Patterns of amino acid incorporation in long-lived genetic strains of Drosophila melanogaster

Experimental Gerontology. Vol. 24, pp. 67-81, 1989 Printed in the USA. All rights reserved.

PATTERNS

OF

GENETIC

0531-5565/89$3.00 + .00 Copyright"~:1989PergamonPress plc

AMINO ACID INCORPORATION IN LONG-LIVED STRAINS OF DROSOPHILA MELANOGASTER

ROBERT PRETZLAFF and ROBERT ARKING Department of Biological Sciences, and Institute of Gerontology, Wayne State University Detroit, Michigan 48202 Abstract - - This study examined the age-dependent alterations in the in vivo incor-

poration of labeled amino acids into protein during the adult life spans of males and females obtained from genetically based long-lived and control strains of Drosophila mehmogaster. All four groups tested showed significant decreases (ca. 50%) in the uptake of labeled amino acids as a function of age. Each of the four groups had their own characteristic temporal pattern of functional decrement. Both the long-lived and the control females have similar patterns of amino acid incorporation, but the onset of these changes is delayed by 10 to 20 days in long-lived animals. These alterations in protein synthesis appear to be related to corresponding changes in the female fecundity patterns of each strain. The male patterns differ from one another and from the female patterns, but they both show periods of high fluctuation early in life followed by a terminal period of relatively low and constant synthesis. These data are consistent with the view that the overall alterations in the longevity of these two strains are likely due to changes in the timing of particular events in the adult life cycle. Key Words: aging, protein synthesis, genetics of aging, Drosophila, senescence

INTRODUCTION ONE OF the more difficult problems in the study of aging is objectively sorting out those physiological activities that are causally involved in the aging process from those other time-dependent processes that are merely correlated with age. It is likely that further insights into the nature of the aging process will depend in large part on the ability to make such distinctions. If it can be demonstrated that at least some portion of the aging process is under genetic control, then the comparison of genetic variants, differing only in life span, may allow us to sort out causal from correlative events and thereby shed some light on the underlying causes involved in senescence. We have previously described the creation and characterization o f a genetically based long-lived strain of Drosophila melanogaster (see Arking 1987b for discussion and references). The comparative analysis of this strain with its control strain has allowed us to demonstrate the value of this approach. For example, we have recently reported that the extended Correspondence to: R. Arking. (Received 9 November 1987;Accepted 15 March 1988) 67

68

R. PRETZI.AFF and R. ARKING

life span is not due to modifications in energy metabolism as were originally predicted by the rate of living theories (for discussion and references, see Arking et al., 1988). Since genetic regulation does appear to be involved in the control of normal aging (Arking 1987a, b), then it would seem reasonable to study the age-dependent role and activity of gene products in the long-lived and control strains ofDros~phila. The examination of changes in gene products can take several forms; for example, one could assay the age-dependent alterations in the amounts and/or activities of specific gene products believed to play key regulatory roles in the aging process, or one could assay changes in the rates of synthesis and degradation of such specific products, or one could conceivably monitor age-dependent changes in the accuracy of synthesis, processing, and turnover associated with these same gene products. We have no information at this time that will allow us to objectively identify any specific gene products as being causally involved in the aging process. Therefore, we have chosen to initiate our biochemical inquiries with a more general question devoid of too many specific assumptions regarding the nature of the mechanisms involved in aging; namely, an examination into the rate of in vivo amino acid incorporation into long- and short-lived strains of l)ro,sophiht m~'la;togast~,r, with particular concern being paid as to how this parameter might vary with respect to age, strain, and sex differences. It is generally accepted, as a result of both in vivo studies and in vitro studies, that the rates of protein synthesis are substantially lower in old insects that they are in young insects (Makrides, 1983; Baker, 1985). Only one relatively early study on another species ofl)r~).~ol?hila (Clarke and Maynard-Smith, 1966) has ever reported an increase in the rate o f protein synthesis with increasing age. In an in vivo study, Bauman and Chen (1972), found about a 60~Z: decrease in the rate of incorporation of three amino acids in male l)r~,s~phila .s'llbo/9,~(lllYt o v e r the life span of the fly. In vitro studies have also observed decreases in the rate of protein synthesis, most notable being the work of Webster (see Webster, 1987 for review and references). Webster and Webster (1979), using the combined sexes of wild-type l)ro,sOl~hila tJz¢'ht,oga,~t~'r, found a 70#/~,decrease in 14C amino acid incorporation into microsomal protein during the first 14 days of adult life followed by a slow decline thereafter. In a later paper (Webster ~,t al., 1980), it was found that this decrease was not uniform within the organism, but was region-specific and represented the mean of a drop in synthesis in the head, thorax, and abdomen of 15%, 95%, and 33% respectively. This result held forth the possibility of tissue-specific gene control and modulation of the aging process, but also pointed out the necessity for a comparative analysis if one were to have any realistic hope of identifying the mechanisms and molecules causally involved in the aging process.

