Mechanisms o f Ageing and Development, 11 (1979) 77-90
77
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
A C O M P A R I S O N O F P R O T E I N SYNTHESIS BY L I V E R P A R E N C H Y M A L CELLS ISOLATED FROM FISCHER F344 RATS OF VARIOUS AGES
JOHN J. CONIGLIO, DANIEL S. H. LIU and ARLAN RICHARDSON Department o f Chemistry, Illinois State University, Normal, Illinois 61761 {U.S.A.)
(Received December 15, 1978)
SUMMARY Rates of protein synthesis by intact liver parenchymal cells isolated from male Fischer F344 rats ranging in age from 2.5 to 30 months were determined by measuring the incorporation of [3H] valine into acid-insoluble material and the specific activity of the extracellular valine. The rate of protein synthesis decreased 44% from 2.5 to 18 months and then increased slightly (18%) from 18 to 30 months. There was no dramatic change in the types of proteins synthesized by isolated liver parenchymal cells isolated from 2-or 18-month-old rats as determined by SDS-polyacrylamide gel electrophoresis. The ribosomal-transit time by liver parenchymal cells isolated from 18-month-old rats was 60% higher than the ribosomal-transit time of liver parenchymal cells isolated from 4-month-old rats. The fidelity of protein synthesis by parenchymal cells isolated from 4and 18-month old rats. was compared by measuring the incorporation ofp-fluorophenylalanine (an analogue of phenylalanine) into acid-insoluble material. Although protein synthesis decreased significantly from 4 to 18 months, the fidelity of protein synthesis remained constant.
INTRODUCTION It is generally believed that the natural life-span of living organisms is at least partially under genetic control. Therefore, it would be expected that one of the mechanisms involved in aging would arise from changes in the genetic apparatus and in protein biosynthesis [1]. Over the past ten to fifteen years, there have been numerous studies on the effect of aging on protein biosynthesis. The majority of these studies have compared protein synthesis in liver tissue of rodents of various ages. Several investigators have compared the protein synthetic activities of liver homogenates from rats of various ages using a variety of cell-free systems. A cell-free system allows an investigator to control carefully the conditions required for protein synthesis and eliminates the problem of measuring the specific activity of the amino acid precursor pool. Although Chen et al. [2] have reported that cell-free protein synthesis
78 does not change with increasing age, all other studies which have compared cell-free protein synthesis by liver homogenates from rats or mice clearly demonstrate that protein synthesis declines with increasing age [3-8]. In general, a 40 to 60% decrease in cell-free protein synthesis has been observed with increasing age. Because the rate of cell-free protein synthesis by the liver is only 1% of the rate observed in vivo [9, 10], it is possible that the age-related changes observed in liver cellfree protein synthesis are not representative of the changes which occur in the whole animal. Although there have been several studies which have compared the rates of in vivo protein synthesis by liver of rodents of various ages, the results of these studies are conflicting. Kanungo et al. [ 11 ] have reported that [14C] leucine incorporation into acidinsoluble material by the liver increased with increasing age. However, the specific activity of the leucine pool was not determined. Ove et al. [12] have reported that the in vivo incorporation of [~4C] leucine into protein by liver was the same for 1- and 17-month-old rats. Recently D u e t al. [13] determined the rate of in vivo protein synthesis in the liver of 12- and 32-month-old mice by measuring the incorporation of [3H]leucine into protein and the specific activity of the intracellular leucine pool. Their data indicated that the rates of protein synthesis for most cellular fractions of liver did not change with age; however, protein synthesis by liver microsomes increased with age and the synthesis of heme proteins decreased with age. The conflicting observations cited above could arise from the fact that it is difficult to control experimental variables in whole animals. In addition, the measurement of in vivo protein synthesis by the incorporation of radioactively labeled amino acids into protein is complicated by the absorption of the amino acids, the compartmentation of the amino acids, and competing reactions [ 14, 15]. The aminoacyl-tRNA, the ultimate amino acid precursor for protein synthesis, has been shown to obtain its amino acids from both the intracellular and extracellutar amino acid pools [15-18]. Therefore, to determine accurately the rate of protein synthesis in vivo, it is necessary to measure the specific activity of the aminoacyl-tRNA. Because the specific activity of the leucyl-tRNA was not determined in any of the previous studies it is impossible to assess accurately the effect of aging on protein synthesis in vivo. Over the past seven years, numerous investigators have reported that isolated liver parenchymal cells (ILPC) are similar metabolically to liver parenchymal cells in vivo and can be used to study a wide variety of biochemical processes [ 19, 20]. In 1972, Schreiber and Schreiber [21 ] determined the conditions required for optimal protein synthesis by ILPC. Recently, our