30 December 2011

Life extension: Resveratrol, spermidine and autophagy (2)

Autophagy is a mechanism for protecting cells and preventing aging(continued, the beginning of the article is here)

In the cytoplasm of cells exposed to stress and on the verge of death, the accumulation of autophagosomes and auto (phago) often occurslysosomes. This observation was (erroneously) interpreted as self-destruction of the cell [50]. Therefore, in hundreds of works, "autophagic" (also known as "type 2 death") cell death is described as programmed cell death, preceded by mass autophagic vacuolization, morphologically different from apoptosis ("type 1 death") and necrosis ("type 3 death") [51-54]. Despite the fact that "autophagic cell death" undoubtedly exists as a morphological event [53], it is only a reflection (at least for mammalian cells) of the mechanism of cell self-destruction by autophagy [50]. In fact, in most cases autophagy is a (sometimes fruitless) mechanism of cellular adaptation to a wide range of adverse conditions, including hypoglycemia, hypoxia, deficiency of essential amino acids, lack of necessary growth factors or sublethal damage to organelles contained in the cytoplasm, including mitochondria and ER [4, 55, 56]. Therefore, genetic inhibition of autophagy by blocking genes associated with autophagy (ATG) often leads to apoptotic or necrotic death of cells that could normally survive nutrient deficiency, lack of growth factors, hypoxia, exposure to ionizing radiation or antitumor chemotherapy [11, 50, 57-60]. Disorders of autophagy are directly involved in the pathogenesis of a number of diseases, including neurodegenerative diseases, heart failure, hereditary myopathies, steatosis/fatty degeneration of the liver and other chronic inflammatory processes [6, 61-64]. It has been shown that autophagy-inducing genetic and pharmacological manipulations in vitro protect cells from death in case of normal fatal injuries [5, 6]. Autophagy contributes to maintaining high levels of ATP inside the cell [22, 65], increases the ability of cells to withstand metabolic stress (hypoxia in combination with nutrient deficiency) [22, 66], prevents genetic instability [60, 67] and limits the accumulation of potentially toxic proteins, including proteotoxins that cause neurodegeneration [10, 38]. From a physiological point of view, aging can be considered as a prolonged gradual deterioration of functions at the cellular and organismic level, which (at least partially) is a reflection of the accumulation of improperly formed protein molecules, oxidized fat molecules, as well as mutations in mitochondrial and nuclear DNA.

The only way to increase the life expectancy of each of the organisms tested to date is a low–calorie diet - reducing the amount of calories entering the body, which does not lead to exhaustion [68]. A low-calorie diet is a powerful inducer of autophagy in cells of almost all species, including mammals [69-71]. In roundworms Caenorhabditis elegans, the initiation of autophagy is necessary to increase life expectancy caused by a low-calorie diet [72-74] or the deletion of the p53 gene [22, 49, 75-77]. At the same time, under the condition of simultaneous application of the RNA interference method against atg genes, nematodes contained on a low-calorie diet do not live longer than individuals of the control group [72-74].

Rapamycin, which activates autophagy by inhibiting (m)TOR, is also credited with a pronounced ability to slow down the aging of model organisms. However, rapamycin cannot increase the chronological lifespan (that is, the lifespan of postmitotic cells in the stationary phase) in yeast mutants that do not have functional genes Atg1, Atg7 or Atg11 [79]. The positive effect of rapamycin on the lifespan of C.elegans is lost when an important autophagy modulator BEC-1 (an orthologue of the mammalian Beclin 1 gene and the yeast Atg6 gene) is blocked [74]. Thus, autophagy is necessary for rapamycin-induced increase in life expectancy and slowing down the chronological aging of yeast and nematodes. Despite the fact that there is no official evidence of rapamycin's ability to increase the lifespan of mice by inducing autophagy, even the administration of this drug to aging genetically heterogeneous (nonlinear) mice increased their lifespan [80]. Rapamycin slows down the age-related deterioration of the functions of hematopoietic cells in mice [81], and its ability to prevent physiological aging of cells has been described in a number of in vitro experiments [82, 83].

In general, these results indicate that the induction of autophagy at the body level with the help of pharmacological agents can increase the duration of a healthy life, at least in the laboratory. This supports the idea that autophagy not only protects cells, but also has an aging-slowing effect at the organizational level.

Autophagy mediates the increase in life expectancy caused by resveratrolThe above reasoning formed the basis of the hypothesis expressed by the authors, according to which autophagy is the main or at least one of the main mechanisms by which life-prolonging drugs have their effect.