MATERIALS AND METHODS

Organisms: Maintenan('e attd ,zeast,'e,tent o.f ,survival chara¢'teristi¢'s The long-lived N D C - L eL) and the random-bred control (R) strains of Drosophila melanogaster were used in this study. The selection and survival characteristics of these strains, when raised under conditions known to optimize longevity (single pairs of flies per vial with changes to fresh media daily), have been previously described (Arking, 1987a). Logistical considerations forced us to substantially alter these conditions and raise both the L and the R strain adults under conditions of high adult density and

AMINO ACID INCORPORATION IN DROSOPHILA MELANOGASTER

69

with vial/bottle changes only once per three days. A preliminary study was made to determine the life span characteristics of adult flies of these two strains when kept at high adult population densities. In this experiment, the larval life of both L and R lines was spent under nondensity controlled (NDC) conditions (Arking, 1987a). The flies kept at high density were kept in food vials under conditions approximating those of flies kept in bottles in this lab (10 pair/vial or ca. 6.67 flies/sq, cm of food surface). The vials were changed every three days. Fifty pair of both L and R strain flies were used to measure life span in the high density series. Longevity was measured by recording the number of male and female deaths at each vial changing period. Density was kept constant in the high adult density regime by using ebony replacement flies. No replacement was made for flies that may have suffered an accidental or presenescent death (see Luckinbill et al., 1984 for description). Fecundity measurements presented in this article were taken from a previous experiment, but are presented here for the first time (see Arking et al., 1988, for a complete review of procedures). The data are presented here (see Fig. 4) to illustrate similarities in fecundity and protein synthesis patterns and because of the similarity of life span characteristics in the two strains in the two experiments. The L and R line flies were also used in the injection experiment. The parents of the flies used in this experiment were the L and R stock lines which were maintained as previously described (Luckinbill et al., 1984). Stock bottles were changed approximately every two days and progeny of both L and R lines were held under NDC conditions. Stock bottles were allowed to develop and the newly emerged adults were collected six to seven times a week. No newly eclosed adults were evident in the stock bottles until nine days after the parental stock had been introduced into those bottles. Stock bottles were then routinely discarded after 17 days to prevent F2 contamination. Newly eclosed flies were collected without anesthesia and placed on fresh media. The flies were considered to be zero days old on the day that the flies were collected. On the infrequent occasion when the flies were not collected on a specific day, a collection of all eclosed adults was made on the following day and the flies were labeled as eclosed on the date collected. This procedure introduced a maximum error of 0.5 day into our life tables. The animals were maintained at an average initial density of about 150 to 200 animals. Flies were raised at 25 °C over their entire life span. Bottles were labeled with the date of eclosure and changed every five days until needed or the cohorts died off. Flies of the appropriate age were placed on fresh media one day prior to injection.

Injection procedure To measure the incorporation of labeled amino acid into protein, flies of the desired age, sex, and strain were injected with a 14C amino acid mixture obtained from N E N (NEC 445E, specific activity 55.0 mCi/matom carbon). The 14C amino acid mixture contained 15 of the 20 c o m m o n amino acids. It was hoped that using this amino acid mixture would overcome any variation that would be expected if only a single labeled amino acid had been used. The stock amino acid solution was dried down in vacuo and resuspended in Ringer's solution for use in the injection. The amino acid mixture had an average specific activity of 59 mCi/mmole per amino acid. Flies were injected with a glass needle having a tip diameter between .06 and .10 mm and made using standard procedures. Flies were anesthetized using ether and held in place during an injection with double-sided tape. All flies were injected on the lower half of the ventral side of the

70

R P R E T Z L A F F and R. A R K I N G

abdomen. Three flies were used per sample in order to obtain an adequate quantity of material for analysis. Up to 12 flies were injected at a time. Flies were nominally injected with 1 ~1 (.02 p.C/p.l) of solution as measured by our injection apparatus, but with the inevitable leakage from the wound site the final net volume probably fell to between 0.25 and 0.5/zl. To find out the amount of label available to the fly after the injection, flies were injected, allowed to incubate for 10 min and then measured for total radioactivity (i.e., both incorporated and nonincorporated amino acids). The R females had a total count (mean + standard deviation) of 3929 _+ 1296 cpm/fly; the L females had a total count of 3828 _+ 799 cpm/fly; the R males had a count of 2247 _+ 521 cpm/fly" and the L males had a total count of 2419 _+ 716 cpm/fly. These measurements were taken on single flies. The total activity of the solution injected was approximately 18 882 cpm//xl. The sex-related differences in the net volume of injection fluid retained are most likely related to the sex-related differences in the size of the abdomen in these animals. Following injection, the flies were placed in standard food vials, allowed to recover from the operation, and incubate for 20 min following the injection unless otherwise indicated. Flies took less than five minutes to recover from the anesthetic following injection, though very old flies were slightly slower to recover than were young flies. When the flies had recovered from the procedure, the flies appeared to behave normally. In control experiments, injected adult flies were seen to survive for at least 24 h after injection. Flies were killed by anesthetizing them with CO2, putting the requisite numbers into microfuge tubes, quick freezing them with dry ice and acetone, and then storing them at 70°C until processed. Assay

