A note on 'aging-pharmacodynamics' with application to the analysis of the age-dynamics of adipocyte response to epinephrine and insulin.

This note aims at clearing some semantic problems in the literature concerning the analysis of the effect of age on dose-response curves as well as standardize the way such data should be presented. In particular it is shown how to assess the age-dynamics (i.e. the pattern of change as a mathematical function of age) of the sensitivity of a receptor system to a given agent, of the quantitative overall changes of the cellular response apparatus which affect response capacity, and of the interaction between different simultaneously acting agents. The concepts and theory are illustrated using as an example the in vitro isolated rat fat cell (or adipocyte) which we have recently studied extensively with regards to the age-dynamics of its lipolytic response to epinephrine and insulin. This 'aging-pharmacodynamics' analysis has led to the surprising conclusion of a rebound effect of age (decrease at maturity followed by increase at old age) on the sensitivity of the fat cell to epinephrine and to its antagonist (with respect to lipolysis) insulin, as well as a negative correlation of these sensitivities with fat cell size.

Developmental temperature and life span in Drosophila melanogaster. I. Constant developmental temperature: evidence for physiological adaptation in a wide temperature range.

The concept of an inverse relationship between life span of adult Drosophila and their developmental temperature is probably the result of an unwarranted generalization. Rather, in a wild-type laboratory strain the present study revealed a plateau phase in this relationship between 16 and 29 degrees C in which life span of both male and female flies was roughly independent of developmental temperature. Below and above this range, life span dropped drastically, development being impossible below 12 and above 32.5 degrees C. Simultaneous study of growth characteristics showed that the plateau phase corresponded to a 'physiological' range of developmental temperature, development being apparently disturbed outside that range. Within that physiological range, the growth rate of the flies varied varied 2-fold, while life span remained constant corroborating our previous conclusion that growth rate per se does not determine life span.

Developmental temperature and life span in Drosophila melanogaster. II. Oscillating temperature.

Alternating developmental temperature within the viable range 13-33 degrees C, with increasing amplitude around a mean value of 23 degrees C but constant period (2 days, a 1 day/1 day rhythm), resulted in increased duration of development, decreased body weight and decreased growth rate of both male and female flies. Life span of female flies also decreased with increasing amplitude of temperature of oscillation but that of males increased (compared to that at the mean temperature) at moderate temperature amplitude. Variation of the period of oscillation (from a rhythm of 6 h/6 h up to 2 days/2 days) with constant amplitude (23 +/- 5 degrees C), on the other hand, did not affect either duration of development or body weight of the flies. However, life span of the females only was unaffected but that of males was increased (above that at mean constant temperature 23 degrees C) at the fast oscillation patterns (6 h/6 h and 1 day/1 day). Finally, life span of both sexes was positively correlated with growth rate in the constant period/varying amplitude case, whereas in previous studies variation of growth rate in the same range by other means showed a biphasic or an invariant pattern of life span dependence on growth rate. These results are in agreement with the hypothesis of epigenetic influence on life span by alternating developmental temperature and corroborate the previous conclusion that growth rate per se does not determine life span.

Growth rate and life span in Drosophila V. The effect of prolongation of the period of growth on the total duration of life (J.H. Northrop, 1917)--revisited.

Sixty-eight years ago Northrop observed a constant life span in Drosophila after a progressive increase of the duration of development of the flies achieved by using a yeastless nutrient medium to which he added yeast with a progressively increasing delay. This evidence against the more recent concept of an increased life span following an experimentally decreased developmental rate has generally been ignored due, presumably, to the imprecise methodology employed by Northrop at a time that Drosophila research was just commencing. We describe here a study that aimed at re-examining and extending Northrop's work by developing the flies in either a yeastless or a lightly yeasted medium. While in a yeastless medium development of flies was virtually arrested until yeast was added, in the yeasted medium a slow growth of the larvae was possible before yeast was added. With another method, larval growth rate was reduced over the entire developmental period by adding a relatively low amount of yeast in four portions and with various delays between portions (the first portion being added without delay). Our study confirmed the "Northrop-effect", i.e. the absence of an effect on life span from increased duration of development by a virtual arrest of growth of the larvae for a number of days. Further, it showed that manipulation of growth rate by portioning the yeast amount did not unequivocally support the concept that a lower growth rate leads to an increased life span.

Growth rate and life spain in Drosophila. IV. Role of cell size and cell number in the biphasic relationship between life span and growth rate.

