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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.

Dolichol (D) levels increase dramatically in older tissue. A better understanding of the fate of cell D and exchange between tissues could be essential for understanding the mechanism of the abnormal accumulation. The fate of red blood cell D was investigated by the use of phenylhydrazine-induced hyperhaemolysis. The effect of atrophy on D tissue levels was studied in the perineal muscles of castrated rats. Influence of D transportation between tissues on the levels of D was studied by the use of age-mismatched heterotopic transplantation of D-rich-hearts from older (22 months old) donor rats in younger (3 months old) D-poor syngenic recipients. Increased red blood cell destruction by splenic macrophages did not cause accumulation but rather a significant depletion of the D content of the spleen. The shrinkage of tissues by endocrine or disuse atrophy did not affect the D content of muscle, where D concentration increased. No significant net redistribution of D was observed from the transplanted older heart to liver and tissues of younger recipients. In conclusion, phagocytosis appears to be the only process resulting in the disposal of tissue D.

Dolichol (D) levels increase dramatically in older tissue. An understanding of the exchangeability of D between tissues may be essential in order to understand the mechanism of the abnormal accumulation associated with aging. The question was investigated by the use of organ transplantation. D-poor hearts donated by 3-mon-old and D-rich by 22-mon-old male Lewis rats were transplanted heterotopically in 3-mon-old syngenic recipients, whose peripheral tissues and liver were poor in D. Native and transplanted hearts were taken 7 and 21 d after surgery. Native hearts of 3-mon- and 22-mon-old male Lewis rats served as control. D concentration and quantity were higher in older than in younger native hearts as expected. In the transplanted hearts, the quantity of D was unchanged, irrespective of the age of the donor and of the time of transplantation, whereas D concentration increased because of the remarkable disuse atrophy. No changes in D were observed in recipients' tissues. It is concluded that dolichol is not redistributed via circulation from the transplanted heart to the tissues and liver of the younger recipient.

The long-lasting depletions of creatine phosphate induced by feeding rats with a beta-guanidinopropionic acid (GPA)-supplemented diet induces specific mitochondrial alterations in skeletal muscles very similar to those observed in human mitochondrial myopathies. The slow-twitch soleus muscle appears to be affected primarily, while the fast-twitch extensor digitorum longus is affected less severely and only after a longer period of treatment (6 months). Changes in the enzyme activities of glucose metabolism appear to be secondary and differ between the two muscles. Withdrawal of GPA from the diet after 2 months of treatment shows that both mitochondrial alterations and biochemical modification are reversible.

In this study, we explored the changes in the rate of protein degradation in liver cells in vivo, using a method based on the physiological stimulation of liver autophagy. Male albino rats 1, 2, 6, 12 and 24 months old were fasted overnight, and then received an injection of the antilipolytic agent 3,5-dimethylpyrazole (DMP) to evoke a sudden shortage of lipid fuel. A comparison was made with the in vivo effects of glucagon by giving the 2-month-old group an intraperitoneal injection of this hormone. Samples of liver were taken after 0, 15, 20, 30, 60 and 150 min and processed for electron microscopy, and groups of rats were subjected to short-term single pass liver perfusion. Results show that in the younger age-groups, the DMP stimulation of liver autophagy and amino acid release is highly significant, and compares favourably with the glucagon model of induction of the autophagic process. With older rats, an age-related longer time-lag of the autophagic response and a decrease in the effect of DMP were observed. In conclusion, hormones may activate autophagy, whereas levels of plasma amino acids may tune down the process to adjust the availability of the substrate to tissue needs.

The results of an electron microscopic study of the changes in hepatocytes induced by chronic intoxication with thioacetamide are reported. During the poisoning aspecific toxic changes are intermingled with progressive, preneoplastic ones. The main cell subpopulations identified are: 1) large hepatocytes with smooth endoplasmic reticulum (SER) hypertrophy, with or without rough endoplasmic reticulum (RER) neoformation and glycogen storage, which is starvation resistant; 2) smaller hepatocytes, where RER hypertrophy and ribosome accumulation are the prominent features. Such a pattern persists for months. After the withdrawal of the drug most of the cell changes disappear. However, during this time a simplification of the liver structure and cell composition takes place, allowing a sequence of cell events which seem relevant for establishment of neoplastic progression. The SER-hypertrophied cell appears first and gives rise, via several intermediate stages, to the RER-hypertrophied one, which is believed to play a key role as the ultimate precursor of cancer cells.

The cell composition of the biliary proliferations induced by thioacetamide administration was investigated. At the end of the intoxication period the main cell types identified among the neoformed bile duct cells were as follows: i) poorly differentiated cells (oval cells) usually arranged in clusters or tiny cords, provided with a great amount of free ribosomes; ii) cells arranged in bile ducts of normal appearance; iii) cells arranged in bile ducts and showing intestinal metaplasia. After withdrawal of TAA most of the biliary proliferations disappeared; in the remaining ones, where the incorporation of 3H-thymidine was still appreciable, significant changes in the bile duct cell composition were evident; in fact whereas the oval cells were no longer identifiable, those suggesting an intestinal metaplasia underwent a relative increment as well as those displaying butyrocholinesterase activity; cells devoid of junctional apparatus and filled with free ribosomes were also seen. Some of the reported finding could support the hypothesis that the biological meaning of the different cell types arisen during intoxication is different; some of them could be due to a reactive hyperplasia, while other could be considered as representing a preneoplastic step.

After administration of thiobenzamide (TB) (2.5 mg/100 g b.w.) by stomach tube to male rats, an increase of liver weight was evident within 2 days. It was associated with an increase of hepatic DNA, in the incorporation of [3H]thymidine into nuclei of both hepatocytes and bile duct cells and also in the mitotic index of both types of cells. Liver water content and morphology as well as serum GPT activity were unchanged. In conclusion, TB administration in a single dose below the necrotic threshold stimulates multiplication of liver cells without evidence of damage.

In rats with phosphoryl-creatine depletion (fed a standard Randoin-Causeret diet containing 1% beta-guanidine propionic acid) abnormal mitochondria were observed in slow skeletal muscles, often containing paracrystalline inclusions very like those induced by ischaemia or mitochondrial poisons and in human mitochondrial myopathy.

Thioacetamide administration at low dose induces minimal changes in rat liver. Following withdrawal of the drug, the extent of the subsequent cholangiofibrosis and hyperplastic hepatocyte proliferation is strongly enhanced by partial hepatectomies performed monthly on the same animal.