NOTCH1: A Novel Player in the Molecular Crosstalk Underlying Articular Chondrocyte Protection by Oleuropein and Hydroxytyrosol.

Osteoarthritis (OA) is the most common joint disease, but no effective and safe disease-modifying treatment is available. Risk factors such as age, sex, genetics, injuries and obesity can concur to the onset of the disease, variably triggering the loss of maturational arrest of chondrocytes further sustained by oxidative stress, inflammation and catabolism. Different types of nutraceuticals have been studied for their anti-oxidative and anti-inflammatory properties. Olive-derived polyphenols draw particular interest due to their ability to dampen the activation of pivotal signaling pathways in OA. Our study aims to investigate the effects of oleuropein (OE) and hydroxytyrosol (HT) in in vitro OA models and elucidate their possible effects on NOTCH1, a novel therapeutic target for OA. Chondrocytes were cultured and exposed to lipopolysaccharide (LPS). Detailed analysis was carried out about the OE/HT mitigating effects on the release of ROS (DCHF-DA), the increased gene expression of catabolic and inflammatory markers (real time RT-PCR), the release of MMP-13 (ELISA and Western blot) and the activation of underlying signaling pathways (Western blot). Our findings show that HT/OE efficiently attenuates LPS-induced effects by firstly reducing the activation of JNK and of the NOTCH1 pathway downstream. In conclusion, our study provides molecular bases supporting the dietary supplementation of olive-derived polyphenols to revert/delay the progression of OA.

Small Extracellular Vesicles from Inflamed Adipose Derived Stromal Cells Enhance the NF-κB-Dependent Inflammatory/Catabolic Environment of Osteoarthritis.

The last decade has seen exponentially growing efforts to exploit the effects of adipose derived stromal cells (ADSC) in the treatment of a wide range of chronic degenerative diseases, including osteoarthritis (OA), the most prevalent joint disorder. In the perspective of developing a cell-free advanced therapy medicinal product, a focus has been recently addressed to the ADSC secretome that lends itself to an allogeneic use and can be further dissected for the selective purification of small extracellular vesicles (sEVs). sEVs can act as "biological drug carriers" to transfer information that mirror the pathophysiology of the providing cells. This is important in the clinical perspective where many OA patients are also affected by the metabolic syndrome (MetS). ADSC from MetS OA patients are dysfunctional and "inflammatory" primed within the adipose tissue. To mimic this condition, we exposed ADSC to IL-1ß, and then we investigated the effects of the isolated sEVs on chondrocytes and synoviocytes, either cultured separately or in co-culture, to tease out the effects of these "IL-1ß primed sEVs" on gene and protein expression of major inflammatory and catabolic OA markers. In comparison with sEVs isolated from unstimulated ADSC, the IL-1ß primed sEVs were able to propagate NF-κB activation in bystander joint cells. The effects were more prominent on synoviocytes, possibly because of a higher expression of binding molecules such as CD44. These findings call upon a careful characterization of the "inflammatory fingerprint" of ADSC to avoid the transfer of an unwanted message as well as the development of in vitro "preconditioning" strategies able to rescue the antiinflammatory/anticatabolic potential of ADSC-derived sEVs.

Basal and IL-1ß enhanced chondrocyte chemotactic activity on monocytes are co-dependent on both IKKα and IKKß NF-κB activating kinases.

IKKα and IKKß are essential kinases for activating NF-κB transcription factors that regulate cellular differentiation and inflammation. By virtue of their small size, chemokines support the crosstalk between cartilage and other joint compartments and contribute to immune cell chemotaxis in osteoarthritis (OA). Here we employed shRNA retroviruses to stably and efficiently ablate the expression of each IKK in primary OA chondrocytes to determine their individual contributions for monocyte chemotaxis in response to chondrocyte conditioned media. Both IKKα and IKKß KDs blunted both the monocyte chemotactic potential and the protein levels of CCL2/MCP-1, the chemokine with the highest concentration and the strongest association with monocyte chemotaxis. These findings were mirrored by gene expression analysis indicating that the lowest levels of CCL2/MCP-1 and other monocyte-active chemokines were in IKKαKD cells under both basal and IL-1ß stimulated conditions. We find that in their response to IL-1ß stimulation IKKαKD primary OA chondrocytes have reduced levels of phosphorylated NFkappaB p65pSer536 and H3pSer10. Confocal microscopy analysis revealed co-localized p65 and H3pSer10 nuclear signals in agreement with our findings that IKKαKD effectively blunts their basal level and IL-1ß dependent increases. Our results suggest that IKKα could be a novel OA disease target.

Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes.

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy "maturation arrested state" and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.

Oxidative stress-induced DNA damage and repair in primary human osteoarthritis chondrocytes: focus on IKKα and the DNA Mismatch Repair System.

During osteoarthritis development, chondrocytes are subjected to a functional derangement. This increases their susceptibility to stressful conditions such as oxidative stress, a characteristic of the aging tissue, which can further provoke extrinsic senescence by DNA damage responses. It was previously observed that IκB kinase α knockdown increases the replicative potential of primary human OA chondrocytes cultured in monolayer and the survival of the same cells undergoing hypertrophic-like differentiation in 3-D. In this paper we investigated whether IKKα knockdown could modulate oxidative stress-induced senescence of OA chondrocytes undergoing a DDR and particularly the involvement in this process of the DNA mismatch repair system, the principal mechanism for repair of replicative and recombinational errors, devoted to genomic stability maintenance in actively replicating cells. This repair system is also implicated in oxidative stress-mediated DNA damage repair. We analyzed microsatellite instability and expression of the mismatch repair components in human osteoarthritis chondrocytes after IKKα knockdown and H2O2 exposure. Only low MSI levels and incidence were detected and exclusively in IKKα proficient cells. Moreover, we found that IKKα proficient and deficient chondrocytes differently regulated MMR proteins after oxidative stress, both at mRNA and protein level, suggesting a reduced susceptibility of IKKα deficient cells. Our data suggest an involvement of the MMR system in the response to oxidative stress that tends to be more efficient in IKKαKD cells. This argues for a partial contribution of the MMR system to the better ability to recover DNA damage already observed in these cells.

Small Extracellular Vesicles from adipose derived stromal cells significantly attenuate in vitro the NF-κB dependent inflammatory/catabolic environment of osteoarthritis.

The therapeutic ability of Mesenchymal Stem/Stromal Cells to address osteoarthritis (OA) is mainly related to the secretion of biologically active factors, which can be found within their secreted Extracellular Vesicles including small Extracellular Vesicles (sEV). Aim of this study was to investigate the effects of sEV from adipose derived stromal cells (ADSC) on both chondrocytes and synoviocytes, in order to gain insights into the mechanisms modulating the inflammatory/catabolic OA environment. sEV, obtained by a combined precipitation and size exclusion chromatography method, were quantified and characterized, and administered to chondrocytes and synoviocytes stimulated with IL-1ß. Cellular uptake of sEV was evaluated from 1 to 12 h. Gene expression and protein release of cytokines/chemokines, catabolic and inflammatory molecules were analyzed at 4 and 15 h, when p65 nuclear translocation was investigated to study NF-κB pathway. This study underlined the potential of ADSC derived sEV to affect gene expression and protein release of both chondrocytes and synoviocytes, counteracting IL-1ß induced inflammatory effects, and provided insights into their mechanisms of action. sEV uptake was faster in synoviocytes, where it also elicited stronger effects, especially in terms of cytokine and chemokine modulation. The inflammatory/catabolic environment mediated by NF-κB pathway was significantly attenuated by sEV, which hold promise as new therapeutic strategy to address OA.

Nutraceutical Activity in Osteoarthritis Biology: A Focus on the Nutrigenomic Role.

