Peroxisomes proliferation and pharmacological stimulation of autophagy in rat liver: evidence to support that autophagy may remove the "older" peroxisomes.

Like mitochondria, peroxisomes produce reactive oxygen species (ROS), compounds which have been implicated to play an important role in many degenerative diseases and aging itself, and an exaggerated ROS production might occur in altered or older organelles. Growing evidence shows that autophagy, a required function in cell housekeeping during fasting, can remove damaged macromolecules, organelles, and membranes selectively. Proliferation of peroxisomes can be enhanced in liver cells by perfluorooctanoic acid (PFOA), which causes a marked increase of the Acyl-CoA oxidase (ACOX) activity and no significant change in urate oxidase (UOX) activity. The administration of antilipolytic drugs to fasted animals was shown to intensify autophagy. Here we tested the hypothesis that autophagy may distinguish and remove older from younger peroxisomes in rat liver. Male Sprague-Dawley rats were given PFOA (150 mg/kg body weight) or vehicle. Animals were sacrificed at different times following PFOA administration, and 3 h after the induction of autophagy with the antilipolytic agent 3,5-dimethyl pyrazole (DMP, 12 mg/kg body weight). The levels of ACOX and UOX activity were measured in the liver tissue. Results showed that autophagy caused a parallel, significant decrease in both enzymes activity in control rats, and that in PFOA-treated rats the effects were different and changed with PFOA time administration. Changes are compatible with the hypothesis that newly formed ACOX-rich peroxisomes are resistant to pexophagy and that sensitivity to pexophagy increases with increasing peroxisomal "age." In conclusion, there is indirect evidence supporting the hypothesis that autophagy may recognize and degrade older peroxisomes.

Dolichol: A Component of the Cellular Antioxidant Machinery.

Dolichol, an end product of the mevalonate pathway, has been proposed as a biomarker of aging, but its biological role, not to mention its catabolism, has not been fully understood. UV-B radiation was used to induce oxidative stress in isolated rat hepatocytes by the collagenase method. Effects on dolichol, phospholipid-bound polyunsaturated fatty acids (PL-PUFA) and known lipid soluble antioxidants [coenzyme Q (CoQ) and α-tocopherol] were studied. The increase in oxidative stress was detected by a probe sensitive to reactive oxygen species (ROS). Peroxidation of lipids was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). Dolichol, CoQ, and α-tocopherol were assessed by high-pressure liquid chromatography (HPLC), PL-PUFA by gas-liquid chromatography (GC). UV-B radiation caused an immediate increase in ROS as well as lipid peroxidation and a simultaneous decrease in the levels of dolichol and lipid soluble antioxidants. Decrease in dolichol paralleled changes in CoQ levels and was smaller to that in α-tocopherol. The addition of mevinolin, a competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoAR), magnified the loss of dolichol and was associated with an increase in TBARS production. Changes in PL-PUFA were minor. These findings highlight that oxidative stress has very early and similar effects on dolichol and lipid soluble antioxidants. Lower levels of dolichol are associated with enhanced peroxidation of lipids, which suggest that dolichol may have a protective role in the antioxidant machinery of cell membranes and perhaps be a key to understanding some adverse effects of statin therapy.

Antiaging therapy: a novel target for antilipolytic drugs.

Aging has been defined as a gradually decreasing ability to maintain homeostasis and increasing risk to die. Growing evidence supports malfunctioning with age of quality control system. At an older age, accumulation of altered macromolecules and membranes may impair cell functioning; accumulation of altered mitochondria and peroxisomes may boost the yield of ROS per unit of produced energy and accelerate the aging process. Evidence produced that autophagy, an essential part in cell housekeeping during fasting, may help removal of altered membranes, mitochondria and peroxisomes selectively and account for the antiaging effects of caloric restriction. Stimulation of autophagy may improve innate and adaptive immunity; decrease the risk of myopathy, heart disease, liver disease, neurodegeneration and cancer; and retard aging. Functioning of autophagy may decline in well fed adults and is almost negligible at older age. Induction of autophagy may result in "cleaner cells" lower in oxidative status and more resistant to injury and disease. The administration of antilipolytic drugs to fasted animals was shown to intensify autophagy in a physiologically appropriate manner, to enhance submaximal antiaging effects of low level of caloric restriction, to rapidly rescue older cells from the accumulation of altered mtDNA and older peroxisomes, to increase urinary 8-OHdG levels, and counteract the age-related hypercholesterolemia in rodents. In conclusion, benefits of long-lasting stimulation of autophagy and protein and organelle turnover shows that antilipolytic drugs might find a novel therapeutic application in antiaging medicine.