laboratory has demonstrated that the specific activities of the intracellular and extracellular valine pools rapidly equilibrate in suspensions of liver parenchymal cells [22]. Therefore, the specific activity of the valyl-tRNA in ILPC would be equal to the specific activity of either the intracellular or extracellular valine because the valyl-tRNA derives its amino acids from these two pools [15-18]. Using the specific activity of the extracellular valine, the rate of protein synthesis by ILPC was found to be comparable to rates of protein synthesis reported for perfused liver and liver in vivo [22]. By measuring albumin immunologically, van Bezooijen et al. [23] have shown that albumin synthesis by ILPC is similar to that reported for liver in vivo. Therefore, the protein synthetic capacity of ILPC seems to be comparable to the liver in vivo and it is
79 possible to measure accurately rates of protein synthesis from the specific activity of either the intracellular or extracellular amino acid pool when using radioactively labeled amino acids. Our laboratory also determined the actual rates of valine incorporation into protein by ILPC prepared from 1- to 18-month-old Sprague-Dawley rats [22]. The rate of protein synthesis by ILPC decreased 46% with increasing age, and this age-related decrease in protein synthesis was found to be representative of the liver in vivo. However, because there were no survival data for the colony of rats used in this study, only limited conclusions could be drawn about the effect of aging on protein synthesis. Van Bezooijen et al. [24] have compared protein synthesis by ILPC prepared from rats of various ages obtained from their aging colony of WAG/RIj rats. From the age of 3 to 24 months a 4 0 50% decrease in protein synthesis was observed. An increase in protein synthesis occurred from age 24 to 36 months. However, in this study, only the incorporation of [~4C]leucine into protein was determined; the specific activity of the leucine pool was not determined. In the study described herein, the rates of protein synthesis were determined for ILPC prepared from 2.5- to 30-month-old male Fischer F344 rats obtained from the animal colony maintained by the National Institute on Aging. The rates of protein synthesis were determined from the amount of [3H] valine incorporated into protein and the specific activity of the extracellular valine.
MATERIALS AND METHODS Animals Male Fischer F344 rats ranging from 2 to 30 months of age were purchased from the animal colony maintained for the National Institute on Aging by Charles River Breeding Laboratory. This particular colony of rats is barrier reared, carefully monitored, and used for aging studies. The incidence and severity of a wide variety of age-related pathological conditions have been determined in these animals [25], The mean survival of these r[ts is 28-29 months and the maximum survival 35 months [25]. Mortality in these rats is negligible until 20 months, at which time the survival of the rats decreases rapidly. After receiving the rats from the Charles River Breeding Laboratory, the rats were maintained in our laboratory in sterilized filter.top cages for two weeks before they were used for the experiments described below. Preparation o f isolated liver parenchymal cells Isolated liver parenchymal cells (ILPC) were obtained from rats by a modification of the method of Howard et al. [26] as described by Ricca et al. [22]. The rats were killed by decapitation between 9 a.m. and 11 a.m. to minimize diurnal variation, and the livers were removed immediately and placed in ice-cold calcium-free Hanks' solution [27]. The livers were rinsed twice with ice-cold calcium-free Hanks' solution and then perfused through the portal vein with 15 to 30 ml of ice-cold enzyme solution containing
80 0.05% collagenase in calcium-free Hanks' solution supplemented with 1 mM pyruvate, a vitamin mixture [28], twice the level of essential and non-essential amino acids as described by Eagle [28], and 50 mM Hepes buffer (pH 7.2). After the livers became completely blanched, they were sliced with a Stadie-Riggs tissue slicer into 0.3 to 0.4 mm thick slices. Approximately 2 to 3 g of liver slices were placed into 10 ml of the enzyme solution and incubated with shaking at 37 °C under an atmosphere of O2--CO2 (95:5). After 30 to 35 min of incubation, 0.1 ml of 1.41% CaCI2 was added to each flask. After an additional 20 min of incubation, the cell suspension was filtered through stocking nylon and then through nylon mesh (61 gm pore size). The cells were collected by differential centrifugation and washed as described by Howard et al. [26]. The yield of cells routinely ranged between 5 and 10 million cells per gram of liver, except for the 30-month-old rats, and the viability of the preparations ranged between 90 and 96% as determined by trypan blue exclusion. This cell yield is comparable to yields reported for this method in the scientific literature [20, 26]. The cell yield for the 30-month-old rats was consistently lower (approximately one-third to one-half) than that observed for the younger rats. Although the cell yield was lower, the viability of hepatocytes isolated from the 30-month-old rats was similar to that obtained with the younger rats. Protein synthesis by 1LPC Protein synthesis by ILPC was measured using the system which we have described previously [22]. Approximately 