Ettore Bergamini

Autophagy and Aging The Importance of Maintaining "Clean" Cells

A decrease in the rate of protein turnover and the intracellular accumulation of altered proteins in cytosol and membranes are features common to all aged cells. Diminished autophagic activity plays a major role in these age-related manifestations. In this work we review the molecular defects responsible for the malfunctioning of two forms of autophagy - macroautophagy and chaperone-mediated autophagy - in old mammals, and highlight general and cell-type specific consequences of dysfunction of the autophagic system with age. Dietary caloric restriction and antilipolytic agents have been proven to efficiently stimulate autophagy in old rodents. These and other possible restorative efforts are discussed.

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.

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma.

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by α-tocopherol. We show here that α-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, α-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3′-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by α-tocopherol. However, α-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-γ (PPARγ; troglitazone), indicating that this pathway is susceptible to α-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and α-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARγ element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARγ and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARγ signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

Application of amino acid analysis by high-performance liquid chromatography with phenyl isothiocyanate derivatization to the rapid determination of free amino acids in biological samples

An amino acid analysis by reversed-phase high-performance liquid chromatography after precolumn derivatization with phenyl isothiocyanate was adapted to the determination of free amino acids in plasma or other biological fluids and in tissue homogenates. Preparation of samples included deproteinization by 3% sulphosalicylic acid, and careful removal under high vacuum of residual phenyl isothiocyanate after derivatization. A Waters Pico-Tag column (15 cm long) was used, immersed in a water-bath at 38 degrees C. In rat or human plasma, separation of 23 individual amino acids, plus the unresolved pair tryptophan and ornithine, was obtained within 13 min. Including the time for column washing and re-equilibration, samples could be chromatographed at 23-min intervals. Variability was tested for each amino acid by calculating the coefficients of variation of retention times (less than 1% in the average) and peak areas (less than 4% for both intra-day and inter-day determinations). The linearity for each standard amino acid was remarkable over the concentration range 3-50 nmol/ml. The mean recovery of amino acid standards added to plasma prior to derivatization was 97 +/- 0.8%, except for aspartate (82%) and glutamate (81%). This method is rapid (almost three samples per hour can be analysed, more than in any other reported technique), with satisfactory precision, sensitivity and reproducibility. Therefore, it is well suited for routine analysis of free amino acids in both clinical and research work.

Alternate-day fasting protects the rat heart against age-induced inflammation and fibrosis by inhibiting oxidative damage and NF-kB activation

The free radical theory of aging is currently one of the most popular. In parallel, many studies have demonstrated the association of fibrosis and increased oxidative stress in the pathogenesis of some chronic human diseases, and fibrosis is often characteristic of aging tissues. One of the few interventions that effectively slow aging is calorie restriction and the protection against the age-associated increase of oxidative stress remains one of the foremost hypotheses to explain this action. As an alternative to traditional calorie restriction, another dietary regimen, termed alternate-day fasting, has also been tested, whose antiaging mechanisms have not been explored so much extensively. We thus studied the effects of alternate-day fasting, started at 2 months of age, on oxidative stress and fibrosis in the heart during aging. In the left ventricle of the heart of elderly (aged 24 months) versus young (aged 6 months) male rats we found a significant increase in oxidative stress paralleled by increased fibrosis. In parallel there was a significant increase in inflammatory cytokine levels and in NF-kB DNA binding activity with advancing age. Alternate-day fasting protected against all these age-related phenomena. These data support the hypothesis that this kind of dietary restriction protects against age-related fibrosis, at least in part by reducing inflammation and oxidative damage, and this protection can thus be considered a factor in the prevention of age-related diseases with sclerotic evolution.

Evidence for selective mitochondrial autophagy and failure in aging.

Autophagy is a major intracellular degradation/recycling system ubiquitous in eukaryotic cells. It contributes to the turnover of cellular components by delivering portions of the cytoplasm and organelles to lysosomes, where they are digested. Starvation-induced autophagy is required for maintaining an amino acid pool for gluconeogenesis and for the synthesis of proteins essential to survival under starvation conditions. In addition, autophagy plays an important role in the degradation of excess or injured organelles, including mitochondria. To test the hypothesis of an involvement of a decrease in autophagy in the process of aging, we explored the antiaging effects of pharmacological stimulation of autophagy on the age-dependent accumulation of 8-OHdG-rich mitochondria in rat liver. Male 3-month and 16-month-old 24 hours-fasted Sprague Dawley rats were injected with the antilipolytic agent [3,5-dimethylpyrazole (DMP)] intraperitoneally. Results showed that drug injection rescued older cells from the accumulation of 8-OHdG in the mtDNA in less than 6 hours, but no significant decrease in the level of cytocrome c oxidase activity was observed. Together, these data provide indirect evidence that 8-OHdG might accumulate in a small pool of mitochondria with increasing age rather than be degraded by the autophagic machinery selectively. Addendum to: Stimulation of Macroautophagy Can Rescue Older Cells from 8-OHdG mtDNA Accumulation: A Safe and Easy Way to Meet Goals in the SENS Agenda A. Donati, M. Taddei, G. Cavallini and E Bergamini Rejuvenation Res 2006; 9:408-12

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.