In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

• MeSH
• autofagie * fyziologie   MeSH
• autofagozomy MeSH
• biologické markery MeSH
• biotest normy   MeSH
• lidé MeSH
• lyzozomy MeSH
• proteiny spojené s autofagií metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• směrnice MeSH

The success of chemotherapy largely depends on the anticancer immune response triggered by tumor cells that succumb to immunogenic cell death (ICD). One of the hallmarks of ICD is premortem autophagy that facilitates the release of adenosine triphosphate from dying cancer cells and acts as a chemoattractant for dendritic cell precursors. Here, we show that the immune response induced by inoculation of cancer cells undergoing ICD in response to the anthracycline mitoxantrone (MTX) can be improved by a short-term fasting regimen (48 hours of starvation) and that this effect is reversed by systemic administration of the autophagy inhibitor dimethyl α-ketoglutarate. Tumor growth reduction by MTX treatment is known to depend on autophagy induction in cancer cells as well as on an intact immune system. We compared the antitumor effects of MTX on autophagy-competent cancers implanted in wild type (WT) or partially autophagy-deficient (Becn1± or Atg4b-/-) mice. While there was no difference in the tumor growth reducing effects of MTX on tumors evolving in WT, Becn1+/- and Atg4b-/- mice, we observed an increase in the toxicity of MTX on Atg4b-/- mice. These results suggest that autophagy in cancer cells (but less so in host cells) is rate-limiting for therapeutically relevant anticancer immune responses, yet has a major role in blunting the life-threatening toxicity of chemotherapy.

• Publikační typ
• časopisecké články MeSH

Ageing constitutes the most important risk factor for all major chronic ailments, including malignant, cardiovascular and neurodegenerative diseases. However, behavioural and pharmacological interventions with feasible potential to promote health upon ageing remain rare. Here we report the identification of the flavonoid 4,4'-dimethoxychalcone (DMC) as a natural compound with anti-ageing properties. External DMC administration extends the lifespan of yeast, worms and flies, decelerates senescence of human cell cultures, and protects mice from prolonged myocardial ischaemia. Concomitantly, DMC induces autophagy, which is essential for its cytoprotective effects from yeast to mice. This pro-autophagic response induces a conserved systemic change in metabolism, operates independently of TORC1 signalling and depends on specific GATA transcription factors. Notably, we identify DMC in the plant Angelica keiskei koidzumi, to which longevity- and health-promoting effects are ascribed in Asian traditional medicine. In summary, we have identified and mechanistically characterised the conserved longevity-promoting effects of a natural anti-ageing drug.

• MeSH
• Angelica chemie   MeSH
• autofagie účinky léků   MeSH
• buněčná smrt účinky léků   MeSH
• buněčné linie účinky léků   MeSH
• Caenorhabditis elegans účinky léků   MeSH
• dlouhověkost účinky léků   fyziologie   MeSH
• Drosophila melanogaster účinky léků   MeSH
• flavonoidy aplikace a dávkování   farmakologie   MeSH
• ischemická choroba srdeční farmakoterapie   MeSH
• lidé MeSH
• mechanistické cílové místo rapamycinového komplexu 1 metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• proteiny přenášející kationty genetika   MeSH
• regulace genové exprese účinky léků   MeSH
• rostlinné extrakty farmakologie   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae účinky léků   metabolismus   MeSH
• signální transdukce MeSH
• sirolimus farmakologie   MeSH
• stárnutí účinky léků   fyziologie   MeSH
• tradiční orientální medicína MeSH
• transkripční faktory GATA účinky léků   MeSH
• transkripční faktory účinky léků   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The capacity of cells and organisms to sustain, and to eventually adapt to, environmental and genetic insults declines with age. Because macroautophagy/autophagy is regarded as one of the major determinants of cellular fitness in vitro and in vivo, maneuvers that aim at promoting autophagy may slow down aging and promote health span. Caloric restriction (CR), a reduction in caloric intake without malnutrition, efficiently counteracts aging-associated features, yet is difficult to be applied to humans. Caloric-restriction mimetics (CRMs) are pharmacological agents that recapitulate the main biochemical properties of CR, namely a global reduction of protein acetylation and the induction of autophagy. We found that the ancient drug aspirin and its active metabolite salicylate stimulate autophagic flux by virtue of their inhibitory action on acetyltransferase EP300. The inhibition of EP300 results from a direct competition between salicylate and acetyl coenzyme A for binding to the catalytic domain of the enzyme. This mode of action appears to be conserved across evolution as it accounts for the induction of autophagy by aspirin in various mouse models and in the nematode Caenorhabditis elegans. In sum, aspirin acts as a CRM.