3 million cells per ml were incubated with shaking at 37 °C under an atmosphere of O2--CO2 (95:5) in a modified Eagle's Minimum Essential Medium [28] that consisted of Hanks' solution supplemented with 1 mM pyruvate, a vitamin mixture, 10 mM Hepes buffer (pH 7.2); and twice the level of essential and nonessential amino acids described by Eagle minus valine. Following a 5 rain preincubation period at 37 °C, L-[2,3-aH] valine (1 mCi/mmole) was added to the incubation at a final concentration of 1.6 raM. These conditions were found to be optimal for protein synthesis, which was linear for at least 2 h [22]. After 30 min of incubation, protein synthesis was terminated by the addition of the cell suspension to an equal volume of 10% trichloroacetic acid. The resulting acid-insoluble material was washed as described by Khairallah and Mortimore [17], and the acid-insoluble material was then air-dried and dissolved in 0.3 N NaOH. The radioactivity present in the NaOH solution and the protein concentration of the NaOH solution was determined by liquid scintillation counting and the method of Lowry et al. [29], respectively. The incorporation of [all] valine into the acid-insoluble material was expressed as dpm per mg of ILPC protein. To determine the actual rates of protein synthesis by ILPC, the specific activity of the extracellular acidsoluble valine was measured as described by Ricca et al. [22]. The rate of protein synthesis was expressed as pmole of valine incorporated into the acid-insoluble material per min per mg of ILPC protein. Determination o f ribosomal half-transit times Ribosomal half-transit times were determined by a modification of the procedure described by Fan and Penman [30]. ILPC were suspended at a concentration of
81 approximately 6 million cells per ml in the modified Eagle's Minimum Essential Medium used to measure protein synthesis except that L-[3H] valine (12.5 Ci/mmole) was added to the medium at a concentration of 1.7 /iCi]ml. Approximately 20 million cells were withdrawn from the suspension after 8, 11, 14 and 17 min of incubation. The incorporation of radioactivity into protein was determined by adding the cell suspension to an equal volume of ice-cold Hanks' solution containing 40 mM valine and cycloheximide (10 /~g/ml). The cells were washed twice in the Hanks' solution and then homogenized with a Potter-Elvehjem homogenizer at 4 °C in a solution containing 50 mM Tris (pH 7.6), cycloheximide (10 g/ml), 1% Triton X-100, and 1% sodium deoxycholate. The postmitochondrial supernatant (PMS) was obtained by centrifuging the homogenate at 16 000 g for 5 min. The acid-insoluble radioactivity in an aliquot of the PMS was determined using a liquid scintillation counter. Another aliquot of the PMS was centrifuged at 100 000 g for 3 h to obtain the ribosomal pellet. The acid-insoluble radioactivity associated with the ribosomal pellet was determined using a liquid scintillation counter. The acid-insoluble radioactivity in the post-ribosomal supernatant (PRS) was determined by subtracting the acid-insoluble radioactivity in the ribosomal pellet from the acid-insoluble radioactivity in the PMS.
Polyacrylamide gel electrophoresis Proteins from ILPC and extracellular medium were analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis as described by Yang et al. [31]. After incubating the ILPC with L-[3H] valine, the acid-insoluble material was obtained by treating the cell suspension with an equal volume of 10% trichloroacetic acid and the precipitate being collected by centrifugation. The acid-insoluble material was washed twice with 5% trichloroacetic acid and once with acetone. The acid-insoluble material was then dried and dissolved in electrophoresis sample buffer (0.01 M phosphate (pH 7.2), 1% 2mercaptoethanol, 1% SDS, 10% glycerol, and 0.05% bromophenol blue). The solutions were heated at 100 °C for 2 min to destroy protease activity. These solutions then were layered on 10 cm SDS-polyacrylamide gels consisting of 7.5% acrylamide, 0.2% N,N'methylene-bisacrylamide, and 0.1% SDS in 0.1 M phosphate buffer (pH 7.2). Electrophoresis was performed at 5 mA/gel for 16 h. The gels then were frozen in dry-iceacetone and sliced into 2 mm fractions. The slices then were transferred to scintillation vials and incubated with 5 ml of Aquasol (New England Nuclear) at 23 °C for 24 h. The radioactivity associated with each slice was determined using a liquid scintillation counter.
Determination of protein, RNA, and DNA concentrations of liver homogenates laver tissue 'was homogenized with two volumes of 0.9% NaCI using a PotterElvehjem homogenizer. The homogenate was treated with an equal volume of 10% trichloroacetic acid and the acid-insoluble material was collected by centrifugation. The acid-insoluble material was washed once with a chloroform-ether (2:1)solution, three times with 0.5 N HC104, and once with H20. The precipitate was then dissolved in an aliquot of 0.3 N NaOH and samples were withdrawn from this solution to determine
82 protein, RNA, and DNA concentrations. Protein concentrations were measured by the Lowry method [29] using bovine serum albumin as the standard. The RNA concentrations were measured by a modification of the Schmidt-Tannhauser procedure [32] using 1 A26o unit = 32 # g RNA per ml. DNA concentrations were measured by the indole method [33] using salmon sperm DNA as the standard.
