Zina Gori

The anti-ageing effects of caloric restriction may involve stimulation of macroautophagy and lysosomal degradation, and can be intensified pharmacologically.

Caloric restriction (CR) and a reduced growth hormone (GH)-insulin-like growth factor (IGF-1) axis are associated with an extension of lifespan across taxa. Evidence is reviewed showing that CR and reduced insulin of GH-IGF-1 axis may exhibit their effects at least partly by their common stimulatory action on autophagy, the cell repair mechanism responsible for the housekeeping of cell membranes and organelles including the free radical generators peroxisomes and mitochondria. It is shown that the life-long weekly administration of an anti-lipolytic drug may decrease glucose and insulin levels and stimulate autophagy and intensify anti-ageing effects of submaximal CR.

Ageing-related changes in the in vivo function of rat liver macroautophagy and proteolysis.

Autophagy is a universal, highly regulated mechanism responsible for the degradation of long-lived proteins, cytomembranes and organelles during fasting and may be the cell repair mechanism that mediates the anti-ageing effects of calorie restriction (Bergamini and Gori, 1995). The function of autophagy was studied in vivo on male Sprague Dawley rats fed ad libitum or 40% food restricted. Autophagy was induced in overnight fasted rats by the injection of an anti-lipolytic agent and was investigated by electron microscopy. Changes in regulatory plasma nutrients and hormones were assessed and rate of proteolysis was calculated from the release of 14C(6)-valine from pre-labelled resident proteins. Results in rats fed ad libitum showed that autophagic-proteolytic response to antilypolitic agents was paramount in one month-old rats; was high but delayed in 2 month-old rats, decreased remarkably in 6 month-old rats and almost negligible at older age. Parallel ageing-related changes were observed in the effects of treatment lowering glucose and insulin plasma levels. Calorie restriction prevented all changes. In view of the known suppressive effects of insulin, it may be concluded that the age-changes of autophagy are secondary to the ageing-related alteration in glucose metabolism and hormone levels, whose appearance is delayed by calorie restriction. Data may support the hypothesis that ad libitum feeding accelerates the rate of ageing by raising insulin plasma levels and suppressing autophagy and membrane maintenance, and that calorie restriction may break this vicious circle.

The role of autophagy in aging: its essential part in the anti-aging mechanism of caloric restriction.

Abstract:  Aging denotes a postmaturational deterioration of cells and organisms with the passage of time, an increased vulnerability to challenges and prevalence of age‐associated diseases, and a decreased ability to survive. Causes of this deterioration may be found in an enhanced production of reactive oxygen species (ROS) and oxidative damage and incomplete “housekeeping.” Caloric restriction is the most robust anti‐aging intervention known so far. Similar beneficial effects on median and maximum life span were obtained by feeding animals a 40%‐reduced diet or by every‐other‐day ad libitum feeding. In both instances, animals are forced to spend a great part of their time in a state of fasting and activated autophagy. Autophagy is a highly conserved process in eukaryotes, in which the cytoplasm, including excess or aberrant organelles, is sequestered into double‐membrane vesicles and delivered to the lysosome/vacuole, for breakdown and eventual recycling of the resulting macromolecules. This process has an essential role in adaptation to fasting and changing environmental conditions, cellular remodeling during development, and accumulation of altered ROS‐hypergenerating organelles in older cells. Several pieces of evidence show that autophagy is involved in aging and is an essential part of the anti‐aging mechanism of caloric restriction. As an application, intensification of autophagy by the administration of an antilipolytic drug rescued older cells from accumulation of altered mtDNA in less than 6 hours. It is concluded that the pharmacologic intensification of autophagy (PISA treatment) has anti‐aging effects and might prove to be a big step toward retardation of aging and prevention of age‐associated diseases in humans.

