the effect of dietary fat on longevity of drosophila melanogaster

6
Exp. Geront. Vol. 14, pp. 95-100. Pergamon Press Ltd. 1979. Printed in Great Britain, THE EFFECT OF DIETARY FAT ON LONGEVITY OF DROSOPHILA MELANOGASTER CHRISI'OPHER J. I. DRIVER* and GEORGE COSOPODIOTIS Department of Environmental Studies, State College of Victoria, Rusden, Blackburn Road, Clayton, 3168, Victoria, Australia (Received 7 October 1978) INTRODUCTION IT is generally believed that high dietary intake of fats, especially saturated fats, is a major factor in the high mortality from cardiovascular diseases in many Western countries. This has been reviewed by many authors, for example, Gotto et al. (1974). In addition, high levels of dietary fats, especially polyunsaturated fats, are associated with deaths from cancers, for example, see Lea (1966) and Rose et al. (1974). Both cardiovascular diseases and cancers are typical diseases of middle and old age and increase exponentially with age. Since high dietary fat affects both of these age dependent diseases, the possibility arises that dietary fat may have a direct effect on the common factor, ageing. In rats, Everitt (1974) has reported that high dietary fat accelerates the rate of collagen ageing and causes a marked reduction in lifespan. As far as we are aware, no other studies of age related changes in response to a high fat diet have been made with experimental animals. Therefore, in order to investigate in more detail a possible role of dietary fat in ageing, we have begun experiments with Drosophila melanogaster. METHODS Two strains of flies were used for this work and for each the techniques used differed, Canton S flies were reared on the following medium: glucose (50 g), maize meal (50 g), rolled oats (25 g), dried yeast (5 g), agar (3 g), methyl 4-hydroxybenzoate (1.5 g) and distilled water (540 cc). The experimental media were either the same as the above in the case of the control, or glucose was replaced isoenergetically where designated with fructose (50 g), glycerol (46 g), lard, margarine, butter or dripping (22 g), or palmitic acid (20 g). In one experiment glucose was replaced by a mixture of glucose (25 g) and palmitic acid (10 g). Male imagoes emerging within a 24 h period were anaesthetised using ether and were transferred four at a time to 25 × 75 mm tubes containing the various media. Thereafter, the flies were maintained at 25°C in a humid atmosphere and transferred to fresh media at approximately weekly intervals. Numbers surviving were recorded at two day intervals. As the numbers fell, survivors from several vials were transferred to one vial to maintain the number of flies at or near four. Oregon R flies were reared on the following medium: maize meal (450 g), brown sugar (250 g), agar (90 g), dried yeast (120 g), methyl 4-hydroxybenzoate (20 g) and water (7'0 dma). The medium was seeded with yeast after it had set. The experimental media differed from the above in that the media were not seeded with yeast and the brown sugar was replaced by glucose (250 g) for the controls, or palmitic acid (100 g). Adults were maintained on the control medium for four days before use. They were then anaesthetised with ether and transferred to 24 mm × 75 mm vials containing the appropriate media. Every second day they were transferred to fresh media and the number of deaths recorded. RESULTS In the first experiments, Canton S flies were fed a range of energy substrates. Table 1 lists these substrates and shows the mean lifespans for the flies fed that diet. Figure 1 *Part of this work was carried out at the Zoology Department, Birkbeck College, London, England 95

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Page 1: The effect of dietary fat on longevity of Drosophila melanogaster

Exp. Geront. Vol. 14, pp. 95-100. Pergamon Press Ltd. 1979. Printed in Great Britain,

THE EFFECT OF DIETARY FAT ON LONGEVITY OF DROSOPHILA M E L A N O G A S T E R

CHRISI'OPHER J. I. DRIVER* and GEORGE COSOPODIOTIS

Department of Environmental Studies, State College of Victoria, Rusden, Blackburn Road, Clayton, 3168, Victoria, Australia

(Received 7 October 1978)

I N T R O D U C T I O N

IT is generally believed that high dietary intake of fats, especially saturated fats, is a major factor in the high mortal i ty from cardiovascular diseases in many Western countries. This

has been reviewed by many authors, for example, Got to et al. (1974). In addit ion, high levels of dietary fats, especially polyunsatura ted fats, are associated with deaths from cancers, for example, see Lea (1966) and Rose et al. (1974). Both cardiovascular diseases and cancers are typical diseases of middle and old age and increase exponentially with age. Since high

dietary fat affects both of these age dependent diseases, the possibility arises that dietary fat may have a direct effect on the c o m m o n factor, ageing.