RESULTS The changes in body weight, liver weight, and the protein, DNA, and RNA contents of the liver from 2- to 30-month-old male Fischer rats are shown in Table I. The body weight of these rats increased with age, reaching a maximum at 12 months. From 12 to 30 months a steady decrease in body weight was observed. Liver weight increased steadily from 2 to 30 months. Although the protein and RNA contents of liver fluctuated with age, no statistically significant age-related trend was observed. Ross [34] has reported that the nitrogen content per gram of liver remained relatively constant from 4 to 20 months. The DNA content per gram of liver increased steadily with age; however, this increase was not significant. Several investigators have reported that the ploidy of liver parenchymal cells increases with age [35-37]. The greatest change in the ploidy of liver cells appears to occur durin~ the first 12 months of life [35, 37]. In addition, Castle et al. [38] have observed that the DNA content of nuclei isolated from rat liver increases as a function of the animal's age. TABLE I COMPARISON OF BODY WEIGHT, LIVER WEIGHT AND THE CONTENT OF PROTEIN, RNA, AND DNA FROM THE LIVER OF RATS OF VARIOUSAGES Each value represents the mean -+S.E.M. of 4 animals Age (months)
Body weight (g)
Liver weight (g)
Liver Protein (mg/g)
RNA (mg/g)
DNA (mg/g)
2-4 6 11-13 18-20 28-30
275 ± 18 340± 8 408-+ 16 387 ± 39 336 ± 10
10.4 ± 0.5 11.6±0.3 12.7-+0.6 13.7 +-0.5 14.6 ± 0.6
148 ± 10 123-+ 11 118-+ 1 132 ± 8.7 124 ± 9
6.42 ± 0.08 5.96+-0.37 5.74-+0.24 6.06 ± 0.45 6.84 ± 0.18
2.03 ± 0.33 2.16+-0.18 2.21-+0.51 2.34 ± 0.44 2.47 ± 0.16
Table II shows the rate of [all] valine incorporation into protein by ILPC prepared from 2- to 30-month-old rats during a 30 min incubation period. We have shown previously that protein synthesis by ILPC in this system is linear for at least 120 min of incubation [22]. Because the specific activities of the intracellular and extracellular valine equilibrate rapidly and remain constant in ILPC suspensions [22], the specific activity of the valyl-tRNA (the ultimate amino acid precursor for protein synthesis) would be
83
TABLE II RATES OF PROTEIN SYNTHESIS BY ISOLATED LIVER PARENCHYMAL CELLS PREPARED FROM RATS OF VARIOUS AGES Age {months}
2.5--4 6-7 12-13 18 28-30
Valine incorporation pmole per min per mg protein
%
120.7 -+ 11.8 99.0 -+ 10.4 85.0 -+ 10.1 67.6-+ 6.99 80.0 -+ 16.3
100 82 70 56 66
The rates of protein synthesis were determined by measuring the incorporation of [3H]valine into acid-insoluble material after 30 min of incubation at 37 °C. The cell viability remained greater than 90% throughout the 30 min incubation period for all experiments. Each value represents the mean -+ S.E.M. of 4 rats. The decrease in protein synthesis between ages 2.5--4 months and 18 months was significant at the p < 0.01 level; while the decrease in protein synthesis between ages 6-7 months and 18 months was significant at the p < 0.1 level as determined by the Student's t-test (unpaired). equivalent to the specific activity of either the intracellular or the extracellular valine [ 1 5 - 1 8 ] . Consequently, it is possible to measure accurately the rate o f valine incorporation into protein by ILPC from the specific activity o f either the intracellular or extracellular valine. In the present study, the rates of protein synthesis were determined using the specific activity of the extracellular valine. The rates of valine incorporation into protein by ILPC prepared from 2.5- to 18-month-old rats decreased from 120.7 to 67.6 pmoles of valine per mg of ILPC protein per min. This represents a 44% decrease in protein synthesis. However, from 18 to 30 months, the rate of protein synthesis increased 18%. This increase was not statistically significant. Using suspensions of ILPC from rats of various ages, it was possible to determine the ribosomal half-transit time as described by Fan and Penman [30]. The ribosomal halftransit time represents the elongation time required for the synthesis of an average halflength of a nascent polypeptide chain. However, it is important t o note that the ribosomal half-transit time does not represent the rate of ribosome attachment to the protein synthetic apparatus [30]. Fig. 1 shows that the ribosomal half-transit times of polypeptide synthesis by ILPC prepared from 4- and 18-month-old rats were 1.46 and 2.4 min, respectively. Feldhoff et al. [39] have reported a ribosomal half-transit time of 1.6 + 1 min for ILPC prepared from 3- to 4-month-old male Sprague-Dawley rats. It is apparent that a 1.6-fold increase in the ribosomal half-transit time occurred between 4and 18.month-old rats. Therefore, the translation of mRNA by ribosomes occurs at a slower rate in ILPC prepared from 18omonth-old rats than compared to ILPC prepared from 4-month-old rats. To determine if the decrease observed in protein synthesis between ages 2 and 18 months was due to a general decrease in the synthesis of all "proteins, or to a decrease in the synthesis o f only certain proteins, the type of proteins synthesized by ILPC from rats of various ages were compared by SDS-polyacrylamide gel electrophoresis. ILPC
84
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77
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
A C O M P A R I S O N O F P R O T E I N SYNTHESIS BY L I V E R P A R E N C H Y M A L CELLS ISOLATED FROM FISCHER F344 RATS OF VARIOUS AGES
JOHN J. CONIGLIO, DANIEL S. H. LIU and ARLAN RICHARDSON Department o f Chemistry, Illinois State University, Normal, Illinois 61761 {U.S.A.)
