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* a n d 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 m a j o r factor in the high mortality from cardiovascular diseases in m a n y Western countries. This has been reviewed by m a n y authors, for example, G o t t o et al. (1974). In addition, high levels of dietary fats, especially p o l y u n s a t u r a t e d fats, are associated with deaths from cancers, for example, see Lea (1966) a n d Rose et al. (1974). Both cardiovascular diseases a n d cancers are typical diseases o f middle a n d old age a n d increase exponentially with age. Since high dietary fat affects both of these age d e p e n d e n t diseases, the possibility arises that dietary fat m a y have a direct effect o n the c o m m o n factor, ageing. I n rats, Everitt (1974) has reported that high dietary fat accelerates the rate of collagen ageing a n d causes a m a r k e d reduction in lifespan. As far as we are aware, n o 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 b e g u n 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 I n the first experiments, C a n t o n S flies were fed a range of energy substrates. Table 1 lists these substrates a n d shows the m e a n 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
96
CHRISTOPHER J, I. DRIVER AND GEORGE COSOPODIOTIS
I00 1 90 80 70 60
~
4(? 50 20 PO
0
c~
~/0
30
40
50
I-irne from beginning of experiment,
60
70
80
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 o b t a i n e d in a subsequent e x p e r i m e n t using much larger numbers o f O r e g o n R flies c o m p a r i n g only the c o n t r o l diet a n d a palmitic acid c o n t a i n i n g medium. In all cases the m e a n lifespans were significantly greater t h a n those o b t a i n e d when the flies were given a m e d i u m c o n t a i n i n g only a g a r 15% a n d methyl 4 - h y d r o x y b e n z o a t e , i n d i c a t i n g that calories were c o n s u m e d . N e i t h e r fructose n o r glycerol gave results significantly different f r o m glucose. H o w e v e r , all the edible fats a n d palmitic acid were significantly life shortening. T h e relative toxicity o f edible fats have been f o u n d to vary from one e x p e r i m e n t to another. This possibility reflects differences o f chemical c o m p o s i t i o n between batches o f n o m i n a l l y the same products. However, variation in response to palmitic acid has also been encountered, as detailed below. TABLE 1, MEAN LIEESPAN OF MALE CANTON S
Drosophilamelanogaster ON DIFFERENT
MEDIA INCLUDING SOME
EDIBLE FATS
Variable energy source Glucose Fructose Glycerol Lard Palmitic acid
Lifespan 42.6 ± 44.3 ± 43.3 ±
± S.E.M. 2.8 days 2.8 days 3.7 days
No. in group 71 ~ 71 ',> 68 J
19.5 ± 2'0 days 17.5 ± 1.8 days
54 '% 66 I
Dripping Butter
6.9 -4- 0.7 days 7.1 2- 0"9 days
37 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 further investigations o f the effects o f a high lipid diet we used palmitic acid because it is chemically stable a n d representative o f the c o m p o s i t i o n o f the usual edible a n d stored fats.
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. If 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
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* a n d 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 m a j o r factor in the high mortality from cardiovascular diseases in m a n y Western countries. This has been reviewed by m a n y authors, for example, G o t t o et al. (1974). In addition, high levels of dietary fats, especially p o l y u n s a t u r a t e d fats, are associated with deaths from cancers, for example, see Lea (1966) a n d Rose et al. (1974). Both cardiovascular diseases a n d cancers are typical diseases o f middle a n d old age a n d increase exponentially with age. Since high dietary fat affects both of these age d e p e n d e n t diseases, the possibility arises that dietary fat m a y have a direct effect o n the c o m m o n factor, ageing. I n rats, Everitt (1974) has reported that high dietary fat accelerates the rate of collagen ageing a n d causes a m a r k e d reduction in lifespan. As far as we are aware, n o 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 b e g u n 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 I n the first experiments, C a n t o n S flies were fed a range of energy substrates. Table 1 lists these substrates a n d shows the m e a n 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
96
CHRISTOPHER J, I. DRIVER AND GEORGE COSOPODIOTIS
I00 1 90 80 70 60
~
4(? 50 20 PO
0
c~
~/0
30
40
50
I-irne from beginning of experiment,
60
70
80
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 o b t a i n e d in a subsequent e x p e r i m e n t using much larger numbers o f O r e g o n R flies c o m p a r i n g only the c o n t r o l diet a n d a palmitic acid c o n t a i n i n g medium. In all cases the m e a n lifespans were significantly greater t h a n those o b t a i n e d when the flies were given a m e d i u m c o n t a i n i n g only a g a r 15% a n d methyl 4 - h y d r o x y b e n z o a t e , i n d i c a t i n g that calories were c o n s u m e d . N e i t h e r fructose n o r glycerol gave results significantly different f r o m glucose. H o w e v e r , all the edible fats a n d palmitic acid were significantly life shortening. T h e relative toxicity o f edible fats have been f o u n d to vary from one e x p e r i m e n t to another. This possibility reflects differences o f chemical c o m p o s i t i o n between batches o f n o m i n a l l y the same products. However, variation in response to palmitic acid has also been encountered, as detailed below. TABLE 1, MEAN LIEESPAN OF MALE CANTON S
Drosophilamelanogaster ON DIFFERENT
MEDIA INCLUDING SOME
EDIBLE FATS
Variable energy source Glucose Fructose Glycerol Lard Palmitic acid
Lifespan 42.6 ± 44.3 ± 43.3 ±
± S.E.M. 2.8 days 2.8 days 3.7 days
No. in group 71 ~ 71 ',> 68 J
19.5 ± 2'0 days 17.5 ± 1.8 days
54 '% 66 I
Dripping Butter
6.9 -4- 0.7 days 7.1 2- 0"9 days
37 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 further investigations o f the effects o f a high lipid diet we used palmitic acid because it is chemically stable a n d representative o f the c o m p o s i t i o n o f the usual edible a n d stored fats.
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. If 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