The patterns of variation of wing cell size and number were studied under developmental conditions leading to a biphasic relationship between life span and growth rate while duration of development remained constant (development on an agar-only medium with a varying added yeast amount, constant temperature (25 degrees C) and constant larval density). Across the yeast range, a 125% increase of body weight was accompanied by a roughly 30% increase in the wing linear dimensions, wing cell size and wing cell number while estimated duration of cell division and its reciprocal mitotic division rate remained constant. Furthermore, cell size (but not cell number) varied with growth rate in a similar biphasic pattern to that observed for life span. Finally, from a simultaneous examination of the covariation patterns of life span, growth rate, cell size and cell number with decreasing yeast amount, it became apparent that there was a "critical" yeast amount, approximately 125 mg/120 eggs, below which: (a) cell number abruptly started to decrease linearly from a roughly constant value; (b) the rate of the slow decrease of cell size now tripled and that of growth rate increased even more; and (c) life span which, in the upper yeast range, increased slowly with decreasing yeast, apparently reached a maximum at the critical yeast level and decreased three times faster below that level. These data taken together suggest that: (i) the decrease of all parameters (including life span) below the critical yeast level results from a presumably suboptimal or disturbed development because of and in proportion to the lack of nutrients and (ii) the increase of life span with decreasing yeast amount above the critical yeast level has not been definitely explained but some possibilities are suggested such as changes in subcellular organelle numbers, size and/or functional properties, or other changes due to a phenomenon equivalent to food restriction in rats, probably without changes in overall metabolic rate of the flies.

Rate of aging, rate of dying and non-Gompertzian mortality - encore....

That the steep increase of mean life expectancy in the developed countries during the first decades of this century has now come to a halt is neither news nor reason for panic. Mean life span cannot exceed the apparent biological limit for the human species, a maximum life span LSmax of 100-110 years, that has remained unchanged across time, races and civilizations; it will moreover remain about 20% less than LSmax even if major diseases were to be eliminated due to the inherent individual differences - only a few individuals are endowed with a genetic-physiologic profile that allows them to reach LSmax. Some concepts on the relation of rates of aging and dying and the mechanism of mortality are here pertinent and are amplified. Finally, though the author has extensively criticized the general applicability of the Gompertz 'law of mortality', he has some reservations concerning the recently expressed view that the course of the rate of increase of force of mortality with age may be different in men and women, chiefly slowing down versus continuously increasing; this conclusion may well be an artifact of the analysis.

Changes in lipolytic activity of isolated adipocytes from rats throughout life span.

The lipolytic response of isolated adipocytes from 1 1/2-, 6-, 24- and 32-month-old rats to various doses of epinephrine was studied. Both the basal and the maximal stimulated glycerol release were largest in the mature rats (6 months old) which had the largest adipocytes. Expressing glycerol release per unit cell surface area (which was drastically reduced with age) eliminated the difference between mature and senescent rats both in absence of epinephrine and at high doses of the hormone. However, at low epinephrine doses the adipocytes of the very young and the very old rats showed an enhanced response per unit surface area. A simple pharmacodynamic analysis based on the occupancy theory of drug-receptor interaction suggests that the sensitivity of rat adipocyte receptors is increased during senescence; this increase may be related to the decreased surface of old adipocytes. On the other hand, the decreased maximal lipolytic response during senescence may be due in part to a reduced number of receptors and to a reduced sensitivity of the cellular enzymatic system underlying lipolysis.

Growth rate and life span in Drosophila. II. A biphasic relationship between growth rate and life span.

The relationship between growth rate and life span was studied in Drosophila by varying the amount of yeast available to each developing larva at constant temperature, 25 degrees C. With one approach the larvae developed in a standard medium at constant larval density and a varying amount of yeast added on the medium. Across the entire growth rate range covered in this way (10-100 micrograms/day, male flies) imaginal life span depended on growth rate in a biphasic way, the relationship having a parabolic form with a maximum at about 55 to 60 micrograms/day. Similar covariation of growth rate and life span was obtained by varying larval density at a constant amount of added yeast. With both these approaches growth rate variation was due to opposite variations of both components of growth rate, i.e. duration of development and body size. However, development in a medium without nutrients but with a varying amount of added yeast at constant larval density led to a similar biphasic relationship between growth rate and life span although duration of development did not vary. Therefore, the present results are not compatible with the hypothesis that there is a single causal negative relationship between growth rate and life span and demonstrate that duration of development is not a causative factor of the biphasic relationship between growth rate and life span established here.

Growth rate and life span in Drosophila. III. Effect of body size and developmental temperature on the biphasic relationship between growth rate and life span.