Samples were assayed for TCA precipitable radioactive material and for whole body protein. Samples were placed on ice and homogenized with 250/M of grinding buffer (3.5 × l0 - 0 2 M tris pH 7.4, 2.5 × l0 - 0 2 M KCI, l × l0 - 0 2 M MgCl2, 5 × l0 - 0 3 M phenyl(thio)urea, 3.4 × l0 - 0 4 M/~-mercaptoethanol) (Rose and Hiilman, 1969). Samples were gound in a 1.5 ml microcentrifuge tube with a microcentrifuge tube homogenizer and a Tri-R Stir-R variable speed homogenizing motor set at speed 3.5. Fifteen strokes of the pestle were used to grind each sample. Samples were then spun in an E p p e n d o r f microcentrifuge for two minutes to remove debris. Two hundred/~l of the supernatant was extracted and placed on ice. Of this, 50/~l of the supernatant was used for the protein assay. Protein was assayed using a Bio-Rad assay kit, with bovine serum albumin (BSA) used as the protein standard. Standards and samples were measured for absorbance at 595 mm on a Beckman DU-40 spectrophotometer with the Quant II software package. The remaining 150/M of sample was used to measure TCA precipitable radioactive material. To the remaining sample, 250/M of cold 10% TCA was added. Following TCA addition, samples were incubated for 15 min at 90°C to disassociate amino-acyl t-RNA's. Samples were cooled and then collected on Whatman GF/A glass fiber filters. Filters were washed with 10% TCA, cold acetone, and cold ether (Egilmez and Rothstein, 1985). Filters were dried under a lamp and placed in scintillation vials with 10 ml of Amersham ACS liquid scintillation fluid and counted on a Beckman LS-230 liquid scintillation counter for one minute using the entire 14C window. Rates of amino acid incorporation into protein were obtained by taking these raw data and using them to calculate the age specific values of cpm/mg protein/fly. All points along the graph of incorporation vs. age are the mean and standard deviation of four samples,

AMINO ACID INCORPORATION IN DROSOPHILA MELANOGASTER

71

TABLE 1. LIFE SPAN PARAMETERS OF ADULT FLIES RAISED AT HIGH DENSITY 1

Age in Days o f Each Strain at

L~]~" span Parameter

R line

L line

L T 10

Male Female

26

48

19

33

44 35

60 56

51 50

71 67

54 54

77 74

L T 50

Male Female L T 90

Male Female L T 100 Male

Female X+SD

Male Female

4 2 . 7 +_ 8.9

6 0 . 0 _+ 11.32

36.3 _+ l l.5

5 4 . 0 _+ 14.0

~High adult density = l0 p a i r o f flies/vial. 2 F o r both sexes separately, a t test shows thatp < 0.0001.

except for the L line day 1 and day 60 values and the R line day 1 and day 40 values. For these time points, the values were taken directly from the 20 min reading of the rate study curves. Each of these points is the result of three samples. RESULTS

Effect of adult density on adult life span In our previous studies the life span of the flies was obtained by a longitudinal study on a small number of paired animals kept under low density. Such a regime would not suffice for the present study which employed a cross-sectional destructive analysis of the animals. It was logistically necessary to maintain large numbers of animals under somewhat different conditions than those employed earlier. Accordingly, a preliminary study designed to test the effect of different adult population densities on adult life span parameters was performed on both sexes of both the R and the L strains of flies. The results are summarized in Table 1. First, in agreement with all our past data, is the obvious fact that the L animals still have higher mean and maximum life spans than do the R animals when kept under the high adult density condition tested, and that those differences are statistically significant. The genetic contribution to the increased life span characteristics of the L line are not overcome by this environmental change. Second, in both strains the males have a higher mean life span relative to their female sibs. Third, the males and females, of both R and L strains, have divergent mean life spans, but convergent, if not identical, maximum life spans.

72

R. P R E T Z L A F F and R. A R K I N G

12.

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0 TIME

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40

60

(MINS)

FtG. I. Kinetic studies of amino acid incorporation into protein during the first hour after injection of the label in young (solid circles) and old (open circles) adults. The results are shown by strain and by sex. Young animals were I-day-old in all four panels. Old animals were 40 days of age in the R (control) strains and 60 days of age in the L (long-lived) strains.

