liver plasma membrane enzymes occurring on hibernation. Biochem. J., 174, 909-919.
124 Kleinfeld, A., Dragsten, P., Klauser, R., Pjura, W. and Matayoshi, F. (1981): The lack of relationship between fluorescence polarization and lateral diffusion in biological membranes. Biochemistry, 13, 3699-3705. Koizumi, A., Weindruch, R. and R. Walford, L. (1987): Influences of dietary restriction and age on liver enzyme activities and lipid peroxidation in mice. J. Nutr., 117, 361-367. Laganiere, S. and Yu, B.P. (1987): Anti-lipoperoxidation action of food restriction. Biochem. Biophys. Res. Comm., 145, 1185-1191. Laganiere, S. and Yu, B.P. (1989): Effect of chronic food restriction in aging rats: II. Liver cytosolic antioxidants and related enzymes. Mech. Ageing. Dev., 48, 221-230. Lee, A.G., Birdsall, N.J.M., Metcalfe, J.C., Toom, P.A. and Warren, G.B. (1974): Clusters in lipid bilayers and interpretation of thermal effects in biological membranes. Biochemistry, 13, 3699-3705. Livingstone, C.J. and Schachter, D. (1980): Lipid dynamics and lipid-protein interactions in rat hepatocyte plasma membranes. J. Biol. Chem., 255, 10902-10908. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951): Proteins measurement with the folin phenol reagent. J. Biol. Chem., 193, 265-275. McVey, E., Yguerabide, J., Hamson, D. and Clark, W. (1981): Cooperative nature of binding of cholesterol onto synaptosomal plasma membrane of dog brain. Biochim. Bioph. Acta, 642, 106-118. Merisko, E.M., Ojakian, G.K. and Widnell, C.C. (1981): Effects of phospholipids on the properties of hepatic 5'-nucleotidase. J. Biol. Chem., 256, 1983-1993. Nelson, W. and Halberg, F. (1986): Meal-timing, circadian rhythms and life span of mice. J. Nutr., 116, 2244-2253. Nokubo, M. (1985): Physical-chemical and biochemical differences in liver plasma membranes in aging F-344 rats. J. Gerontol., 40, 409-414. Pieri, C., Recchioni, R., Moroni, F., Marcheselli, F., Falasca, M. and Piantanelli, L. (1990): Food restriction in female Wistar rats. I. Survival characteristics, membrane microviscosity and proliferative response in lymphocytes. Arch. Gerontol. Geriatr, 11, 99-108. Shinitzky, M. and Barenholz, S. (1978): Fluidity parameters of lipid regions determined by fluorescence polarization. Biochim. Biophys. Acta, 515, 367-394. Sottocasa, G.L., Kuylenstierna, B., Ernster, L. and Bergstrand, A. (1967): An electron-transport system associated with the outer membrane of liver mitochondria. J. Cell. Biol., 32, 415-438. Widnell, C.C. (1974): Purification of liver 5'-nucleotidase as a complex with sphingomyelin. Methods Enzymol., 32, 368-374. Wunderlich, F., Ronai, A., Speth, V., Seelig, J. and Blume, A. (1975): Thermotropic lipid clustering in Tetrahymena membranes. Biochemistry, 14, 3730-3735.
117
AGG 00342
Food restriction in female Wistar rats. III. Thermotropic transition of membrane lipid and 5'-nucleotidase activity in hepatocytes C. Pieri, M. Falasca, F. Moroni, F. Marcheselli and R. Recchioni Cytology Center, Gerontological Research Department LN.R.C.A., Via Birarelli 8, 60121 Ancona, Italy
(Received 9 April 1990; revised version received 9 July 1990; accepted 10 July 1990)
Summary The effect of diet restriction was measured on the anisotropy parameter of 1,6-diphenyl-l,3,5hexatriene (DPH) and 5'-nucleotidase enzyme activity in liver plasma membrane preparates. Diet restriction was applied to rats 3.5 months old on an every-other-day schedule (EOD) and the rats were killed at the age of 28-29 months. Six months and 24 month rats, fed ad libitum (AL), were used as controls. The Arrhenius plots of anisotropy parameter of liver membranes from young, old AL and old EOD animals exhibited well defined breakpoints at 16.3° C, 19.5°C and 16.7o C, respectively. The breakpoint temperature of 5'-nucleotidase activity was lower in samples from young rats as compared to those from old AL rats, whereas no difference was observed comparing young and EOD fed rats. Present results support the hypothesis that diet restriction modifies lipid composition of liver plasma membranes in such a way that the appearance of age-dependent alterations is delayed. Undernutrition; Hepatocyte membrane; DPH anisotropy; 5'-Nucleotidase
Introduction Aging of the liver has largely b e e n investigated because its pivotal role i n the metabolism. Liver p l a s m a m e m b r a n e , i n particular, is the p r i m a r y interface i n the homeostatic b a l a n c e b e t w e e n exogenous influence, e.g. diet, a n d e n d o g e n o u s c o n t r o l over the biosynthesis or utilization of various substrates ( C l a n d i n i n et al., 1983). F o r these reasons, a t t e n t i o n has b e e n paid to the a g e - d e p e n d e n t m o d i f i c a t i o n s occurring i n the liver p l a s m a m e m b r a n e s a n d a n increased microviscosity of lipid m a t r i x was d e m o n s t r a t e d i n old a n i m a l s as c o m p a r e d to y o u n g ones (Hegner, 1980; N o k u b o , 1985).