The previous finding of a biphasic relationship between life span and growth rate of Drosophila, developed at 25 degrees C, was confirmed at other temperatures in the usual range (19-28 degrees C) and for development in either a standard medium or one deprived of nutrients but with varying amounts of yeast added on the medium. The role of body size in this relationship was studied by developing flies in a nutrient-less medium with a constant, submaximal amount of added yeast and varying temperature. It was found that under these conditions, which abolished the usual inverse relationship between body size and developmental temperature, body size variations did not account for the observed variations in life span. Thus, corroborating previous studies from this laboratory, body size was ruled out as a causative factor in the life span-growth rate relationship.

Growth rate and life span in Drosophila. I. Methods and mechanisms of variation of growth rate.

It has been suggested that development and ageing may be linked and it has been shown, in Drosophila, under conditions of varying developmental temperature and larval crowding that the rate of development may be inversely related with the duration of adult life. In order to test this hypothesis systematically, precise methods were devised for varying, in Drosophila, either growth rate or each of its components, i.e. body weight and duration of development, while holding the other constant. These methods are described in the present paper. Moreover, we report studies that shed some light on the mechanisms underlying the effects of temperature and larval crowding on Drosophila development. The major novel findings from these studies were: (a) the restriction of the amount of yeast per larva as larval density increases accounts entirely for the effect of larval crowding on duration of development but only for about two-thirds of its effect on body size; and (b) the increased size of flies grown at lower temperatures may be due to assimilation of more food rather than to more efficient assimilation of food.

Effects of simulated increased gravity on the rate of aging of rats: implications for the rate of living theory of aging.

Ever since Pearl proposed the rate of living theory of aging numerous studies have demonstrated its validity in poikilotherms. In mammals, however, satisfactory experimental demonstration is still lacking because an externally imposed increase of basal metabolic rate of these animals (e.g. by placement in the cold) is usually accompanied by general homeostatic disturbance and stress. The present study was based on the finding that rats exposed to slightly increased gravity are able to adapt with little chronic stress but at a higher level of basal metabolic expenditure (increased 'rate of living'). The rate of aging of 17-mth-old rats that had been exposed to 3.14 times normal gravity in an animal centrifuge for 8 mth was larger than of controls as shown by apparently elevated lipofuscin content in heart and kidney, reduced numbers and increased size of mitochondria of heart tissue, and inferior liver mitochondria respiration (reduced 'efficiency': 20% larger ADP: 0 ratio, P less than 0.01; reduced 'speed': 8% lower respiratory control ratio, P less than 0.05). On the other hand, steady-state food intake per day per kg body weight, which is presumably proportional to 'rate of living' or specific basal metabolic expenditure, was about 18% higher than in controls (P less than 0.01) after an initial 2-mth adaptation period. Finally, though half of the centrifuged animals lived only a little shorter than controls (average about 343 vs. 364 days on the centrifuge, difference statistically nonsignificant), the remaining half (longest survivors) lived on the centrifuge an average of 520 days (range 483-572) compared to an average of 574 days (range 502-615) for controls, computed from onset of centrifugation, or 11% shorter (P less than 0.01). Therefore, these results show that a moderate increase of the level of basal metabolism of young adult rats adapted to hypergravity compared to controls in normal gravity is accompanied by a roughly similar increase in the rate of organ aging and reduction of survival, in agreement with Pearl's rate of living theory of aging, previously experimentally demonstrated only in poikilotherms.

Antioxidants, metabolic rate and aging in Drosophila.

In line with the (metabolic) rate-of-living theory of aging, previous work from this laboratory showed that the life-prolonging effect of the antioxidant thiazolidine carboxylic acid (TCA) in Drosophila was paralleled by a similar reduction of the oxygen consumption rate of the flies. To assess the generality of this phenomenon, several life-prolonging antioxidants were dietarily administered to the flies (in standard medium with 1% w/v of tocopherol-stripped corn oil) and their effects on metabolic rate and life span were determined. Respiration rate of groups of continuously agitated flies was measured in the Gilson respirometer. The studied antioxidants were as follows: (the numbers in parentheses are consecutively the antioxidant concentration in the medium in % wt/vol.; mean life span in days; and metabolic rate in microliter O2/mg fly per 24 h): vitamin E (0.4; 46.3; 58.5); 2,4-dinitrophenol (0.1; 45.7; 66.2); nordihydroguaiaretic acid (0.5; 45.6; 69.1); thiazolidine carboxylic acid (0.3; 53.1; 55.8); and control with no antioxidant added (0; 40.7; 73.3). All of these antioxidants at the tested concentrations reduced oxygen consumption rate and increased mean life span; there was a significant negative linear correlation (r = -0.87) between mean life span and metabolic rate. These data suggest that some antioxidants may inhibit respiration rate in addition to their protective effect against free radical-induced cellular damage.

Rate of aging, rate of dying and the mechanism of mortality.