Profile of the rate o f amino acid incorporation A s t u d y w a s m a d e to e x a m i n e the c h a n g e in t h e e x t e n t o f 14C a m i n o a c i d i n c o r p o r a tion into p r o t e i n o v e r t i m e . T h e d a t a p r e s e n t e d in Fig. 1 s h o w t h e d i f f e r e n c e in the r a t e o f 14C a m i n o a c i d i n c o r p o r a t i o n into T C A p r e c i p i t a b l e m a t e r i a l in y o u n g ( l - d a y - o l d ) a n d s e n e s c e n t ( 4 0 - d a y - o l d R o r 6 0 - d a y - o l d L) m a l e a n d f e m a l e a d u l t flies. A s e x p e c t e d , all f o u r d a t a sets s h o w t h a t y o u n g flies i n c o r p o r a t e the 14C l a b e l m o r e q u i c k l y than d o t h e i r s e n e s c e n t c o u n t e r p a r t s . W i t h the u n e x p l a i n e d e x c e p t i o n o f the R f e m a l e s , the o l d e r a n i m a l s a c c u m u l a t e s i g n i f i c a n t l y less r a d i o l a b e l e d p r o t e i n e v e n a f t e r a o n e - h o u r

AMINO ACID INCORPORATION IN DROSOPHILA MELANOGASTER

73

13. 12

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11

0 '-

10

x

~-

9

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8

,.,I ul~

I,-

0

• \\

7

/

Q.

6 0 ~

5

U

4 3

1'0

2'o

3'0

ADULT

4'o

so

go

7'0

AGE

FIG. 2. Age-related alterations in the amino acid incorporation in females of the L (solid circles) and R (open circles) strain. Each individual point is the mean (_+ SD) of at least three independent samples. See text for discussion and interpretation.

labeling period. It was decided to allow the flies to incubate for 20 min after injection and to use this as the time point with which to compare the animals at the several different ages. This decision was based on the fact that this time point was near the time at which all of the incorporation curves began to plateau and appeared to represent the end of the linear phase of amino acid incorporation. Additionally, it was felt that there would be minimal protein turnover within the 20-min incorporation period, thus avoiding problems with protein degradation.

Study of the changes in amino acid incorporation with increasing age The results of an examination of the age dependent changes in the rate of amino acid incorporation into protein for female flies of the L and R lines are given by Fig. 2 and Table 2. Examination of the data show that the amino acid incorporation abilities of R and L females are statistically equivalent at most of the shared time points, as judged by the overlapping standard deviations. Significant differences between the L and R strains are found only at day 1 and day 50. The R line female flies have a higher rate of incorporation than do the L line females at

74

R.

PRETZLAFF and R. ARKING

TABLE 2, AGE-RELATED CHANGES IN AMINO ACID INCORPORATION IN FEMALES

Strain

Age I day lOdays 20 d a y s 30 days 40 days 50 days 60 days 70 d ays Overall Decrease ~

R ('prnlmg protein(lly ± SD 9355 8613 8009 6481 5345 7634

+ m + ± ± ± 2 2

1140 1838 1235 950 1805 695

43%

L cprnlmg protein/lly +_ SD 6986 8103 7419 7840 5929 5328 4496 7671

± ± ± ± ± ± ± ±

988 1236 1218 1524 1051 1425 348 1939

44%

~From peak value to lowest value. 2No data; R animals have died by this time.

day 1. From this peak the incorporation level of the R line females declines until day 40 and then suddenly increases during the last 10 days of life. The L line female flies show a plateau from day 1 to day 30, with perhaps a peak at day 10. This plateau period is followed by a decrease from day 40 to day 60 and then sharply increases over the last 10 days of life. There is a decrease in the ability of R females to incorporate amino acids of 43% from peak to minimum values. The L females exhibit a decrease of 44% from their peak to minimum values. The upward trend observed in both lines during the last 10 days of the examination period may probably be attributed as an artifact arising from a large drop in the amount of whole body protein measured in the fly at the last time point. Whole body protein obtained from R female flies from 1 to 40 days averaged 32.1 _+ 5.9/xg of protein, while at day 50 their sibs only contained 25.8 _ 2.7/zg of total protein. This 20% drop in total protein is accompanied by a 30% rise in cpm/mg protein/fly in the R females (Table 2). The average amount of total protein obtained from L females between day 1 and 60 was 24.6 -4- 4.9/xg of protein, but at day 70 the animals only contained 10.8 _ 2.1 /xg of protein. This 40% decrease in the amount of available protein is accompanied by a 41% rise in cpm/mg protein/fly in the L females (Table 2). During the injection procedure, it was observed that the oldest females used in each line appeared to have substantially smaller abdomens when compared with those of younger females. The males of neither line displayed this condition. This condition apparently results from what appears to be a decreased volume and/or activity of the ovaries in these senescent females (unpublished data). This decreased ovarian volume and/or activity appears to be related to the onset of the decreased fecundity previously observed in these animals (Arking, 1987a, b), as shown in Fig. 4. The changes in rate of amino acid incorporation for male flies of the L and R lines are presented in Fig. 3 and Table 3. Examination of the data show that the incorporation levels of the L and R male are statistically equivalent at all but one of the shared time points. The only significant difference was observed at day 10, which is the time at