• MeSH
• acetylkoenzym A MeSH
• Aspirin MeSH
• autofagie * MeSH
• Caenorhabditis elegans MeSH
• kalorická restrikce * MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH
• práce podpořená grantem MeSH

The age-associated deterioration in cellular and organismal functions associates with dysregulation of nutrient-sensing pathways and disabled autophagy. The reactivation of autophagic flux may prevent or ameliorate age-related metabolic dysfunctions. Non-toxic compounds endowed with the capacity to reduce the overall levels of protein acetylation and to induce autophagy have been categorized as caloric restriction mimetics (CRMs). Here, we show that aspirin or its active metabolite salicylate induce autophagy by virtue of their capacity to inhibit the acetyltransferase activity of EP300. While salicylate readily stimulates autophagic flux in control cells, it fails to further increase autophagy levels in EP300-deficient cells, as well as in cells in which endogenous EP300 has been replaced by salicylate-resistant EP300 mutants. Accordingly, the pro-autophagic activity of aspirin and salicylate on the nematode Caenorhabditis elegans is lost when the expression of the EP300 ortholog cpb-1 is reduced. Altogether, these findings identify aspirin as an evolutionary conserved CRM.

• MeSH
• acetylkoenzym A metabolismus   MeSH
• Aspirin farmakologie   MeSH
• autofagie účinky léků   genetika   MeSH
• kalorická restrikce * MeSH
• lidé MeSH
• metabolom účinky léků   MeSH
• metabolomika MeSH
• myši inbrední C57BL MeSH
• nádorové buněčné linie MeSH
• protein p300 asociovaný s E1A metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

In the last years, autophagy has been revealed as an essential pathway for multiple biological processes and physiological functions. As a catabolic route, autophagy regulation by nutrient availability has been evolutionarily conserved from yeast to mammals. On one hand, autophagy induction by starvation is associated with a significant loss in body weight in mice. Here, we demonstrate that both genetic and pharmacological inhibition of the autophagy process compromise weight loss induced by starvation. Moreover, autophagic potential also impacts on weight gain induced by distinct hypercaloric regimens. Atg4b-deficient mice, which show limited autophagic competence, exhibit a major increase in body weight in response to distinct obesity-associated metabolic challenges. This response is characterized by the presence of larger adipocytes in visceral fat tissue, increased hepatic steatosis, as well as reduced glucose tolerance and attenuated insulin responses. Similarly, autophagy-deficient mice are more vulnerable to experimentally induced type-I diabetes, showing an increased susceptibility to acute streptozotocin administration. Notably, pharmacological stimulation of autophagy in wild-type mice by spermidine reduced both weight gain and obesity-associated alterations upon hypercaloric regimens. Altogether, these results indicate that systemic autophagic activity influences the resilience of the organism to weight gain induced by high-calorie diets, as well as to the obesity-associated features of both type-1 and type-2 diabetes.

• MeSH
• autofagie * MeSH
• beta-buňky metabolismus   patologie   MeSH
• buněčná smrt účinky léků   genetika   MeSH
• cysteinové endopeptidasy nedostatek   MeSH
• dieta škodlivé účinky   MeSH
• experimentální diabetes mellitus genetika   metabolismus   patologie   MeSH
• myši knockoutované MeSH
• myši MeSH
• obezita chemicky indukované   genetika   metabolismus   patologie   MeSH
• proteiny spojené s autofagií nedostatek   MeSH
• tukové buňky metabolismus   patologie   MeSH
• ztučnělá játra chemicky indukované   genetika   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Starvation is a strong physiological stimulus of macroautophagy/autophagy. In this study, we addressed the question as to whether it would be possible to measure autophagy in blood cells after nutrient deprivation. Fasting of mice for 48 h (which causes ∼20% weight loss) or starvation of human volunteers for up to 4 d (which causes <2% weight loss) provokes major changes in the plasma metabolome, yet induces only relatively minor alterations in the intracellular metabolome of circulating leukocytes. White blood cells from mice and human volunteers responded to fasting with a marked reduction in protein lysine acetylation, affecting both nuclear and cytoplasmic compartments. In circulating leukocytes from mice that underwent 48-h fasting, an increase in LC3B lipidation (as assessed by immunoblotting and immunofluorescence) only became detectable if the protease inhibitor leupeptin was injected 2 h before drawing blood. Consistently, measurement of an enhanced autophagic flux was only possible if white blood cells from starved human volunteers were cultured in the presence or absence of leupeptin. Whereas all murine leukocyte subpopulations significantly increased the number of LC3B+ puncta per cell in response to nutrient deprivation, only neutrophils from starved volunteers showed signs of activated autophagy (as determined by a combination of multi-color immunofluorescence, cytofluorometry and image analysis). Altogether, these results suggest that white blood cells are suitable for monitoring autophagic flux. In addition, we propose that the evaluation of protein acetylation in circulating leukocytes can be adopted as a biochemical marker of organismal energetic status.