(Received December 15, 1978)
SUMMARY Rates of protein synthesis by intact liver parenchymal cells isolated from male Fischer F344 rats ranging in age from 2.5 to 30 months were determined by measuring the incorporation of [3H] valine into acid-insoluble material and the specific activity of the extracellular valine. The rate of protein synthesis decreased 44% from 2.5 to 18 months and then increased slightly (18%) from 18 to 30 months. There was no dramatic change in the types of proteins synthesized by isolated liver parenchymal cells isolated from 2-or 18-month-old rats as determined by SDS-polyacrylamide gel electrophoresis. The ribosomal-transit time by liver parenchymal cells isolated from 18-month-old rats was 60% higher than the ribosomal-transit time of liver parenchymal cells isolated from 4-month-old rats. The fidelity of protein synthesis by parenchymal cells isolated from 4and 18-month old rats. was compared by measuring the incorporation ofp-fluorophenylalanine (an analogue of phenylalanine) into acid-insoluble material. Although protein synthesis decreased significantly from 4 to 18 months, the fidelity of protein synthesis remained constant.
INTRODUCTION It is generally believed that the natural life-span of living organisms is at least partially under genetic control. Therefore, it would be expected that one of the mechanisms involved in aging would arise from changes in the genetic apparatus and in protein biosynthesis [1]. Over the past ten to fifteen years, there have been numerous studies on the effect of aging on protein biosynthesis. The majority of these studies have compared protein synthesis in liver tissue of rodents of various ages. Several investigators have compared the protein synthetic activities of liver homogenates from rats of various ages using a variety of cell-free systems. A cell-free system allows an investigator to control carefully the conditions required for protein synthesis and eliminates the problem of measuring the specific activity of the amino acid precursor pool. Although Chen et al. [2] have reported that cell-free protein synthesis
78 does not change with increasing age, all other studies which have compared cell-free protein synthesis by liver homogenates from rats or mice clearly demonstrate that protein synthesis declines with increasing age [3-8]. In general, a 40 to 60% decrease in cell-free protein synthesis has been observed with increasing age. Because the rate of cell-free protein synthesis by the liver is only 1% of the rate observed in vivo [9, 10], it is possible that the age-related changes observed in liver cellfree protein synthesis are not representative of the changes which occur in the whole animal. Although there have been several studies which have compared the rates of in vivo protein synthesis by liver of rodents of various ages, the results of these studies are conflicting. Kanungo et al. [ 11 ] have reported that [14C] leucine incorporation into acidinsoluble material by the liver increased with increasing age. However, the specific activity of the leucine pool was not determined. Ove et al. [12] have reported that the in vivo incorporation of [~4C] leucine into protein by liver was the same for 1- and 17-month-old rats. Recently D u e t al. [13] determined the rate of in vivo protein synthesis in the liver of 12- and 32-month-old mice by measuring the incorporation of [3H]leucine into protein and the specific activity of the intracellular leucine pool. Their data indicated that the rates of protein synthesis for most cellular fractions of liver did not change with age; however, protein synthesis by liver microsomes increased with age and the synthesis of heme proteins decreased with age. The conflicting observations cited above could arise from the fact that it is difficult to control experimental variables in whole animals. In addition, the measurement of in vivo protein synthesis by the incorporation of radioactively labeled amino acids into protein is complicated by the absorption of the amino acids, the compartmentation of the amino acids, and competing reactions [ 14, 15]. The aminoacyl-tRNA, the ultimate amino acid precursor for protein synthesis, has been shown to obtain its amino acids from both the intracellular and extracellutar amino acid pools [15-18]. Therefore, to determine accurately the rate of protein synthesis in vivo, it is necessary to measure the specific activity of the aminoacyl-tRNA. Because the specific activity of the leucyl-tRNA was not determined in any of the previous studies it is impossible to assess accurately the effect of aging on protein synthesis in vivo. Over the past seven years, numerous investigators have reported that isolated liver parenchymal cells (ILPC) are similar metabolically to liver parenchymal cells in vivo and can be used to study a wide variety of biochemical processes [ 19, 20]. In 1972, Schreiber and Schreiber [21 ] determined the conditions required for optimal protein synthesis by ILPC. Recently, our laboratory has demonstrated that the specific activities of the intracellular and extracellular valine pools rapidly equilibrate in suspensions of liver parenchymal cells [22]. Therefore, the specific activity of the valyl-tRNA in ILPC would be equal to the specific activity of either the intracellular or extracellular valine because the valyl-tRNA derives its amino acids from these two pools [15-18]. Using the specific activity of the extracellular valine, the rate of protein synthesis by ILPC was found to be comparable to rates of protein synthesis reported for perfused liver and liver in vivo [22]. By measuring albumin immunologically, van Bezooijen et al. [23] have shown that albumin synthesis by ILPC is similar to that reported for liver in vivo. Therefore, the protein synthetic capacity of ILPC seems to be comparable to the liver in vivo and it is
