Maria Pollera

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 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.

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.

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.

Increased degradation in rat liver induced by antilipolytic agents: a model for studying autophagy and protein degradation in liver?

A dramatic increase in the plasma glucagon/insulin ratio can be induced by treating fasted rats with antilipolytic drugs (e.g., with 3,5-dimethylpyrazole, 12 mg/kg body wt). These hormone changes are the physiologically appropriate response to a rapid decrease in free fatty acids and glucose plasma levels. Under this experimental condition, many vacuolated lysosomes can be observed at the electron microscopic level as early as 30 min and autophagic vacuoles are detectable in the liver cells 1 hr after the administration of the drug. By 1 hr and 45 min, vacuoles often contain recognizable peroxisomes. At the biochemical level, liver proteolysis in vitro is increased significantly. Very interestingly, changes in peroxisomal (but not mitochondrial or reticulum or cytosolic) enzyme activities are detected that are preventable by the administration of glutamine (i.e., of an inhibitor of proteolysis in vivo) but not by an isocaloric amount of glycine or alanine. It is concluded that the administration of antilipolytic agents to fasted animals may provide a convenient (i.e., an inexpensive, highly reproducible and timable) physiologic model to study hormone-induced autophagy in liver cells.

The Regulation of Liver Protein Degradation by Aminoacids in vivo. Effects of Glutamine and Leucine

The effects in vivo of the two major in vitro regulatory aminoacids, leucine and glutamine, on liver protein degradation were explored in male young adult Sprague Dawley rats. Protein degradation was stimulated by the injection of the antilipolytic drug 3,5 dimethylpyrazole (DMP), which rises glucagon and lowers insulin plasma levels. At the appropriate time-points (20 and 40 min) after the injection of DMP, glutamine or leucine (12.5 mg/kg b.w.) were injected intraperitoneally. The rate of liver protein breakdown was evaluated 60 min after the injection of DMP on the basis of the release of valine into the perfusate during a short term single pass liver perfusion. The aminoacid was assayed by an HPLC procedure. Results show that the administration of glutamine inhibited the DMP-induced increase in the rate of valine release from the perfused liver whereas the administration of leucine did not; neither of the aminoacids appeared to have any effect on the metabolic or endocrine changes that are required for the induction of liver autophagy and protein breakdown by DMP. It is concluded that the aminoacid glutamine has a powerful action on the in vivo regulation of liver protein breakdown, which is not apparent with leucine.

Liver cell proliferation induced by a single administration of thiobenzamide

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.

Dolichol levels in younger and older rat hearts heterotopically transplanted in younger recipients

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 fate of dolichol in rat cells and tissues.

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.

The brown adipose tissue of hyperthyroid rats. A biochemical and ultrastructural study

Male Sprague-Dawley rats were treated with triiodothyronine (100 micrograms/100 g/day) for 2, 4, 7, 14 and 21 days and the biochemical and ultrastructural changes of the brown adipose tissue were investigated. Results showed that the tissue weight, DNA and phospholipid content increased very early (by day 2 or 4) and that triglycerides increased later. These hormonal effects are not inhibited by the beta 1-antagonist propranolol. From the morphological point of view, triiodothyronine administration induced the early proliferation and maturation of adipocyte precursors (interstitial cells and preadipocytes). It is concluded that triiodothyronine administration causes a very early hyperplasia in the brown adipose tissue similar to that observed during exposure to cold by mechanisms that may not be secondary to the involvement of norepinephrine.