Use of red blood cell membranes to evaluate the antioxidant potential of plant extracts.

Antioxidant phytochemicals in fruits and vegetables of a vegetarian diet may account for the reduced risk of aging and stress oxidative associated diseases. In this study, a simple, rapid and accurate new bioassay for the determination of the antioxidant activity of purified or crude plant extracts and thier interactions is described, based on the fluorimetric determination of thiobarbituric acid reactive substances (TBARS) released by UV-B radiated red blood cell (RBC) ghosts. Pure resveratrol, white and red wine and pomegranate juice (PJ) were used as antioxidant source to test the biological method. TBARS production is a function of radiation time, the number of RBC ghosts in the radiated sample and the loaded antioxidant. The antioxidant activity of resveratrol was detected at a submicromolar concentration range [0.02 µg/mL-0.1 µmol/L]. The activity of red wine was almost 10 times higher than that of white wine, and PJ juice had the highest activity. Submaximal protective effects of PJ and red wine were additive.

Effects of stimulation of autophagy on the urinary excretion of biomarkers of the oxidative status.

Autophagy, a health-promoting lysosomal degradation pathway that controls the quality of the cytoplasm by eliminating protein aggregates and damaged organelles including 8-OHdG-rich mitochondria, is under investigation as a target for prevention and/or treatment of several human diseases and decelerating aging. Stimulation of autophagy was shown to rescue older liver cells from accumulation of 8-OHdG-rich mitochondria and to increase urinary 8-OHdG levels. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a recently recommended biomarker for monitoring oxidative status over time. In order to rule out the possibility that the in vivo stimulation of autophagy may cause an increase in the oxidative status, in this study we compared the effects of the stimulation of autophagy by two different procedures (the administration of antilipolytic drug and everolimus, an mTOR inhibitor in clinical use) on the urinary levels of 8-OHdG and 15-isoprostane F2t, another well-known biomarker of the oxidative status. Results show that both procedures increased the urinary 8-OHdG levels without any change in urinary 15-isoprostane F2t; this increase in urinary 8-OHdG levels after the antilipolytic drug was fully suppressed by the simultaneous injection of glucose to make rats transiently incompetent for the endocrine stimulation of autophagy. Conclusions are that the in vivo stimulation of autophagy does not affect the oxidative status and that the increasing effect on urinary 8-OHdG may be secondary to an increased degradation of previously accumulated 8-OHdG-rich (mt)DNA. The authors are aware that findings may open the way to a safe, easy, highly desirable non-invasive test for successful in vivo activation of autophagy after pharmacological stimulation.

Effects of aging, antiaging calorie restriction and in vivo stimulation of autophagy on the urinary excretion of 8OHdG in male Sprague-Dawley rats.

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.

Searching for the fountain of autophagy-dependent youth: Ettore Bergamini. Interview by Daniel J. Klionsky.

One of the most exciting aspects of autophagy is its connection with aging, and the possibility of manipulating autophagy to extend life span. Many studies now demonstrate a general decrease in autophagy with age and offer some very interesting indications that reversing this trend, either genetically or pharmacologically, can have a beneficial effect with regard to aging. One of the first researchers to consider the connection between diet, aging and autophagy is Ettore Bergamini, the subject of this interview.

Alternate-day fasting reverses the age-associated hypertrophy phenotype in rat heart by influencing the ERK and PI3K signaling pathways.