79 possible to measure accurately rates of protein synthesis from the specific activity of either the intracellular or extracellular amino acid pool when using radioactively labeled amino acids. Our laboratory also determined the actual rates of valine incorporation into protein by ILPC prepared from 1- to 18-month-old Sprague-Dawley rats [22]. The rate of protein synthesis by ILPC decreased 46% with increasing age, and this age-related decrease in protein synthesis was found to be representative of the liver in vivo. However, because there were no survival data for the colony of rats used in this study, only limited conclusions could be drawn about the effect of aging on protein synthesis. Van Bezooijen et al. [24] have compared protein synthesis by ILPC prepared from rats of various ages obtained from their aging colony of WAG/RIj rats. From the age of 3 to 24 months a 4 0 50% decrease in protein synthesis was observed. An increase in protein synthesis occurred from age 24 to 36 months. However, in this study, only the incorporation of [~4C]leucine into protein was determined; the specific activity of the leucine pool was not determined. In the study described herein, the rates of protein synthesis were determined for ILPC prepared from 2.5- to 30-month-old male Fischer F344 rats obtained from the animal colony maintained by the National Institute on Aging. The rates of protein synthesis were determined from the amount of [3H] valine incorporated into protein and the specific activity of the extracellular valine.
MATERIALS AND METHODS Animals Male Fischer F344 rats ranging from 2 to 30 months of age were purchased from the animal colony maintained for the National Institute on Aging by Charles River Breeding Laboratory. This particular colony of rats is barrier reared, carefully monitored, and used for aging studies. The incidence and severity of a wide variety of age-related pathological conditions have been determined in these animals [25], The mean survival of these r[ts is 28-29 months and the maximum survival 35 months [25]. Mortality in these rats is negligible until 20 months, at which time the survival of the rats decreases rapidly. After receiving the rats from the Charles River Breeding Laboratory, the rats were maintained in our laboratory in sterilized filter.top cages for two weeks before they were used for the experiments described below. Preparation o f isolated liver parenchymal cells Isolated liver parenchymal cells (ILPC) were obtained from rats by a modification of the method of Howard et al. [26] as described by Ricca et al. [22]. The rats were killed by decapitation between 9 a.m. and 11 a.m. to minimize diurnal variation, and the livers were removed immediately and placed in ice-cold calcium-free Hanks' solution [27]. The livers were rinsed twice with ice-cold calcium-free Hanks' solution and then perfused through the portal vein with 15 to 30 ml of ice-cold enzyme solution containing
80 0.05% collagenase in calcium-free Hanks' solution supplemented with 1 mM pyruvate, a vitamin mixture [28], twice the level of essential and non-essential amino acids as described by Eagle [28], and 50 mM Hepes buffer (pH 7.2). After the livers became completely blanched, they were sliced with a Stadie-Riggs tissue slicer into 0.3 to 0.4 mm thick slices. Approximately 2 to 3 g of liver slices were placed into 10 ml of the enzyme solution and incubated with shaking at 37 °C under an atmosphere of O2--CO2 (95:5). After 30 to 35 min of incubation, 0.1 ml of 1.41% CaCI2 was added to each flask. After an additional 20 min of incubation, the cell suspension was filtered through stocking nylon and then through nylon mesh (61 gm pore size). The cells were collected by differential centrifugation and washed as described by Howard et al. [26]. The yield of cells routinely ranged between 5 and 10 million cells per gram of liver, except for the 30-month-old rats, and the viability of the preparations ranged between 90 and 96% as determined by trypan blue exclusion. This cell yield is comparable to yields reported for this method in the scientific literature [20, 26]. The cell yield for the 30-month-old rats was consistently lower (approximately one-third to one-half) than that observed for the younger rats. Although the cell yield was lower, the viability of hepatocytes isolated from the 30-month-old rats was similar to that obtained with the younger rats. Protein synthesis by 1LPC Protein synthesis by ILPC was measured using the system which we have described previously [22]. Approximately 3 million cells per ml were incubated with shaking at 37 °C under an atmosphere of O2--CO2 (95:5) in a modified Eagle's