In rats, Everitt (1974) has reported that high dietary fat accelerates the rate of collagen

ageing and causes a marked reduction in lifespan. As far as we are aware, no other studies of age related changes in response to a high fat diet have been made with experimental animals. Therefore, in order to investigate in more detail a possible role of dietary fat in

ageing, we have begun experiments with Drosophila melanogaster.

M E T H O D S

Two strains of flies were used for this work and for each the techniques used differed, Canton S flies were reared on the following medium: glucose (50 g), maize meal (50 g), rolled oats (25 g),

dried yeast (5 g), agar (3 g), methyl 4-hydroxybenzoate (1.5 g) and distilled water (540 cc). The experimental media were either the same as the above in the case of the control, or glucose was replaced isoenergetically where designated with fructose (50 g), glycerol (46 g), lard, margarine, butter or dripping (22 g), or palmitic acid (20 g). In one experiment glucose was replaced by a mixture of glucose (25 g) and palmitic acid (10 g).

Male imagoes emerging within a 24 h period were anaesthetised using ether and were transferred four at a time to 25 × 75 mm tubes containing the various media. Thereafter, the flies were maintained at 25°C in a humid atmosphere and transferred to fresh media at approximately weekly intervals. Numbers surviving were recorded at two day intervals. As the numbers fell, survivors from several vials were transferred to one vial to maintain the number of flies at or near four.

Oregon R flies were reared on the following medium: maize meal (450 g), brown sugar (250 g), agar (90 g), dried yeast (120 g), methyl 4-hydroxybenzoate (20 g) and water (7'0 dma). The medium was seeded with yeast after it had set. The experimental media differed from the above in that the media were not seeded with yeast and the brown sugar was replaced by glucose (250 g) for the controls, or palmitic acid (100 g).

Adults were maintained on the control medium for four days before use. They were then anaesthetised with ether and transferred to 24 mm × 75 mm vials containing the appropriate media. Every second day they were transferred to fresh media and the number of deaths recorded.

RESULTS

In the first experiments, Can ton S flies were fed a range of energy substrates. Table 1 lists these substrates and shows the mean lifespans for the flies fed that diet. Figure 1

*Part of this work was carried out at the Zoology Department, Birkbeck College, London, England

95

Page 2: The effect of dietary fat on longevity of Drosophila melanogaster

96 CHRISTOPHER J, I. DRIVER AND GEORGE COSOPODIOTIS

I00 1

90

80

70

60

~ 4(?

50

20

PO

0 c~ ~/0 30 40 50 60 70 80

I-irne from beginning of experiment, doys

FIG. 1. Survival of Drosophila melanogaster (Oregan R) on a palmitic acid medium (left) and a glucose medium (right). The arrows show the times at which transfers from one medium to another were made in

the experiments in Figs. 3 and 4~

shows the survival curves ob ta ined in a subsequent exper iment using much larger numbers o f Oregon R flies compar ing only the cont ro l diet and a palmit ic acid conta in ing medium. In all cases the mean lifespans were significantly greater than those ob ta ined when the flies were given a medium conta in ing only agar 15% and methyl 4 -hydroxybenzoa te , indica t ing that calories were consumed. Nei ther fructose nor glycerol gave results sig- nificantly different f rom glucose. However , all the edible fats and palmit ic acid were sig- nificantly life shortening. The relat ive toxici ty of edible fats have been found to vary f rom one exper iment to another . This possibi l i ty reflects differences o f chemical compos i t ion between batches o f nomina l ly the same products . However , var ia t ion in response to palmit ic

acid has also been encountered, as deta i led below.