The age-related increased impedance in large arteries overloads the senescent heart, and the myocardial phenotype is hypertrophic. Together with qualitative changes observed in the senile heart, this can be responsible for impaired diastolic function. A restricted diet providing adequate nutrient intake, e.g. alternate-day fasting (ADF), has been shown to extend life-span and decrease incidence and progression of age-associated diseases in laboratory rodents, and to ameliorate some metabolic markers of aging in rhesus monkeys and humans. This study reports an age-related increase of some biological and morphological hypertrophy markers in the rat heart, together with increased plasma BNP, a well known marker of heart failure. The tissue modifications might likely be related to hyper-activation of two of the signaling pathways associated with myocardial pathological hypertrophy: ERK1/2 and PI3Kγ. Increased ERK1/2 activation might be in part related to the disturbance of STAT3, with a consequent decrease of SOCS3. In this context, the down-modulation of ERK1/2 and PI3Kγ signaling, together with the restoration of STAT3 activity and SOCS3 content, both observed with ADF, might help to reduce pathological hypertrophy stimuli and to rescue an important cardioprotective pathway, possibly opening new preventive and therapeutic perspectives in age-related heart failure.

Acipimox reduces circulating levels of insulin and associated neutrophilic inflammation in metabolic syndrome.

Metabolic syndrome is a proatherosclerotic condition clustering cardiovascular risk factors, including glucose and lipid profile alterations. The pathophysiological mechanisms favoring atherosclerotic inflammation in the metabolic syndrome remain elusive. Here, we investigated the potential role of the antilipolytic drug acipimox on neutrophil- and monocyte-mediated inflammation in the metabolic syndrome. Acipimox (500 mg) was orally administered to metabolic syndrome patients (n = 11) or healthy controls (n = 8). Serum and plasma was collected before acipimox administration (time 0) as well as 2-5 h afterward to assess metabolic and hematologic parameters. In vitro, the effects of the incubation with metabolic syndrome serum were assessed on human neutrophil and monocyte migration toward the proatherosclerotic chemokine CCL3. Two to five hours after acipimox administration, a significant reduction in circulating levels of insulin and nonesterified fatty acid (NEFA) was shown in metabolic syndrome patients. At time 0 and 2 h after acipimox administration, metabolic syndrome serum increased neutrophil migration to CCL3 compared with healthy controls. No effect was shown in human monocytes. At these time points, serum-induced neutrophil migration positively correlated with serum levels of insulin and NEFA. Metabolic syndrome serum or recombinant insulin did not upregulate CCR5 expression on neutrophil surface membrane, but it increased intracellular JNK1/2 phosphorylation. Insulin immunodepletion blocked serum-induced neutrophil migration and associated JNK1/2 phosphorylation. Although mRNA expression of acipimox receptor (GPR109) was shown in human neutrophils, 5-500 µM acipimox did not affect insulin-induced neutrophil migration. In conclusion, results suggest that acipimox inhibited neutrophil proatherosclerotic functions in the metabolic syndrome through the reduction in circulating levels of insulin.

Biogerontology in Italy.

In this paper experimental gerontology in Italy is reviewed on the basis of research developed in Academic and non Academic Centres. There are several groups across Italy working actively on basic science of aging producing high impact papers with a significant contribution to biogerontology. Some distinguished Italian scientist working abroad is also mentioned. Interesting issues on longevity and interventions on aging (including caloric restriction) and on aging brain are quoted. Relevant studies encompass the (glyco-)oxidative stress as direct damage mechanism and main process of theory of aging, other research lines include IGF-1, mitochondria DNA, obesity/sarcopenia and exercise and also an animal model for aging studies is reported. Notwithstanding financial restrictions and structure deficit the biogerontology research in Italy could be judged as good, but additional resources are necessary to keep this good rank.

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.

Stimulation of autophagy by antilipolytic drugs may rescue rodents from age-associated hypercholesterolemia.