Correspondence to: Dr. Carlo Pieri, Cytology Center, Gerontological Research Department, I.N.R.C.A., Via Birarelli, 8, 60121 Ancona, Italy. 0167-4943/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
118 Recently it has been reported that food restriction reduced the age-related peroxidative membrane deterioration of liver microsomal and mitochondrial fractions (Laganiere and Yu, 1987; 1989; Koizumi et al., 1987). Liver plasma membrane composition can be influenced by the quality of the diet (Clandinin et al., 1983; Brivio-Haughland et al., 1976), but no data are available on the effect of the quantity of food on the chemical composition and biophysical properties of liver plasma membranes. The aim of the present work was to evaluate the effect of undernutrition on the biophysical properties of liver plasma membrane and the activity of 5'-nucleotidase, an enzyme which is regulated mainly by the physicochemical properties of the plasma membrane, the activity of which can be influenced by diet (Gonzales-Calvin et al., 1985).
Material and Methods
Animals Female Wistar rats of our breed were used. The diet restriction applied; the breeding conditions, as well as the survival and body weight curves were reported in our previous paper (Pied et al., 1990). Briefly, the diet restriction was applied to the animals from the age of 3.5 months, feeding them on an every-other-day schedule (EOD) with the same laboratory chow given ad libitum to the control rats (AL). This treatment was able to increase the lifespan of EOD animals when compared to the AL ones (Goodrick et al., 1982; Pieri et al., 1990). The EOD and AL rats were killed at 28-29 and 24 months of age, respectively, together with young control animals 6 months old. In the present experiment six animals of each group were used.
Liver plasma membrane preparation Liver plasma membranes were prepared by differential centrifugation followed by discontinuous gradient centrifugation according to Croze and Morr6 (1984) and washed in 1 mM NaHCO3, pH 7.4. Purity of the membranes was monitored by electron microscopic examination and contamination with mitochondria was assessed by the assay of succinic dehydrogenase (Sottocasa et al., 1967).
Fluorescence polarization studies Liver plasma membranes were suspended in phosphate-buffered saline (PBS; 5 mM phosphates, 145 mm NaC1, 4 mM KC1, 1 mM CaC12, pH 7.4) to a final concentration of 75 #g protein/ml. The membranes were loaded with the fluorescent lipid probe 1,6-diphenyl-l,3,5-hexatriene (DPH). Steady state fluorescence polarization was determined by means of SFM 25 spectrofluorimeter (Kontron) and expressed as the fluorescence anisotropy parameter [(ro/r ) - 1 ] -1 where r is the measured fluorescence anisotropy and r o is the limiting anisotropy. The latter is
119 assumed to be 0.362 for DPH (Shinitzky and Barenholz, 1978). The temperature dependency of [ ( r o / r ) - 1] -1 was determined over the range of 4 - 4 0 ° C and the logarithm of the anisotropy parameters was plotted against 1 / o K to detect thermotropic transitions. 5 '-Nucleotidase assay
The assay of 5'-nucleotidase (EC 3.1.3.5) was performed by means of the coupled spectrophotometric method of Dipple and Houslay (1978). Plasma membranes (75-150/~g protein) were suspended in 1 ml of 5 mM Tris-HC1 buffer pH 7.6 (pH adjusted at each temperature) containing 75 /~M adenosine 5'-monophosphate, 50 mM glycerol phosphate, 0.8 units of adenosine deaminase and 0.5 mM MgSO4. The reaction was monitored by the decrease in absorbance at 265 nm in a Beckman DU-7 spectrophotometer equipped with a temperature controlled older sample, in the temperature range 6-40 o C. Protein determination
The protein content of membrane preparations was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. Data analysis
Arrhenius plot parameters, transition temperatures and energies of activities were calculated using nonlinear regression analysis (Duggleby, 1984).
