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Aging is accompanied by a decline in the healthy function of multiple organs, leading to increased incidence and mortality from diseases such as cancer and inflammatory, cardiovascular and neurodegenerative diseases. Dietary restriction is the most effective experimental intervention known to consistently slow the aging process and with positive effects on health span in different organisms, from invertebrates to mammals. Age is also associated with progressive decline in physical activity levels in a wide range of animal species: therefore, regular physical exercise could represent a safe intervention to antagonize aging. In this research we explore the effects of exercise training initiated in late middle aged rats fed with different lifelong dietary regimens: one group was fed ad libitum and the second group was subjected to every-other-day fasting. These two groups might represent examples of "normal" aging and "successful" aging. The study shows the effects of exercise and food restriction and their interaction on plasma levels of total antioxidant capacity, lactate, amino acids, and on products of protein oxidation in soleus and tibialis anterior muscles. In addition, we evaluated body composition measurement by bioelectrical impedance analysis and muscle strength by grasping test. Results show that late-onset exercise training has the potential to improve some metabolic and physiological parameters in rats with the same "chronological age" but different "biological age", without negative effects, and highlight the relevance of a personalised and selected exercise protocol, since the responsiveness to exercise may depend on the individual's "biological age".

Ageing has been defined as a progressive decrease in physiological capacity and a reduced ability to respond to environmental stresses. It has been observed that diet-restricted animals show a minor morbidity in age-related disease. Among these age-related diseases, hypercholesterolemia is the most recurring one and it is often associated with cardiac failure. Several studies have been published indicating age-dependent changes in circulating levels of cholesterol in both humans and in rodents; recently changes have also been reported in the proteins involved in cholesterol homeostasis, that is, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR), Insig-induced gene (Insig) protein, SREBP cleavage activating protein (SCAP), sterol regulatory element binding protein (SREBP), and low density lipoprotein receptor (LDLr). Most age-related modifications of biochemical parameters are normalized or very improved in food-restricted animals, so the aim of this work is to examine whether or not alterations of the factors involved in cholesterol homeostasis which occur during ageing could be counteracted by caloric restriction (CR). The data show that the diet restrictions used attenuate the age-related effects on the factors involved in the synthesis and the degradation rate of HMG-CoAR; in spite of this, CRs have a good effect on the age-related hypercholesterolemia whose reduction seems to depend both on the correct membrane LDLr localization and on the proper restored HMG-CoAR activity. J. Cell. Biochem. 101: 235–243, 2007. © 2007 Wiley-Liss, Inc.

The physiological role of dolichol is not yet known but its accumulation in several tissues has been extensively reported in various physiological states or pathological conditions. Increased dolichol concentration in mammalian tissues during ageing has been also reported; in particular, we have previously indicated dolichol accumulation in liver as a new biomarker of ageing. However, the mechanism and the role of this accumulation is unknown. The aim of this work was to study the mechanism of the age-dependent dolichol accumulation analysing, in the liver and in the brain, the activity of the rate-limiting enzyme of isoprenoid biosynthesis, the 3-hydroxy 3-methylglutaryl CoA reductase, the dolichol and cholesterol synthesis on aged rats both fed ad libitum and caloric restricted. Furthermore, the dolichol and cholesterol levels in the plasma were assayed. The data shows that during ageing, the tissue dolichol accumulation is connected with the increase of 3-hydroxy 3-methylglutaryl CoA reductase activity and only in liver affected by diet restriction. In addition the aged rats maintain the capability to regulate their tissue cholesterol content by modifying cholesterol delivery into the blood. The amount of the 3-hydroxy 3-methylglutaryl CoA reductase enzyme detectable in liver and brain by Western blot analysis does not show significant changes during ageing. The presented data show that the accumulation of dolichol is related to the loss of enzymatic regulation characteristic of ageing. In fact, a higher mevalonate availability deriving from an increased expressed activity of HMGCoA-R could cause an increased production of dolichol.

8-Hydroxy-2-deoxyguanosine (8OHdG) excreted into the urine is considered a marker of oxidative stress effect on DNA, and it is reported to be mainly produced by the DNA repair system. In previous works, we showed that autophagy was also involved in 8OHdG disposal through the degradation of oxidatively altered mitochondria. Here, we show that aging in Sprague–Dawley male rats is associated with a decline in the in vitro function of liver autophagy and a slight and not significant decrease in the urinary excretion of 8OHdG. In addition, we demonstrate that anti-aging caloric restriction maintains levels of both liver autophagy and urinary excretion of 8OHdG at very high levels throughout life. Finally, we show the in vivo stimulation of autophagy by the administration of an antilipolytic agent or everolimus, which rescues rats from the accumulation of 8OHdG in the liver mtDNA, also causes a dramatic increase in the urinary excretion of 8OHdG. The intensification of autophagy by the administration of the antilipolytic drugs to fasting rats faded progressively with increasing age, together with a reduced increase in 8OHdG output into the urine. It is concluded that the process of autophagy may play a major role in the disposal of 8OHdG with urine, and that the assay of 8OHdG levels in the urine before and after the stimulation of autophagy may provide a novel, non-invasive and safe procedure to monitor the in vivo functioning of the process.

Proteasome activity is known to decrease with aging in ad libitum (AL) fed rats. Severe caloric restriction (CR) significantly extends the maximum life-span of rats, and counteracts the age-associated decrease in liver proteasome activities. Since few investigations have explored whether lower CR diets might positively counteract the age associated decrease in proteasome activity, we then investigated the effects of a mild CR regimen on animal life-span, proteasome content and function. In addition, we addressed the question whether both CR regimens might also affect the expression of Hsc70 protein, a constitutive chaperone reported to share a role in the function of proteasome complex and in the repair of proteotoxic damage, and whose level decreased during aging. In contrast to severe CR, mild CR had a poor effect on life-span; however, it better counteracted the decrease of proteasome activities. Both regimens, however, maintain Hsc70 in liver of old rats at level comparable to that of young rats. Interestingly, the effects of aging and CRs on liver proteasome enzyme activities did not appear to be associated with parallel changes in the amount of proteasome proteins suggesting that the quality (molecular activity of the enzymes) rather than the quantity are likely to be modified with age. In conclusion, the results presented in this work show that a mild CR can have beneficial effects on liver function of aging rats because is adequate to counteract the decrease of proteasome function and Hsc70 chaperone level.

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.

8-hydroxy-deoxyguanosine adducts (8-OHdG), indices of oxidative DNA damage, were measured by immunohystochemistry with diaminobenzidine detection in the brain, skeletal muscle, heart, liver, tenuum mucosa and lymphocytes from young (4 months) and aged (24 months) Sprague-Dawley rats fed ad libitum or held on two different caloric restriction diets (alternate day ad libitum feeding or daily feeding with 40% reduced calories). In the absence of caloric restriction the levels of oxidative DNA damage increased as a function of age in all tissues examined, with a maximum approximately 3-fold increase being detected in the peripheral lymphocytes and the heart and a minimum approximately 2-fold increase being detected in the liver and brain tissues. Caloric restriction regimens effectively reduced age-dependent increase of oxidative DNA damage in all tissues examined; in particular, the brain and small intestine did not exhibit any age-related increase of oxidative DNA damage. We propose that the levels of 8-OHdG in peripheral lymphocytes may serve a biochemical index of age-related whole organism oxidative DNA damage. Immunohistochemistry might be exploited as a rapid and simple techniques for measuring lymphocytes oxidative DNA damage in large scale studies.