TABLE 1, MEAN LIEESPAN OF MALE CANTON S Drosophila melanogaster ON DIFFERENT MEDIA INCLUDING SOME EDIBLE FATS

Variable energy source Lifespan ± S.E.M. No. in group Glucose 42.6 ± 2.8 days 71 ~ Fructose 44.3 ± 2.8 days 71 ',> Glycerol 43.3 ± 3.7 days 68 J

Lard 19.5 ± 2'0 days 54 '% Palmitic acid 17.5 ± 1.8 days 66 I

Dripping 6.9 -4- 0.7 days 37 Butter 7.1 2- 0"9 days 19 )

Margarine 4.8 ± 0.5 days 28

Significance of differences: The bracketed groups show no significant differences from one another. Differences between groups is significant with P < 0-001 except that the dripping-butter vs margarine difference

is significant only at the 0.01 level.

F o r fur ther invest igat ions o f the effects of a high l ipid diet we used palmit ic acid because it is chemical ly stable and representat ive o f the compos i t ion of the usual edible and stored fats.

Page 3: The effect of dietary fat on longevity of Drosophila melanogaster

DIETARY FAT AND LONGEVITY OF Drosophila 97

One of the explanations of the life shortening effects of the 'fat containing' diets is that the complete absence of a sweetening agent made the food unpalatable to the flies, which therefore ate less and died early as a result of partial starvation. I f this were so, it would be possible to reverse the effects of a high fat diet by making the medium sweet. We tested this by comparing the lifespan of the flies maintained on three diets. The control diet contained glucose as before, the second, palmitic acid as before, and in the third, half as much glucose as in the control was used, and an energetically equivalent amount of palmitic was used. The results of this experiment are shown in Table 2. It is clear that in spite of the presence of glucose the palmitic acid diet is life shortening. In fact the amount of life shortening is proportional to the concentration of pahnitic acid. It seems unlikely, therefore, that the effect of palmitic acid is due to unpalatability.

TABLE 2. MEAN LIFESPAN OF MALE CANTON S Drosophila melcmogaster ON DIETS CONTAINING DIFFERENT

AMOUNTS OF GLUCOSE AND PALMIrIC ACID. ALL DIFFERENCES ARE SIGNIFICANT ( P <~ 0"001)

Variable energy source Mean lifespan ± S.E.M. No. in group Glucose, 40 ~ of calories 44.2 ± 2.0 days 155

Glucose, 20~ of calories t 32-5 ± 1-8 days 168

Palmitic acid, 20 ~ of calories

Palmitic acid, 40}£ of calories 23"7 ± 1.4 days 172

Differences in lifespan may not reflect differences in the rate of ageing, i.e. an acceleration of degenerative changes. As had been discussed by Clark and Maynard-Smith (1961), Lamb (1977) and others, the differences in lifespan found in two environments could be due either to different rates of ageing (in their terminology, to different rates of loss of vitality) or to differences in the level of environmental stress imposed on the old flies. In the latter case the rate of ageing may be the same in both environments, but in the ter- minology of Clarke and Maynard-Smith, the threshold level of vitality necessary for life is higher in one environment than in the other.

An examination of Fig. 1 suggests that a high fat diet may accelerate the ageing process since not only is the high fat curve shifted to the left, but it also shows a more rapid increase in the death rate. However, a more critical test was needed to determine whether, in spite of a possible increase in the threshold on the high fat diet, there was, in addition, an accelera- tion of the ageing process. In this test, flies were maintained on a high fat diet for variou:~ periods of time and then transferred to a low fat diet where the environmental threshold is the same as in the control. Under these conditions, any difference in lifespan would be due to an irreversible damage sustained while on a high fat diet. The combined results of two such experiments are shown in Fig. 2. In these experiments flies were transferred from a high fat diet to a low fat diet at 6, 12, 18, 24 or 30 days after commencing the experiment. Since the mean lifespan of flies kept continuously on the high fat diet was only 29.6 days, flies transferred to the low fat medium at 24 and 30 days had lost 25 ~ and 50 ~ , respectively, of the original flies. To avoid any bias due to deaths prior to transfer from one medium to another, for all groups the further mean expectation of life at 30 days has been plotted. Figure 2 shows that the longer the period spent on the high fat diet, the shorter the lifespan. This suggests that on the high fat diet, there is a progressive accumulation or loss of some