AMINO ACID INCORPORATION IN DROSOPHILA MELANOGASTER

75

15

14..I

d

13.

,,,2.! _°

i

11. >=

i0.

r

tL

Z

9

= p.

7

0 ~

6_

~

5

1

10

20

30

ADULT

40

50

60

70

AGE

FIG. 3. Age-related alterations in the amino acid incorporation in males of the L (solid circles) and R (open circles) strain. Each individual point is the mean (_+ SD) of at least three independent samples. See text for discussion and interpretation.

which the R line males have their peak incorporation rate, R line males have a fairly large increase from day 1 to day 10 followed by a sharp drop until day 30 where they have a more gentle decline until day 50. The pattern in the drop of the rate of 14C amino acid incorporation in the males of the L line is strikingly different from that of the controls. The L line males begin at a lower level of amino acid incorporation than do the R line males, and this low level holds constant for the first 10 days of life. This level then drops gradually until day 40 at which time there appears to be a transient peak in synthesis followed by a terminal decline to day 70. Neither strain exhibits the terminal upsurge in amino acid incorporation observed in their female sibs. There is a 63% decrease in the level of amino acid incorporation in the R line males from the time of their peak value at day 10 to their lowest point at day 50. The L line males show a qualitatively similar decrease in the amino acid incorporation level, dropping 54% from day 1 through 70. Measurements of whole body protein over the course of the experiment indicates that R and L line males are about the same size and do not appear to

76

R. PRETZLAFF and R. ARKING

T A B L E 3. A G E - R E L A T E D CHANGES IN AMINO ACID INCORPORATION IN MALES

Strain R cpm/mg protein/fly ± SD

Age I day 5 days 10 days 15 days 20 days 30 days 35 days 40 days 45 days 50 days 60 days 70 days

9913 ± 12015 ~ 13742 ± 10204 ± 9102 + 5634 ± 2 5198 ± 2 5036 ± 3 --:~

Overall Decrease'

L cpm/mg protein//ly +_ SD

1955 2372 2140 2815 2031 973

8612 + = 8663 ± 2 6783 + 6080 ± 5215 ± 7587 ± 4149 ~ 5500 ± 4901 + 4015 ±

991 1288

63%

2449 1373 1281 546 1293 1635 898 850 216 823

54%

1From peak value to lowest value. 2No data: measurements not made at these times. 3No data: R animals have died by this time.

70 0 N,

,u 60 o.

5O

0 0

30

lal 2 0

10

o

20

40

60

80 AGE

FIG. 4. Age-related changes in the fecundity of females of the R (open circles) and L (solid circles) strains as measured in a separate longitudinal study (Arking et al., 1988a) in which the animals were raised under conditions comparable to those of the present study. Each point represents the mean n u m b e r of eggs laid/3 days of individually housed mated females. There was an initial population of 60 females for each strain. See text for discussion and interpretation.