Results
Representative Arrhenius plots of the logarithms of anisotropy parameters of DPH are illustrated in Fig. 1. The values were obtained from liver plasma membrane preparations from the three experimental models: old EOD, old AL and young female Wistar rats. The measured parameters are summarized in Table I. The Arrhenius plot of anisotropy parameters of liver membranes from young, old AL and EOD animals exhibited well defined breakpoints at 16.3, 19.5 and 16.7 o C, respectively. The breakpoint temperature observed in young animals is in close agreement with other reported results obtained by using the same method based on changes in DPH polarization. This method is able to detect only the lower critical temperature of the broad transitions observed with differential scanning calorimetry (Livingstone and Schachter, 1980). Breakpoints in Arrhenius plots are closely associated with lipid phase separations occurring within the membrane and the temperatures at which they occur are closely related to the membrane lipid compositions (Lee et al., 1974; Wunderlich et al., 1975).
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3.6
1 / T x l O ~ (*K)
Fig. 1. Representative Arrhenius plots of anisotropy parameter of liver plasma membranes from young (zx), old EOD (0) and old AL (n). The clear increase in break temperature observed in old A L as c o m p a r e d with y o u n g rats indicates changes in m e m b r a n e composition during aging. I m p o r t a n t l y enough, the undernourished rats showed breakpoints very close to those of y o u n g animals, supporting the view that the age-dependent alterations of liver m e m b r a n e composition was prevented by diet restriction. The anisotropy values of liver m e m b r a n e suspension from old A L rats were higher over the entire temperature range of 4 - 4 0 ° C as c o m p a r e d to the E O D animals and the difference was approximately 3 ° C (Fig. 1). As opposed to the pattern of breakpoints, no significant changes of the activation energies of the phase transitions were observed u p o n aging and diet restriction. Typical Arrhenius plots of 5'-nucleotidase activity f r o m isolated hepatic plasma membranes from rats, in the experimental conditions considered, are shown in Fig. 2 and the Arrhenius plot parameters are summarised in Table II.
TABLE I Temperature dependence of DPH fluorescence polarization in liver plasma membrane Models
Young Old AL Old EOD
Temperature induced kinetics Breakpoint ( ° C)
Activation energies (kcal/mol)
16.3 + 0.3 19.5 + 0.6 a 16.7 + 1.4 b
Above b.p. 6.43 + 0.21 5.96 + 1.33 6.39 + 0.21
Below b.p. 7.39 + 0.32 6.93+ 0.23 7.28 + 0.25
Entries are the means+S.E.M, of six separate experiments for each model. Statistical comparisons: a p < 0.01 from young; b p < 0.01 from old AL.
121 40
30
I
I
I
20 I
I
10 ( ° C ) I
2.0.
0
& E
1.5
o')
o
1.0
i
i
3.18
3.28
i
3.38
3.48
1 / T 103 (°K) Fig. 2. Arrhenius plots of 5'-nucleotidase activity of liver plasma membranes. Symbols as in Fig. 1.
TABLE II Arrhenius plot parameters of 5'-nucleotidase activity of liver plasma membrane Temperature induced kinetics Breakpoint ( o C) Young Old AL Old EOD
25.1 +0.6 28.0 + 0.7 a 25.7 5:0.4 b
Activation energies (kcal/mol) Above b.p.
Below b.p.
10.345:0.91 7.37 5:0.43 a 9.99 5:0.73 b
21.2 +0.83 18.77 5:1.1 20.94 + 0.66
Entries are the means+S.E.M, of six separate experiments for each model. Statistical comparisons: a p < 0.02 from young; b p < 0.02 from old AL.
Even in this case the b r e a k p o i n t t e m p e r a t u r e was l o w e r in s a m p l e s f r o m y o u n g a n i m a l s as c o m p a r e d to those of old, whereas n o difference was o b s e r v e d c o m p a r i n g s a m p l e s f r o m y o u n g a n d o l d E O D fed rats. T h e a c t i v a t i o n energies a b o v e the b r e a k p o i n t were higher for the 5 ' - n u c l e o t i d a s e f r o m y o u n g a n d o l d E O D rats as c o m p a r e d those of the o l d A L animals.
Discussion T h e e x p e r i m e n t d e s c r i b e d in the p r e s e n t p a p e r was d e s i g n e d to e x p l o r e the p h y s i c o - c h e m i c a l p r o p e r t i e s of liver p l a s m a m e m b r a n e of rats d u r i n g aging a n d following diet restriction. T h e A r r h e n i u s p l o t s of D P H fluorescence p o l a r i z a t i o n as well as of 5 ' - n u c l e o t i d a s e activity were m e a s u r e d a n d analyzed.