Aging is characterized by several metabolic changes responsible for the decline of certain functions and the appearance of age-related diseases, including hypercholesterolemia, which is the main risk factor for atherosclerosis and cardiovascular disease. Similar changes in a number of morphological and biochemical parameters were observed in rats. Caloric restriction (CR) was shown to increase longevity and prevent age-related diseases in various organisms, and to counteract the age-associated increase in plasma cholesterol. CR was thought to operate through the stimulation of the process of macroautophagy. The aim of this work was to investigate the effect of the stimulation of macroautophagy on age-associated cholesterolemia. Mature Sprague-Dawley rats were fasted overnight and given the antilipolytic agent 3,5-dimethylpyrazole (DMP; 12 mg/kg b.w. in 0.2 mL of saline, intraperitoneally). The age-related changes in cholesterol plasma level, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA-R) activity, and lipoperoxidation were determined. Low-density lipoprotein (LDL) receptor expression was determined by immunoblot of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated liver membranes. Results show that the stimulation of macroautophagy reduces the total LDL and high-density lipoprotein (HDL) cholesterol plasma level to juvenile values, and triglycerides levels even lower. The hypocholesterolemic action of DMP requires neither the counteraction of the age-related changes in the HMG-CoA-R activation state and regulation, nor the counteraction of the age-related increase in lipoperoxidation, and only involves a restoration of the numbers of LDL receptors on liver membranes to juvenile levels.

Methods for inducing and monitoring liver autophagy relative to aging and antiaging caloric restriction in rats.

The functioning of macroautophagy declines with increasing age in the liver of ad libitum fed animals, whereas it is preserved in rats submitted to antiaging caloric restriction. In this perspective, monitoring autophagy during aging may provide a useful biomarker of aging. Here we describe a procedure for the quantification of the ex vivo functioning of autophagy by the use of single-pass liver perfusion to measure the rate of degradation of prelabeled long-lived proteins. The maximum rate of autophagy can be measured after the pharmacological suppression of the supply of free fatty acids during fasting, which intensifies the activation of autophagy by a physiological mechanism. The effects of treatment on the plasma level of branched chain amino acids may be used as a minimally invasive indicator of the intensification of autophagy. The effects of aging on autophagic proteolysis and on amino acid and hormone control can also be assessed by measuring the rate of the 3-methyladenine-sensitive valine released from isolated liver cells incubated in vitro.

Deficiency and supplementation of PUFA in the diet have similar effects on the age-associated changes in rat-plasma cholesterol levels.

Levels of plasma cholesterol, particularly LDL cholesterol, increase with increasing age in humans and rodents. Feeding a fish oil-rich diet may exert hypocholesterolemic effects. The aim of this work was to examine the effects of a life-long administration of a PUFA-enriched diet and of a PUFA-deficient diet in male Sprague-Dawley rats on the age-associated increases in plasma cholesterol and triglycerides. Diet had small effects on body-weight, and had dramatic effects on liver phospholipids-fatty acids. Surprisingly, both diets counteracted the age-associated changes in plasma cholesterol and triglycerides similarly and benefits were already visible in adult rats.

Effect of aging and anti-aging caloric restriction on the endocrine regulation of rat liver autophagy.

Autophagy is a process that sequesters and degrades altered organelles and macromolecular cytoplasmic constituents for cellular restructuring and repair, and as a source of nutrients for metabolic use in early starvation it may be involved in anti-aging mechanisms of caloric restriction. The effects of 40% daily dietary restriction (DR) and intermittent feeding (EOD) on the age-related changes in the endocrine regulation of autophagic proteolysis were studied by monitoring the rate of valine release from isolated rat liver cells. Results show that in ad libitum-fed rats sensitivity of autophagy to glucagon and insulin declines by one order of magnitude in older rats. Both DR and EOD maintain the sensitivity to glucagon at juvenile levels, whereas only EOD can fully maintain response to insulin. It is concluded that changes in the sensitivity to glucagon may have a role in the aging process.

Age-related changes in endothelin-1 receptor subtypes in rat heart.

Density, affinity, and subtype distribution of endothelin-1 (ET-1) binding sites were determined in rat cardiac tissue as a function of age in order to evaluate the association of alterations in the endothelin receptor system and aging in the heart. A significant decrease in the receptor subtype ET-A, which represents 70% to 80% of the total receptor population in cardiac tissue of 3- and 12-month-old rats, was observed in 24-month-old rats with respect to the younger groups. These findings indicate an alteration in ET-1 cardiac receptors associated with aging, mainly due to a variation in the receptor subtype distribution.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver.

Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5'-dimethylpyrazole, DMP, 12mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis.

Low-level caloric restriction rescues proteasome activity and Hsc70 level in liver of aged rats.

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.

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.