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98 CHRISTOPHER J. I . DRIVER AND GEORGE COSOPODIOTIS

factor which leads to a reduction in lifespan. I f the lifespan differences were due solely to the severity of the stresses imposed by the two media, all groups of flies would have the same lifespan, regardless of time spent on the high fat medium. To determine whether the slope of the regression line was in fact non-zero, the mean life expectancy of each tube was used, although for clarity in the diagram, only the means and the standard error of these groups are shown. The slope of the regression line is significantly non-zero (P < 0.01), in spite of an apparently large scatter of points about the line.

Q_

F

g.

25

24

22

2 t - -

2 0 - -

1 9 - -

I I / I i 6 12 18 24 30

Days on polmitic acid medium before transferring to glucose

Fla. 2. Effect on life expectancy on glucose medium at 30 days after the beginning of the experiment, of a previous period on a palmitic acid containing medium. The regression line Y = 23"5 -- 0"14 X has a slope

which is significantly non-zero (P < 0'01). Bars are standard errors.

To confirm the conclusion that there is a cumulative, irreversible damage on a high fat medium, an experiment essentially the reverse of the above was performed. Flies were maintained on a low fat diet for the same times and transferred to a high fat diet, otherwise the protocol was as above. This experiment has two advantages over the previous experi- ment. Firstly deaths prior to transfer from one medium to another are negligible. Secondly, the differences expected are the same as above, but the total lifespans are smaller. Con- sequently, the precision of the results should be rather higher. Figure 3 shows that this prediction is correct. There is obviously a progressive increase in life expectancy for longer periods on a low fat diet. The slope of this graph is numerically equivalent to, but opposite in sign to the graph on Fig. 3, within the error of the experiment, and the slope is sig- nificantly non-zero (P < 0.001).

The extrapolated lifespan at zero time on the test medium is significantly different in both cases from the observed values, being lower in Fig. 2 and higher in Fig. 3 (see Table 3). This difference was found to be quantitatively variable. We suggest that this effect may be due to a response to palmitic acid of the associated microflora, possibly allowing one set of organisms to displace another. However, the variability of this response has made critical testing of this hypothesis difficult.

Page 5: The effect of dietary fat on longevity of Drosophila melanogaster

DIETARY FAT AND LONGEVITY OF Drosophila 99

0

.= -~ > , 0

03~ oJ

LL

6

5

4

2

I I 6 12 18

I J 24 30

Days on glucose medium before transferr ing to palmit ic acid medium

FIG. 3. Effect on life expectancy on palmitic acid medium at 30 days after the beginning of the experiment, of a previous period on a glucose containing medium. The regression line Y = 0'5 + 0"13 X has significantly

non-zero slope (P < 0.001). Bars are standard errors.

TABLE 3. COMPARISON OF EXTRAPOLATED LIFESPANS AFTER ZERO TIME ON THE TEST MEDIA AND OBSERVED

VALUES, FROM THE EXPERIMENTS SHOWN IN FIGS. 3 AND 4

First medium, palmitic acid, transferred to glucose medium Extrapolated life expectancy 23'5 4- 0.2 days

(P < 0"001) Observed life expectancy 18.5 ::k 0.7 days

First medium, glucose, transferred to palmitic acid media

Extrapolated life expectancy 3.5 ~ 0-5 days (P < 0.001)

Observed life expectancy 0"5 :k 0.1 days

DISCUSSION AND CONCLUSIONS

These results indicate that high levels of dietary fat may significantly reduce the lifespan of Drosophila melanogaster. Investigations into the irreversible processes affected by the model compound palmitic acid showed a complex effect with two phases of response. In the first six days there is an irreversible beneficial effect of palmitic acid. This highly variable response may account for the majority of the variation of response to palmitic acid. The most likely reason for this response seems to be due to an antibiotic action of palmitic acid. Similar responses, possibly due to the antibiotic action of radiation, have been ob- served under virtually identical conditions by Lamb (1964, 1965). After the first six days, progressively longer periods on the palmitic acid medium produces irreversible life shorten- ing compared with the glucose medium.