The protection of rat liver autophagic proteolysis from the age-related decline co-varies with the duration of anti-ageing food restriction

Restricting caloric intake (CR) well below that of ad libitum (AL) fed animals retards and/or delays many characteristics of ageing and the occurrence and progression of age-associated diseases, efficacy depending on duration. The hypothesis that the anti-ageing effect of CR might involve stimulation of the cell-repair mechanism autophagy was tested. The effects of ageing and duration of anti-ageing CR on liver autophagic proteolysis (AP) were explored in male AL Sprague-Dawley rats aged 2-, 6-, 12- and 24-months; and 24-month-old rats on a CR diet initiated at 2-, 6- and 12-month of age or initiated at age 2-months and interrupted at age 18 months. The age-related changes in the regulation of AP were studied by monitoring the rate of valine release in the incubation medium from isolated liver cells by an HPLC procedure. Results show that the maximum attainable rate and the regulation of AP decline with increasing age; that changes are prevented by anti-ageing CR initiated at young age, that the protective effects of CR change with the duration of diet. It is concluded that the data are compatible with the hypothesis that AP and improved membrane maintenance might be involved in the antiageing mechanism of CR.

Ageing and oxidative stress: A role for dolichol in the antioxidant machinery of cell membranes?

Dolichol is a polyprenol compound broadly distributed in membranes, biosynthetized by the general isoprenoid pathway from acetate via mevalonate and farnesyl pyrophosphate. Dolichol lays inside the membrane between the two leaflets of the lipid bilayer very close to the tail of phospholipid fatty acids. No definite catabolic pathways for this molecule have yet been identified. Evidence is produced that dolichol levels increase dramatically with increasing age; that anti-ageing caloric restriction retards this age-associated change; that dolichol may act as a radical scavenger of peroxidized lipids belonging to the cell membranes. In view of the polyunsaturated fatty acids (PUFA), dolichol and Vitamin E location and stechiometry, it is proposed that molecules might interact each-other to form a highly matched free-radical-transfer chain, whose malfunctioning might be involved in statin toxicity and neurodegenerative diseases.

Anti-aging effects of anti-lipolytic drugs.

Genetic disruption of insulin and insulin-like signaling pathways may extend lifespan. Hyperinsulinemia and insulin resistance may accelerate aging. The hypothesis was tested that a once-a-week life-long inhibition of insulin secretion by the administration of anti-lipolytic drugs might have anti-aging effects. Groups of 3-month-old male Sprague-Dawley rats were (a) given standard laboratory food ad libitum (AL); (b) fed AL 6 days and fasted 1 day every week (FW); (c) fed AL every other day (EOD), (d) fed like FW and given Acipimox (50 mg/kg b.w.) on the day of fasting (FWA) by the gastric tube. The AL, FW and EOD groups received saline intragastrically. Treatment with ACIPIMOX transiently decreased plasma free fatty acids, glucose and insulin and increased valine plasma levels, and had no long-term effect on food consumption and body weight. By age 6, 12 and 24 months subgroups were taken and the age-related changes in liver dolichol and autophagic proteolysis--which are correlated with life-expectancy--were measured. Liver dolichol levels increased and autophagic proteolysis decreased in mature and older AL rats; EOD and FWA fully counteracted these changes; FW rats had significant but smaller beneficial effects. It is concluded that life-long weekly-repeated transient inhibition of insulin secretion by antilipolytic drugs may have an anti-aging effect, additive to the anti-aging effect of a milder caloric restriction. Speculation is that transiently lower plasma insulin levels might stimulate the anti-aging cell-repair mechanism autophagy, which has longer lasting effects on cell housekeeping.

The castration atrophy of the dorsal bulbocavernosus muscle of rat: An electron microscopic study

Abstract The castration atrophy of the dorsal bulbocavernosus (‘levator ani’) muscle of male rats has been studied, mainly by electron microscopy. The fibers of this muscle are white. The rate of weight decrease of dorsal bulbocavernosus muscle after castration is similar to that of denervation atrophy in rat skeletal muscles. The main process at work in the fibers during atrophy is a reduction of the diameter of the myofibrils caused by loss of myofilaments at the periphery, while the center of the fibrils shows the normal myofilaments pattern. Sometimes a peculiar degenerative process which brings about rapid destruction of the contractile material has been detected. The sarcolemma shows an increased pinocytotic activity and contributes actively, by deep branching invaginations, to the release from the fiber of excess sarcoplasm. The mitochondria disappear in parallel with the contractile material. Lysosomes which are very rare in the normal muscle increase in number and size and are widely distributed in the fiber. Overproduction of the sarcoplasmic reticulum and of the T tubules takes place and also leads to the appearance of ‘pentads.’ The same regressive processes already observed in denervation atrophy are at work, although with some, mainly quantitative, differences also in this type of atrophy.