Minimum Essential Medium [28] that consisted of Hanks' solution supplemented with 1 mM pyruvate, a vitamin mixture, 10 mM Hepes buffer (pH 7.2); and twice the level of essential and nonessential amino acids described by Eagle minus valine. Following a 5 rain preincubation period at 37 °C, L-[2,3-aH] valine (1 mCi/mmole) was added to the incubation at a final concentration of 1.6 raM. These conditions were found to be optimal for protein synthesis, which was linear for at least 2 h [22]. After 30 min of incubation, protein synthesis was terminated by the addition of the cell suspension to an equal volume of 10% trichloroacetic acid. The resulting acid-insoluble material was washed as described by Khairallah and Mortimore [17], and the acid-insoluble material was then air-dried and dissolved in 0.3 N NaOH. The radioactivity present in the NaOH solution and the protein concentration of the NaOH solution was determined by liquid scintillation counting and the method of Lowry et al. [29], respectively. The incorporation of [all] valine into the acid-insoluble material was expressed as dpm per mg of ILPC protein. To determine the actual rates of protein synthesis by ILPC, the specific activity of the extracellular acidsoluble valine was measured as described by Ricca et al. [22]. The rate of protein synthesis was expressed as pmole of valine incorporated into the acid-insoluble material per min per mg of ILPC protein. Determination o f ribosomal half-transit times Ribosomal half-transit times were determined by a modification of the procedure described by Fan and Penman [30]. ILPC were suspended at a concentration of
81 approximately 6 million cells per ml in the modified Eagle's Minimum Essential Medium used to measure protein synthesis except that L-[3H] valine (12.5 Ci/mmole) was added to the medium at a concentration of 1.7 /iCi]ml. Approximately 20 million cells were withdrawn from the suspension after 8, 11, 14 and 17 min of incubation. The incorporation of radioactivity into protein was determined by adding the cell suspension to an equal volume of ice-cold Hanks' solution containing 40 mM valine and cycloheximide (10 /~g/ml). The cells were washed twice in the Hanks' solution and then homogenized with a Potter-Elvehjem homogenizer at 4 °C in a solution containing 50 mM Tris (pH 7.6), cycloheximide (10 g/ml), 1% Triton X-100, and 1% sodium deoxycholate. The postmitochondrial supernatant (PMS) was obtained by centrifuging the homogenate at 16 000 g for 5 min. The acid-insoluble radioactivity in an aliquot of the PMS was determined using a liquid scintillation counter. Another aliquot of the PMS was centrifuged at 100 000 g for 3 h to obtain the ribosomal pellet. The acid-insoluble radioactivity associated with the ribosomal pellet was determined using a liquid scintillation counter. The acid-insoluble radioactivity in the post-ribosomal supernatant (PRS) was determined by subtracting the acid-insoluble radioactivity in the ribosomal pellet from the acid-insoluble radioactivity in the PMS.
Polyacrylamide gel electrophoresis Proteins from ILPC and extracellular medium were analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis as described by Yang et al. [31]. After incubating the ILPC with L-[3H] valine, the acid-insoluble material was obtained by treating the cell suspension with an equal volume of 10% trichloroacetic acid and the precipitate being collected by centrifugation. The acid-insoluble material was washed twice with 5% trichloroacetic acid and once with acetone. The acid-insoluble material was then dried and dissolved in electrophoresis sample buffer (0.01 M phosphate (pH 7.2), 1% 2mercaptoethanol, 1% SDS, 10% glycerol, and 0.05% bromophenol blue). The solutions were heated at 100 °C for 2 min to destroy protease activity. These solutions then were layered on 10 cm SDS-polyacrylamide gels consisting of 7.5% acrylamide, 0.2% N,N'methylene-bisacrylamide, and 0.1% SDS in 0.1 M phosphate buffer (pH 7.2). Electrophoresis was performed at 5 mA/gel for 16 h. The gels then were frozen in dry-iceacetone and sliced into 2 mm fractions. The slices then were transferred to scintillation vials and incubated with 5 ml of Aquasol (New England Nuclear) at 23 °C for 24 h. The radioactivity associated with each slice was determined using a liquid scintillation counter.
Determination of protein, RNA, and DNA concentrations of liver homogenates laver tissue 'was homogenized with two volumes of 0.9% NaCI using a PotterElvehjem homogenizer. The homogenate was treated with an equal volume of 10% trichloroacetic acid and the acid-insoluble material was collected by centrifugation. The acid-insoluble material was washed once with a chloroform-ether (2:1)solution, three times with 0.5 N HC104, and once with H20. The precipitate was then dissolved in an aliquot of 0.3 N NaOH and samples were withdrawn from this solution to determine
82 protein, RNA, and DNA concentrations. Protein concentrations were measured by the Lowry method [29] using bovine serum albumin as the standard. The RNA concentrations were measured by a modification of the Schmidt-Tannhauser procedure [32] using 1 A26o unit = 32 # g RNA per ml. DNA concentrations were measured by the indole method [33] using salmon sperm DNA as the standard.