The progressive irreversible damage sustained on the palmitic acid medium has the characteristics of an ageing process. It is impossible to say whether a single ageing process is accelerated, or the fat accelerated process is only one of several progressive and de- generative processes. However, fat is a normal food constituent and a normal metabolite and so it seems reasonable that the fat accelerated process occurs to some extent at least on a low fat diet.

Page 6: The effect of dietary fat on longevity of Drosophila melanogaster

100 CHRISTOPHER J. I. DRIVER AND GEORGE COSOPODIOTIS

Only a fraction of the differences in lifespan are due to irreversible effects. For example, after 30 days on a palmitic acid medium, the survivors lived another 18.5 days on a low fat diet, compared with the extrapolated life expectancy of 3.5 days on a high fat, and the 23-5 days they would have lived if they never had been fed a high fat diet. In addition the irreversible components o f the damage caused by a high fat diet are themselves complex and variable. Consequently conclusions about the effects of dietary fat on one or more processes cannot readily be drawn from simple lifespans, and satisfactory progress in studying ageing processes will probably ultimately depend on simplification of the re- sponses, and reduction of variability by more rigorous control o f the conditions o f culture.

S U M M A R Y

The effects on the lifespan of adult male Drosophila melanogaster of replacing dietary glucose by fructose, glycerol or one o f a variety of fats has been studied. Isocaloric re- placement o f glucose by fructose or glycerol has no significant effect on lifespan but sub- stituting lard, butter, margarine, dripping or palmitic acid for glucose led to a significant reduction in lifespan.

A detailed study of the nature o f the life shortening produced by one o f the fats, palmitic acid, suggested that most o f the life shortening effects are due to rapidly reversible pro- cesses, but approximately 2 0 ~ of the life shortening effects persist irreversibly when trans- ferred back to a low fat medium. This suggests that a high fat medium may accelerate the ageing process. In contrast, a short exposure to palmitic acid early in the life of the flies produces an irreversible increase in the life expectancy which is very variable. This may be due to an action on endogeneous microttora.

Acknowledgements--We are indebted to Dr. Marion J. Lamb for her assistance with the Oregan R systems, her interest in this work and help in preparation of this manuscript. We would also like to acknowledge the late Norman Wakefield and Dr. David Stokes, Department Heads, for their interest and encouragement. Ms. Barbara Lythgo participated in the early experimental work as part of her undergraduate course.

R E F E R E N C E S

CLARKE, J. M. and MAYNARD-SMITH, J. (1961) Nature, Land. 190, 1027. EDERER, F., LEREN, P., TURPEINEN, O. and FRANTZ, 1. D. (1971) Lancet 2, 203. EVERITr, A. V. (1970) Proc. Aust. Ass. Geront. 1, 65. EVERITT, A. V. and PORTER, B. (1975) Nutrition and ageing. In : Hypothalamtts, Pituitary and Ageing (Edited

by A. V. EVERITT and J. A. BUR~3ESS). Charles C. Thomas, Springfield, Illinois. GOTTO, A. M., JR., SCOTT, L. and MANIS, E. (1974) Geriatrics 29, 109. LAMa, M. J. (1964) J. Insect. Physiol. 10, 487. LAMB, M. J. (1965) Exp. Geront. 1, 181. LAMa, M. J. (1977) Biology of Ageing, p. 3. Blackie, Glasgow. LEA, A. J. (1966) Lancet 1,332. PEARCE, M. L. and DAYTON, S. (1971) Lancet 1,464. ROSE, G., BLACKBURN, H., KEYS, A., TAYLOR, H. L., KAMMELL, W. B., OGLESBY, P., REID, D. D. and STAM-

LER, J. (1974) Lancet 1, 181.