• MeSH
• acetylace MeSH
• autofagie MeSH
• dospělí MeSH
• hladovění krev   metabolismus   MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• lysin metabolismus   MeSH
• metabolom MeSH
• metabolomika MeSH
• mladý dospělý MeSH
• myši inbrední C57BL MeSH
• neutrofily metabolismus   MeSH
• omezení příjmu potravy krev   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky MeSH

Cancer can be viewed in 2 rather distinct ways, namely (i) as a cell-autonomous disease in which malignant cells have escaped control from cell-intrinsic barriers against proliferation and dissemination or (ii) as a systemic disease that involves failing immune control of aberrant cells. Since macroautophagy/autophagy generally increases the fitness of cells as well as their resistance against endogenous or iatrogenic (i.e., relating to illness due to medical intervention) stress, it has been widely proposed that inhibition of autophagy would constitute a valid strategy for sensitizing cancer cells to chemotherapy or radiotherapy. Colliding with this cell-autonomous vision, however, we found that immunosurveillance against transplantable, carcinogen-induced or genetically engineered cancers can be improved by pharmacologically inducing autophagy with caloric restriction mimetics. This positive effect depends on autophagy induction in cancer cells and is mediated by alterations in extracellular ATP metabolism, namely increased release of immunostimulatory ATP and reduced adenosine-dependent recruitment of immunosuppressive regulatory T cells into the tumor bed. The combination of autophagy inducers and chemotherapeutic agents is particularly efficient in reducing cancer growth through the stimulation of CD8+ T lymphocyte-dependent anticancer immune responses.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• autofagie * MeSH
• imunitní dozor MeSH
• lidé MeSH
• myši MeSH
• nádory imunologie   patologie   terapie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Caloric restriction mimetics (CRMs) mimic the biochemical effects of nutrient deprivation by reducing lysine acetylation of cellular proteins, thus triggering autophagy. Treatment with the CRM hydroxycitrate, an inhibitor of ATP citrate lyase, induced the depletion of regulatory T cells (which dampen anticancer immunity) from autophagy-competent, but not autophagy-deficient, mutant KRAS-induced lung cancers in mice, thereby improving anticancer immunosurveillance and reducing tumor mass. Short-term fasting or treatment with several chemically unrelated autophagy-inducing CRMs, including hydroxycitrate and spermidine, improved the inhibition of tumor growth by chemotherapy in vivo. This effect was only observed for autophagy-competent tumors, depended on the presence of T lymphocytes, and was accompanied by the depletion of regulatory T cells from the tumor bed.

• MeSH
• Atg5 genetika   MeSH
• autofagie MeSH
• citráty aplikace a dávkování   farmakologie   MeSH
• experimentální nádory dietoterapie   farmakoterapie   imunologie   MeSH
• kalorická restrikce metody   MeSH
• lidé MeSH
• methotrexát aplikace a dávkování   farmakologie   MeSH
• monitorování imunologické MeSH
• mutace MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• protoonkogenní proteiny p21(ras) genetika   MeSH
• regulační T-lymfocyty účinky léků   MeSH
• spermidin aplikace a dávkování   farmakologie   MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• MeSH
• autofagie * fyziologie   MeSH
• biotest metody   normy   MeSH
• lidé MeSH
• počítačová simulace MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• směrnice MeSH