AMINO ACID INCORPORATION IN DROSOPHILA MELANOGASTER

77

obviously decrease in size over time nor do they display the age-dependent drop in total protein characteristic of the females. The R line males have, on average, 15.9 _+ 2.9/zg of measured whole body protein while the L line males have, on average, 15.4 __+3.6/zg of protein. There is a certain similarity between the sexes of each strain in that the older animals of either sex have a significantly lower level of amino acid incorporation than do the younger animals of either sex. An inspection of Figs. 2 and 3 reveals that the pattern of decline may be somewhat different for females than for males. In both lines the males exhibited a greater drop in protein synthesis over their life span than did the females. However, this apparent sex limited phenotype appears to be an artifact resulting from the decrease in ovarian function and volume associated with senescence in the female. A comparison of the overall incorporation levels for both sexes and both strains in Figs. 2 and 3 shows that there is no significant difference between any of these four overlapping values. Furthermore, the males of either strain tend to show transient spikes in incorporation levels, while the females of either strain tend to show a stable plateau for the first half of their life spans. DISCUSSION It was necessary for us to first determine the life span characteristics of the L and R adults when raised under conditions somewhat different than those employed in our previous studies. Based on the data shown in Table 1, we conclude that the life span characteristics of the R and L strains do not appear to be affected in any major way by increased adult density. The regime used in this study is known to slightly alter the longevity of the strains, but does not disturb the pattern of senescence established in previous studies for these two strains. Overall it was found that flies raised under conditions approximating those of the high density experimental flies (i.e., the flies used in our incorporation studies) exhibited life span parameters comparable to those reported elsewhere in the literature for these strains. Our main interest in doing this study was to determine if the age-dependent, strainspecific patterns of amino acid incorporation would shed any light on the physiological and genetic mechanisms presumed to underlie the observed strain-specific differences in life span. Within each strain, a decrease in the quantitative levels of labeled amino acid incorporation was observed over the course of the adults' life span. An interstrain comparison showed that the long-lived and the control strain of either sex differ from each other mainly in the pattern and the timing of this decline in incorporation levels. The results are generally comparable with those obtained in other studies on Drosophila (for review see Richardson, 1981; Baker et al., 1985; Makrides, 1983). The early studies of Clarke and Maynard-Smith (1966) suggested the existence of an increase in protein synthetic ability with increased age. However, all subsequent studies in Drosophila (and in most other organisms) agree in reporting a decline in protein synthesis with age. Chen (Bauman and Chen, 1968; Chen, 1972), in a detailed study on amino acid pools and protein synthesis in young, middle, and old age flies, demonstrated the existence of about a 60% age-dependent decrease in the amino acid incorporation rates in vivo. This decrease was partially compensated for by his demonstration that there was concomitant age-dependent decrease in protein turnover as well. These results have been supported by a series of experiments reported by Webster and his colleagues (see Webster, 1987, for review and references) in which they have demon-

78

R. P R E T Z L A F F and R. A R K I N G

strated a dramatic decrease in the in vitro levels of protein synthesis during the first 14 days of adult life followed thereafter by a gentle decline till death. Their data also suggest that the decline is tissue-specific, for the head decreased by 15%, the thorax by 95%, and the abdomen by 33% in their respective levels of protein synthetic ability (Webster et al., 1981). More recent work by Webster and his colleagues has indicated that this decline follows and may be due to a decreased synthesis of elongation factor- 1 (EF-1). Interestingly enough, this decreased synthesis appears to be specific in that it apparently results from the decreased transcription of the EF-1 gene itself (Webster, 1987). Since the EF-I product is involved in the elongation and translation of all proteins, it would be expected that the age-dependent decrease in protein synthesis should affect all proteins equally. Work by Fleming, Quattrocki, et al., (1986), based on the densitometric analysis of two dimensional gel patterns of labeled proteins obtained from young, middle-aged and old flies, concluded that the age-dependent decline is manifested through quantitative but not qualitative changes in protein synthesis. In an analysis of ribosomal proteins no qualitative changes were found with increasing age in male Drosophila (Schmidt and Baker, 1979). It was also shown by Fleming, Melnikoff et al., (1986), that a similar pattern of age-dependent quantitative but not qualitative changes were observed in the expression of mitochondrial proteins. These observations of quantitative changes in protein synthesis do not preclude an earlier qualitative regulatory change. The data presented in the present study is consistent with the results of these prior studies. Our analysis of the data has led us to conclude that the long-lived and the control strains differ from each other mainly in the pattern and the timing of their decrease in protein synthesis. While both strains undergo similar quantitative declines in their amino acid incorporation ability in vivo, they differ primarily in the timing of the declines. The difference in the LT 50 life span parameters for the R and L females is 21 days and the difference in their mean life span is 17.7 days (Table 1). These time intervals correspond very nicely to the 20-day differential in the timing of the terminal upswing noted in the female incorporation data (Fig. 2). Using this terminal upswing as a physiologic parameter with which to synchronize the two strains leads us to conclude that the increased life span of the L strain appears to be brought about by the extension of a portion of the early and/or mid-adult phase of the life cycle rather than by the extension of the late adult, or senescent, phase. This conclusion corresponds well with our life table and fecundity data. One can interpret the data of Fig. 2 so as to suggest that the 20-day life span differential might be due to an extension of the early (day 1 through day 10) phase and of the middle phase (day 30 through day 40) of the normal R line life cycle. The newly eclosed L females have a lower level of amino acid incorporation than their R counterparts during the first days of adult life, thus delaying the time when they attain their peak incorporation levels with respect to the R females. The L females also exhibit a plateau of high protein synthesis extending for about 10 days past the time at which the R females have declining incorporation values. The importance of this decrease to the onset of senescence is not known, although it is believed by many that this decrease should be a major contributor to the senescent changes observed in many aging organisms. We have attempted to investigate this point by correlating these decreases with changes in physiological events of evolutionary importance. These alterations in levels and times of protein synthesis correspond well with previously observed age-dependent alterations in the female fecundity. These data,