Towards an Understanding of the Anti-Aging Mechanism of Caloric Restriction

Accumulation of oxidatively altered cell components may play a role in the age-related cell deterioration and associated diseases. Caloric restriction is the most robust anti-aging intervention that extends lifespan and retards the appearance of age-associated diseases. Autophagy is a highly conserved cell-repair process in which the cytoplasm, including excess or aberrant organelles, is sequestered into double-membrane vesicles and delivered to the degradative vacuoles. Autophagy has an essential role in adaptation to fasting and changing environmental conditions. Several pieces of evidence show that autophagy may be an essential part in the anti-aging mechanism of caloric restriction: 1. The function of autophagy declines with increasing age; 2. The temporal pattern of the decline parallels the changes in biomarkers of membrane aging and in amino acid and hormone signalling. 3. These age-dependent changes in autophagy are prevented by calorie restriction. 4. The prevention of the changes in autophagy and biomarkers of aging co-varies with the effects of calorie restriction on life-span. 5. A long-lasting inhibition of autophagy accelerates the process of aging. 6. A long-lasting stimulation of autophagy retards the process of aging in rats. 7. Stimulation of autophagy may rescue older cells from accumulation of altered mtDNA. 8. Stimulation of autophagy counteracts the age-related hypercholesterolemia in rodents. It is suggested that the pharmacological intensification of suppression of aging (P.I.S.A. treatment) by the stimulation of autophagy might prove to be a big step towards retardation of aging and prevention of age-associated diseases in humans.

The hypertrophy of levator ani muscle of rat induced by testosterone: An electron microscope study

Abstract The recovery process of levator ani muscle of rat atrophic by castration has been studied by electron microscope after testosterone treatment. Muscles have been observed 1–5, 7, 15, and 30 days after the beginning of treatment. The testosterone treatment produces in the atrophic muscle a rapid increase of the weight that returns to the normal values after a month. The diameter of the fibers increases above the normal values between day 5 and 7. Besides, the myofibrils diameter shows a progressive increase and the maximum values are reached between day 5 and 15. The electron microscopy shows that already after 24 hours of treatment several changes are detected in the fibers. The glycogen amount increases until day 5 so that large masses of granules are found whether at the fiber periphery or in the interfibrillar spaces. After day 5 of treatment, the glycogen amount slowly decreases. The ribosomes and polysomes are very abundant especially in the first periods of treatment. The nucleoli show a concomitant increase. Clusters of mitochondira appear at the periphery of the fibers and are still numerous after 15 and 30 days of treatment. The disorganized areas which have been found in the atrophy gradually disappear. They are scarce after 15 days and absent after a month. At this stage they appear to be substituted by contractile material. In fact, the fibers undergo an almost complete restoration of the atrophy lesions so that the recovery process of levator ani muscle by the hormonal influence can be considered accomplished after a month.

The age-related accumulation of dolichol in rat liver may correlate with expectation of life

In order to test the hypothesis that the ageing-related alteration in membrane lipids might reflect the biological age of rodents, the levels of liver dolichol were assayed by the HPLC procedure in male ad-libitum fed (AL) Sprague-Dawley rats aged 2, 6, 12 and 24months, and in 24-month-old rats on anti-aging food-restrictions (FR) differing in duration and in their effects on longevity. Results showed that the effects on liver dolichol of FR initiated at 2, 6 and 12 months of age, or initiated at 2 and interrupted at 18 months of age were significantly different, and reflected the differences in the effects of FR on expectation of life (the longer the expected residual lifespan the lower the content in liver dolichol). The conclusion is that assay of the quantity of dolichol in the liver tissue may be used as a marker of the biological age of the animal and therefore as an important biomarker of ageing.