RESULTS The changes in body weight, liver weight, and the protein, DNA, and RNA contents of the liver from 2- to 30-month-old male Fischer rats are shown in Table I. The body weight of these rats increased with age, reaching a maximum at 12 months. From 12 to 30 months a steady decrease in body weight was observed. Liver weight increased steadily from 2 to 30 months. Although the protein and RNA contents of liver fluctuated with age, no statistically significant age-related trend was observed. Ross [34] has reported that the nitrogen content per gram of liver remained relatively constant from 4 to 20 months. The DNA content per gram of liver increased steadily with age; however, this increase was not significant. Several investigators have reported that the ploidy of liver parenchymal cells increases with age [35-37]. The greatest change in the ploidy of liver cells appears to occur durin~ the first 12 months of life [35, 37]. In addition, Castle et al. [38] have observed that the DNA content of nuclei isolated from rat liver increases as a function of the animal's age. TABLE I COMPARISON OF BODY WEIGHT, LIVER WEIGHT AND THE CONTENT OF PROTEIN, RNA, AND DNA FROM THE LIVER OF RATS OF VARIOUSAGES Each value represents the mean -+S.E.M. of 4 animals Age (months)
Body weight (g)
Liver weight (g)
Liver Protein (mg/g)
RNA (mg/g)
DNA (mg/g)
2-4 6 11-13 18-20 28-30
275 ± 18 340± 8 408-+ 16 387 ± 39 336 ± 10
10.4 ± 0.5 11.6±0.3 12.7-+0.6 13.7 +-0.5 14.6 ± 0.6
148 ± 10 123-+ 11 118-+ 1 132 ± 8.7 124 ± 9
6.42 ± 0.08 5.96+-0.37 5.74-+0.24 6.06 ± 0.45 6.84 ± 0.18
2.03 ± 0.33 2.16+-0.18 2.21-+0.51 2.34 ± 0.44 2.47 ± 0.16
Table II shows the rate of [all] valine incorporation into protein by ILPC prepared from 2- to 30-month-old rats during a 30 min incubation period. We have shown previously that protein synthesis by ILPC in this system is linear for at least 120 min of incubation [22]. Because the specific activities of the intracellular and extracellular valine equilibrate rapidly and remain constant in ILPC suspensions [22], the specific activity of the valyl-tRNA (the ultimate amino acid precursor for protein synthesis) would be
83
TABLE II RATES OF PROTEIN SYNTHESIS BY ISOLATED LIVER PARENCHYMAL CELLS PREPARED FROM RATS OF VARIOUS AGES Age {months}
2.5--4 6-7 12-13 18 28-30
Valine incorporation pmole per min per mg protein
%
120.7 -+ 11.8 99.0 -+ 10.4 85.0 -+ 10.1 67.6-+ 6.99 80.0 -+ 16.3
100 82 70 56 66
The rates of protein synthesis were determined by measuring the incorporation of [3H]valine into acid-insoluble material after 30 min of incubation at 37 °C. The cell viability remained greater than 90% throughout the 30 min incubation period for all experiments. Each value represents the mean -+ S.E.M. of 4 rats. The decrease in protein synthesis between ages 2.5--4 months and 18 months was significant at the p < 0.01 level; while the decrease in protein synthesis between ages 6-7 months and 18 months was significant at the p < 0.1 level as determined by the Student's t-test (unpaired). equivalent to the specific activity of either the intracellular or the extracellular valine [ 1 5 - 1 8 ] . Consequently, it is possible to measure accurately the rate o f valine incorporation into protein by ILPC from the specific activity o f either the intracellular or extracellular valine. In the present study, the rates of protein synthesis were determined using the specific activity of the extracellular valine. The rates of valine incorporation into protein by ILPC prepared from 2.5- to 18-month-old rats decreased from 120.7 to 67.6 pmoles of valine per mg of ILPC protein per min. This represents a 44% decrease in protein synthesis. However, from 18 to 30 months, the rate of protein synthesis increased 18%. This increase was not statistically significant. Using suspensions of ILPC from rats of various ages, it was possible to determine the ribosomal half-transit time as described by Fan and Penman [30]. The ribosomal halftransit time represents the elongation time required for the synthesis of an average halflength of a nascent polypeptide chain. However, it is important t o note that the ribosomal half-transit time does not represent the rate of ribosome attachment to the protein synthetic apparatus [30]. Fig. 1 shows that the ribosomal half-transit times of polypeptide synthesis by ILPC prepared from 4- and 18-month-old rats were 1.46 and 2.4 min, respectively. Feldhoff et al. [39] have reported a ribosomal half-transit time of 1.6 + 1 min for ILPC prepared from 3- to 4-month-old male Sprague-Dawley rats. It is apparent that a 1.6-fold increase in the ribosomal half-transit time occurred between 4and 18.month-old rats. Therefore, the translation of mRNA by ribosomes occurs at a slower rate in ILPC prepared from 18omonth-old rats than compared to ILPC prepared from 4-month-old rats. To determine if the decrease observed in protein synthesis between ages 2 and 18 months was due to a general decrease in the synthesis of all "proteins, or to a decrease in the synthesis o f only certain proteins, the type of proteins synthesized by ILPC from rats of various ages were compared by SDS-polyacrylamide gel electrophoresis. ILPC
84
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