AMINO ACID INCORPORATION IN DROSOPHILA MELANOGASTER

79

presented in Fig. 4, were obtained from flies raised under similar though not identical experimental conditions (for full description see Arking et al., 1988). It may be seen that the newly eclosed L females lay fewer eggs per day than do their newly eclosed R counterparts, thus slightly delaying peak production levels in the L females (also see Luckinbill et al., 1984). The middle-aged L females maintain their high fecundity levels for about 7 to l0 days longer than do the R females. Finally, once the R females can no longer maintain their peak levels of egg production, then their fecundity decays exponentially to zero (Fig. 4). However, the L females do not decay in a similar manner. Instead, when the L females can no longer maintain their peak levels of egg production, their fecundity decays to an intermediate plateau level where it maintains itself for a substantial period of time (Fig. 4). Thus, we believe that the temporal variations in amino acid incorporation levels observed in Fig. 2 are mirrored in the real functional and physiological changes taking place within the animals as depicted in Fig. 4. It should be clear that the changes seen in amino acid incorporation are not the result of fecundity changes, in which case the former might be considered trivial; but rather they precede the fecundity changes, suggesting that they might bear a causal relationship towards the control of reproduction. It is clear that, in both cases, the long-lived and the control strains perform the same physiological processes - - they simply differ in the times during which these processes are done. It is much more difficult to decipher a comparable pattern in the L and R male flies. The observation that young R females have a higher level of amino acid incorporation than do the young L females also holds true for the males of these two strains. In the absence of a defined marker of male physiological activity (comparable to fecundity measurements in females), it is much more difficult to analyze the male amino acid incorporation data. The transient peak seen late in the L line males at 40 days seems to be largely inexplicable at this time. The difference between the R peak, at day 10, and the L peak, at day 40, is 30 days, which is outside the range of expected age-related factors established in these two strains by the female data as analyzed above. Although this discrepancy does not rule out the possibility of there being a strain-dependent shift in timing of some physiological event, it does make that interpretation more strained. The most that can be reasonably said is that in both lines there appears to be significant activity and fluctuation in amino acid incorporation levels prior to the last 20 days of life in both strains, but that the last two weeks of the L and the R males' life span is characterized by a fairly constant but much decreased level of incorporation. This differs considerably from the upswing seen in the females over the last l0 days of life (Fig. 2) and suggests the existence of sex specific patterns of metabolic aging. Such sex specific patterns were previously suggested by Samis et al. (1971). Webster's data (Webster, 1987) suggests the existence of a specific age during which there is a striking decrease in the animals' ability to synthesize proteins. The protein synthesis data obtained from this study can be interpreted as possibly also showing a step wise drop in amino acid incorporation rather than a simple straight line decrease. Table 4 shows an attempt to delineate between young and old flies, for each strain and sex, on the basis of the amino acid incorporation data obtained in this study. The mean value of the samples included in the specified range for each strain was averaged and then compared using a t test. All four groups show significant differences in amino acid incorporation between the young and the old groups. This chart is not intended to specify an exact demarcation time between young and old, but merely demonstrates

80

R. PRETZLAFFand R. ARKING TABLE 4. MEAN VALUES OF AMINO ACID INCORPORATION NOTED IN DIFFERENT PHASES OF THE ADULT LIFE CYCLE

Sex

Strain

Young 1

Old j

Decrease

pZ

Female

R L

8596 7623

5994 5320

30% 30%

p < .002 p < .1301

Male

R L

11520 7563

5314 5237

54% 30%

p < .001 p < .001

~Visual inspection of Fig. 2 led us to adapt 25 days as the timepoint dividing the R strain females and males into young and old, and 35 days as the respective point for the L strain females and males. ZResults of t test comparing indicated groups.

that with regard to amino acid incorporation there could exist an aging event that measurably divides old adults from young adults into two qualitatively different groups. One other point of interest can be gathered from the data. The females have a higher level of amino acid incorporation than do the males (see Materials and Methods), but the sex differences disappear when these data are normalized for whole body protein content. This suggests that the incorporation activity of male and female cells is essentially equivalent, the higher female total body levels stemming from the fact that females have a higher absolute amount of protein per fly than do the males (see Results). This data is consistent with that of Samis et al. (1971). This comparative study has shown that the overall alterations in the longevity of the long-lived and the control strains are probably due to changes in the timing of particular events in the adult life cycle. The changes in the timing of these events have not affected the physiology of these animals with respect to one another during any particular period of the adult life span. In other words, young animals or mature animals or senescent animals of either strain appear to be physiologically similar to one another despite the significant chronological differences in their ages. This conclusion is consistent with our suggestion that aging may be a multiphasic, independently regulated process (Arking, 1987b). A similar conclusion was recently reached by Johnson (1987) based on his genetic analysis of aging in the nematode. A c k n o w l e d g m e n t s - - This w o r k was generously supported by a WSU President's Excellence Award and by a grant form the WSU Institute of Gerontology to R.A. The WSU Institute of Gerontology also awarded a Graduate Research Fellowship to R.P. We thank Dr. Matthew Witten tot his advice regarding the statistical analysis of the fly populations and Mr. Robert A. Wells for his technical assistance.