The history of the search for the law and the mechanism of mortality is reviewed. Recent evidence is summarized showing that the Gompertz law of exponentially increasing force of mortality is only an approximate model of mortality kinetics; various other models also provide a more or less satisfactory fit of mortality kinetics data. In particular, a simple model proposed by the author contains the Gompertz model as a special case and is of general validity: it consists of exponentially increasing cumulative mortality in an initial age range followed by exponentially decreasing survivorship. The various proposed mechanisms underlying mortality kinetics are reviewed, with emphasis on their origin and similarities, and a mechanism is proposed mending two basic classical ideas which are only partially valid: (1) Gompertz's accelerated decline of vitality coupled with identical aging rates of the individuals of a population; and (2) Simms' idea of statistically distributed individual aging rates with a uniform average aging rate (linear decline of physiological vitality). This theory provides a basis for analyzing the relationship between rates of aging and rates of dying.

Evolution of developmental rate in mammals.

The allometric relationship between gestation period and body mass in terrestrial placental mammals was analyzed using data from 147 species. Important differences were found among the various orders. These differences can be explained if it were assumed that the order-specific lifestyle and environment in which the young are born has caused different selection pressures on the newborn young in the various orders and thus influenced the number of young per litter and duration of gestation. As proposed elsewhere, such evolutionary effects on embryonic development may have indirectly influenced the evolution of mammalian life span.

Mammalian design and rate of living.

Terrestrial placental mammals are physiologically and biochemically similar. However, though some of their design and life history characteristics vary in a uniform way with body mass across the mammalian class (e.g. basal metabolic rate, body length, liver or adrenal mass, etc.), others follow less uniform "laws" with apparent differences among the various orders (e.g. brain mass, life span, etc.). Based on data from 38 species, it is shown that despite the absence of a uniform law describing body mass, my, of young at birth, or litter size, n, their product, i.e. total body mass of litter at birth, m lambda, does follow such a law: It appears that, like basal metabolic rate, m lambda is proportional to the 3/4-power of body mass of the mother; no inter-order differences are apparent. Nevertheless, despite their remarkably similar metabolic apparatus, mammals cannot be functional scale models of each other, i.e. the metabolic machinery has to be set at a lower level as body size increases, leading to reduced heart rate, developmental rate, and "rate of living"--and thus prolonged life span.

Is cell aging caused by respiration-dependent injury to the mitochondrial genome?

Though intrinsic mitochondrial aging has been considered before as a possible cause of cellular senescence, the mechanisms of such mitochondrial aging have remained obscure. In this article we expand on our hypothesis of free-radical-induced inhibition of mitochondrial replenishment in fixed postmitotic cells. We maintain that the respiration-dependent production of superoxide and hydroxyl radicals may not be fully counteracted, leading to a continuous production of lipoperoxides and malonaldehyde in actively respiring mitochondria. These compounds, in turn, can easily react with the mitochondrial DNA which is in close spatial relationship with the inner mitochondrial membrane, producing an injury that the mitochondria may be unable to counteract because of their apparent lack of adequate repair mechanisms. Mitochondrial division may thus be inhibited leading to age-related reduction of mitochondrial numbers, a deficit in energy production with a concomitant decrease in protein synthesis, deterioration of physiological performance, and therefore, of organismic performance.

Accelerated aging of fasted Drosophila. Preservation of physiological function and cellular fine structure by thiazolidine carboxylic acid (TCA).

Thiazolidine carboxylic acid (TCA) is a natural liver metabolite whose Mg-salt increased lifespan of flies and mice (Miquel and Economos, 1979, Exp. Geront. 14: 279). We studied the physiological and cellular fine structural effects of various concentrations of TCA in the food of male Drosophila. Flies on 0.3% TCA at 27 degrees C had a reduced oxygen consumption rate (about 20% less than controls) at 3 wks of age while their mating capacity and speed of mating were preserved; the flies lived in various experiments 20-30% longer than controls. Apparently TCA improved the metabolic efficiency of the flies (possibly from less "waste" of energy due to improved mitochondrial coupling). However, flies on 0.9% TCA had a reduced mating capacity and lifespan (:toxicity) while at 0.1% TCA was ineffective. A similar dose-response relationship was found in young flies treated with TCA for 1 week and then deprived of food and water, a procedure found to induce accelerated physiological aging. TCA at the 0.3% and 0.6% level reduced the speed of development and the size of the enclosed flies. Electron microscopic investigation of wing muscle showed that 0.3% TCA had a protective effect on cellular fine structure. Though in starved controls (40% survivors after 24 hours of starvation) there was a total absence of glycogen granules, and a striking shrinkage and densification of mitochondria, TCA to a large extent protected muscle cells from these effects of starvation.