Therefore, they studied the supposed ability of resveratrol, a well–studied aging–retarding agent [84], to increase the lifespan of model organisms through the induction of autophagy. Despite its ability to influence mitochondrial functions [85], resveratrol is a strong allosteric activator of sirtuin 1, a phylogenetically conserved deacetylase enzyme that reacts to changes in the NAD+ ratio/NADF [84]. Resveratrol increases the lifespan of yeast, nematodes and fruit flies (Drosophila melanogaster), and also slows down the aging of mice kept on a fat-rich diet [84, 86]. Indirect evidence suggests that resveratrol can induce autophagy in yeast (although the cause of this was recognized as the oxidation of mitochondrial fats [87]) and human malignant cells, in which resveratrol-induced autophagy often precedes cell death [88]. Sirtuin 1 is the first protein whose ability to increase life expectancy has been demonstrated in yeast (followed by animals, including C.elegans nematodes and fruit flies) [89]. Subsequently, its ability to trigger autophagy in cultured human and rodent cells was also shown [90].

The authors confirmed that overexpression of sirtuin 1 enhances autophagic processes in human malignant cells in vitro and that this effect is neutralized by adding EX527, a pharmacological inhibitor of its catalytic activity, to the culture medium [91, 92]. Similarly, a transgen encoding SIR-2.1 (belonging to the C.elegans orthologue of human sirtuin 1) triggered autophagy in nematodes. This fact indicates that the relationship between the activation of sirtuin 1 and autophagy has been preserved during evolution [91, 92]. It is also important that the need for sirtuin 1 was proved for the induction of autophagy in conditions of nutrient deficiency (provided by cell culture in the absence of serum, amino acids and glucose), which did not extend to the induction of autophagy using other stimuli. Thus, in human cells, suppression of expression (using RNA interference) or inhibition (using EX527) of sirtuin 1 completely neutralized the pro-autophagic effect of nutrient deprivation, without affecting the stimulation of autophagy by inhibiting mTOR (using rapamycin), inhibition of p53 (using pythitrin-alpha) or stress effects on the ER (triggered by the addition of tunicamycin to the medium). Similarly, mutations leading to the loss of the functionality of the sir-2.1 C.elegans gene prevented the initiation of autophagy in a low-calorie diet, but did not affect autophagy caused by the action of rapamycin or tunicamycin [91, 92]. Transgenic overexpression of the sir-2.1 gene increased the average and maximum life expectancy of nematodes compared to individuals of nontransgenic control lines with the same genotype. This effect disappeared when the expression of an important autophagy modulator BEC-1 was suppressed by RNA interference [91, 92]. The knockout of the C.elegans CEP-1 gene, which is an orthologue of p53, caused by RNA interference, a manipulation that increases life expectancy by stimulating autophagy [77], did not enhance the positive effect of sir-2.1 overexpression on life expectancy [91, 92]. The results of this epistatic analysis indicate that the accumulation of SIR-2.1 protein and the removal of CEP-1 protein increase life expectancy by triggering a common end mechanism based on the induction of autophagy.

Another genetic intervention aimed at indirect activation of sirtuin 1 (or its orthologue in the SIR-2.1 nematode genome) consists in transgenic overexpression of a gene encoding the enzyme pyrazinamidase/nicotinamidase PNC-1, cleaving nicotinamide, which is a negative regulator of sirtuin 1/SIR-2.1. Transgenic overexpression of the pnc-1 gene really induced autophagy in nematode cells, and this reaction was neutralized by suppressing the expression of the SIR-2.1 protein using RNA interference. Accordingly, the life-prolonging effect of PNC-1 disappeared when knocking out the gene encoding SIR-2.1, as well as any of the genes encoding important autophagy modulators BEC-1 and ATG-5 [77]. Thus, both overexpression and metabolic activation of sirtuin 1/SIR-2.1 increase life expectancy through the induction of autophagy.

After that, the authors studied the potential ability of resveratrol to induce autophagy in C.elegans by activating SIR-2.1. The addition of resveratrol to the nematode growth medium really stimulated autophagy, and this effect was lost when the expression of SIR-2.1 protein was suppressed by RNA interference. Similarly, resveratrol reduced the age-related mortality of C.elegans, except in the absence of sir-2.1 or bec-1 gene products [77]. Based on the results of these experiments, the authors concluded that resveratrol increases the lifespan of human cells and nematodes through the induction of autophagy, which is the result of resveratrol-mediated activation of sirtuin 1/SIR-2.1 (and not the result of any unforeseen effect).

Part 3: Autophagy mediates the increase in life expectancy caused by spermidinePortal "Eternal youth" http://vechnayamolodost.ru


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