REFERENCES Drosophila: Analysis of the survival data and presentation of a hypothesis on the genetic regulation of longevity. Exp. Gerontol. 22, 199-220, 1987a. A R K I N G , R. Genetic and environmental determinants of longevity in Drosophila. In: Evolution o f Longevity in Animals: A Comparative Approach, W o o d h e a d , A.D. and Thompson, K.H. (Editors), Brookhaven Symposium No. 34, pp 1-22, Plenum Press, N e w York, 1987b. A R K I N G , R., B U C K , S., W E L L S , R.A., and P R E T Z L A F F , R. Metabolic rates in genetically based long-lived strains of Drosophila. Iz:~p. Gerontol. 23, 5%76, 1988. A R K I N G , R. Successful selection for increased longevity in

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BAKER, G.T. 111, JACOBSON, M., and MOKRYNSKI, G. Aging in Drosophila. In: Handbook o f Cell Biology in Aging, Cristofalo, V. (Editor), pp. 511-578, CRC Press, Boca Raton, FL, 1985. BAUMANN, P.A., and CHEN, P.S. Alterunk und Proteinsynthesis bei Drosophila melanogaster. Rev. Suisse Zool. 75, 1051, 1968. CHEN, P.S. Amino acid pattern and rate of protein synthesis in aging Drosophila. In: Molecular Genetic Mechanisms in Development and Aging, Rockstein, M. and Baker, G.T., ii1, (Editors), pp. 19%296, Academic Press, New York, NY, 1972. CLARKE, J.M. and MAYNARD-SMITH, J.M. increase in the rate of protein synthesis with age in Drosophila subobscura. Nature (Lond) 209, 627, 1966. EQIMEZ, N.K. and ROTHSTEIN, M. The effect of aging on cell-free protein synthesis in the free-living nematode Turbatrix aceti. Biochern. Biophys. Acta. 840, 355-363. FLEMING, J.E., QUATTROCKI, E., LATTER, G.I., eI al. Age dependent changes in proteins of Drosophila melanogaster. Science 231, 1157-1159, 1986. FLEMING, J., MELNIKOFF, P.S., LATTER, G.I., CHANDRA, D., and BENSCH, K.G. Age dependent changes in the expression of Drosophila mitochondria proteins. Mech. Ageing Dev. 34, 63-72, 1986. JOHNSON, T.E. Aging can be genetically dissected into component processes using long-lived lines Caenorhabditis elegans. Proe. Natl. Acad. Sci. USA 84, 3777-3781, 1987. LUCKINB1LL, L.S., ARKING, R., CLARE, M.J., CIROCCO, W.C., and BUCK, S.A. Selection for delayed senescence in Drosophila melanogaster. Evolution 38, 996-1003, 1984. MAKRIDES, S.C. Protein synthesis and degradation in aging. Biol. Rev. 58, 344--422, 1983. RICHARDSON, A. The relationships between aging and protein synthesis. In: Handbook o f Bioehernistr3' in Aging, Florini, J.R. (Editor), pp. 7%101, CRC Press, Boca Raton, FL, 1981. ROSE, R. and HILLMAN, R. In vitro studies of protein synthesis in Drosophila. Biochem. Biophys. Res. Cornmun. 35, 197-204, 1969. SAMIS, H.V., ERK, F.C., and BAIRD, M.B. Senescence in Drosophila - - I. Sex differences in nucleic acid, protein and glycogen levels as a function of age. Exp. Gerontol. 6, %18, 1971. SCHMIDT, T. and BAKER, G.T. Analysis of ribosomal protein from adult Drosophila melanogaster in relation to age. Mech. Ageing Dev. 11, 105-112, 1979. WEBSTER, G.C. Effect of aging on the components of the protein synthesis system. In: Insect Aging: Strategies and Mechanisms, Collatz, K.G. and Sohal, R.S. (Editors), pp. 207-216, Springer-Verlag, Berlin, 1987. WEBSTER, G.C. and WEBSTER, S.L. Decreased protein synthesis by microsomes from aging Drosophila melanogaster. Exp. Gerontol. 14, 343-348, 1979. WEBSTER, G.C., BEACHELL, V.T., and WEBSTER, S.L. Differential decrease in protein synthesis by microsomes from aging Drosophila melanogaster. Exp. Gerontol. 15, 495-497, 1980.