122 In general," breakpoints of Arrhenius plots detected by enzymatic or physical techniques are associated with lipid phase transitions in the membranes and the temperatures at which these phenomena occur are closely related to the chemical composition of the membranes (Lee et al., 1974; Wunderlich et al., 1975). The interpretation of fluorescence polarization of DPH as a measure of membrane microviscosity has been questioned (Kleinfeld et al., 1981; Mc Vey et al., 1981). However, there is agreement that polarization of this type of probe reflects the local properties of its microenvironment (Wunderlich et al., 1975) and here we use the term 'microviscosity' only to indicate changes in the local lipid packing. The results obtained in our experimental models clearly suggest that DPH was partitioned in lipid environment of different composition in the young and old AL liver plasma membranes, their phase separation occurring at 16.3 and 19.5°C, respectively. Moreover, the fact that diet-restricted animals showed about the same breakpoint as the young rats suggests that undernutrition induced changes of liver membrane chemical composition which resulted in a decrease of microviscosity. These conclusions are in agrement with recently reported results on liver mitochondria and microsomes showing that the age-dependent decrease of the unsaturation/saturation ratio of lipids was reversed by diet restriction (Laganiere and Yu, 1987; 1989). Further support to these conclusions comes from the analysis of the data of 5'-nucleotidase activity. This enzyme is a glycoprotein located primarily in the plasma membrane. It is an ectoenzyme, i.e., its active site is exposed at the external surface of the membrane (Evans, 1974), and its activity is regulated by the composition and microviscosity of the external half of the bilayer (Dipple et al., 1982). Even in this case, changes in the Arrhenius plot break temperatures have been related to changes in phospholipid and/or fatty acid composition of the interacting lipids (Widnell, 1974; Merisko et al., 1981). Thus the observation that the Arrhenius break temperature of 5'-nucleotidase is enhanced during aging and that this increase is reversed by underfeeding again supports the hypothesis that diet restriction modifies the liver membrane lipid composition. The way in which undernutrition modifies the physico-chemical properties of liver membranes is not well understood. Laganiere and Yu (1987; 1989) hypothesized that the antiaging mechanism of food restriction was due to its stabilizing and antilipoperoxidative action on cellular membranes. Although we agree with this hypothesis (Pied et al., 1990b), we are of the opinion that another factor of great importance should be taken into account, i.e., the body temperature, which was lower in undernourished animals as compared to the ad libitum fed ones (Nelson and Halberg, 1986; Duffy et al., 1989). Indeed the existence of a relationship between body temperature and lipid composition of cell membranes has been found not only in poikilothermic animals (Hazel, 1979) but also in homeotherms (Houslay and Palmer, 1978). As an example of the latter case, the body temperatures of genetically obese mice were lower than that of their lean littermates (Bray and York, 1979) as a result of defective thermogenesis. However, the body temperature could be normalized by housing the animals at 34 °C or by thyroid hormone treatment (French et al., 1983).
123 A c o m p l e t e l y different p i c t u r e e m e r g e d f r o m the n o r m a l i z a t i o n of b o d y t e m p e r a ture of these mice c o n c e r n i n g b o t h liver m e m b r a n e c o m p o s i t i o n a n d b r e a k p o i n t s o f A r r h e n i u s plots o f b o t h D P H p o l a r i z a t i o n a n d 5 ' - n u c l e o t i d a s e activity ( F r e n c h et al., 1983). T h u s we can h y p o t h e s i z e that u n d e r n u t r i t i o n m o d i f i e s m e m b r a n e l i p i d c o m p o s i t i o n s t i m u l a t i n g the cells to react a g a i n s t the l o w e r i n g of b o d y t e m p e r a t u r e to m a i n t a i n the cellular m e m b r a n e in a p r o p e r f u n c t i o n a l state. T o g e t h e r with this, the m a i n t e n a n c e of the activity o f e n z y m e s which p r o t e c t against p e r o x i d a t i o n is the o t h e r key event which prevents the a g e - d e p e n d e n t d e t e r i o r a t i o n of cell m e m b r a n e s . T h e c o n t r i b u t i o n o f these two events m a y e x p l a i n w h y m e m b r a n e m i c r o v i s c o s i t y of o l d E O D a n i m a l s was lower t h a n t h a t of A L l i t t e r m a t e s even if one assumes a d e c r e a s e of b o d y t e m p e r a t u r e o f 2 ° C ( T a b l e I).
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