Osteoarthritis (OA) is a disease associated to age or conditions that precipitate aging of articular cartilage, a post-mitotic tissue that remains functional until the failure of major homeostatic mechanisms. OA severely impacts the national health system costs and patients' quality of life because of pain and disability. It is a whole-joint disease sustained by inflammatory and oxidative signaling pathways and marked epigenetic changes responsible for catabolism of the cartilage extracellular matrix. OA usually progresses until its severity requires joint arthroplasty. To delay this progression and to improve symptoms, a wide range of naturally derived compounds have been proposed and are summarized in this review. Preclinical in vitro and in vivo studies have provided proof of principle that many of these nutraceuticals are able to exert pleiotropic and synergistic effects and effectively counteract OA pathogenesis by exerting both anti-inflammatory and antioxidant activities and by tuning major OA-related signaling pathways. The latter are the basis for the nutrigenomic role played by some of these compounds, given the marked changes in the transcriptome, miRNome, and methylome. Ongoing and future clinical trials will hopefully confirm the disease-modifying ability of these bioactive molecules in OA patients.

Spermidine rescues the deregulated autophagic response to oxidative stress of osteoarthritic chondrocytes.

Oxidative stress (OS) contributes to Osteoarthritis (OA) pathogenesis and its effects are worsened by the impairment of homeostatic mechanisms such as autophagy in OA chondrocytes. Rescue of an efficient autophagic flux could therefore reduce the bulk of damaged molecules, and at the same time improve cell function and viability. As a promising dietary or intra-articular supplement to rescue autophagy in OA chondrocytes, we tested spermidine (SPD), known to induce autophagy and to reduce OS in several other cellular models. Chondrocytes were obtained from OA cartilage and seeded at high-density to keep their differentiated phenotype. The damaging effects of OS and the chondroprotective activity of SPD were assessed by evaluating the extent of cell death, oxidative DNA damage and caspase 3 activation. The autophagy promoting activity of SPD was evaluated by assessing pivotal autophagic effectors, i.e. Beclin-1 (BECN-1), microtubule-associated protein 1 light chain 3 II (LC3-II) and p62. BECN-1 protein expression was significantly increased by SPD and reduced by H2O2 treatment. SPD also rescued the impaired autophagic flux consequent to H2O2 exposure by increasing mRNA and protein expression of LC3-II and p62. SPD induction of mitophagy was revealed by immunofluorescent co-localization of LC3-II and TOM20. The key protective role of autophagy was confirmed by the loss of SPD chondroprotection upon autophagy-related gene 5 (ATG5) silencing. Significant SPD tuning of the H2O2-dependent induction of degradative (MMP-13), inflammatory (iNOS, COX-2) and hypertrophy markers (RUNX2 and VEGF) was revealed by Real Time PCR and pointed at the SPD ability of reducing NF-κB activation through autophagy induction. Conversely, blockage of autophagy led to parallel increases of oxidative markers and p65 nuclear translocation. SPD also increased the proliferation of slow-proliferating primary cultures. Taken together, our findings highlight the chondroprotective, anti-oxidant and anti-inflammatory activity of SPD and suggest that the protection afforded by SPD against OS is exerted through the rescue of the autophagic flux.

Modulation of Fatty Acid-Related Genes in the Response of H9c2 Cardiac Cells to Palmitate and n-3 Polyunsaturated Fatty Acids.

While high levels of saturated fatty acids are associated with impairment of cardiovascular functions, n-3 polyunsaturated fatty acids (PUFAs) have been shown to exert protective effects. However the molecular mechanisms underlying this evidence are not completely understood. In the present study we have used rat H9c2 ventricular cardiomyoblasts as a cellular model of lipotoxicity to highlight the effects of palmitate, a saturated fatty acid, on genetic and epigenetic modulation of fatty acid metabolism and fate, and the ability of PUFAs, eicosapentaenoic acid, and docosahexaenoic acid, to contrast the actions that may contribute to cardiac dysfunction and remodeling. Treatment with a high dose of palmitate provoked mitochondrial depolarization, apoptosis, and hypertrophy of cardiomyoblasts. Palmitate also enhanced the mRNA levels of sterol regulatory element-binding proteins (SREBPs), a family of master transcription factors for lipogenesis, and it favored the expression of genes encoding key enzymes that metabolically activate palmitate and commit it to biosynthetic pathways. Moreover, miR-33a, a highly conserved microRNA embedded in an intronic sequence of the SREBP2 gene, was co-expressed with the SREBP2 messenger, while its target carnitine palmitoyltransferase-1b was down-regulated. Manipulation of the levels of miR-33a and SREBPs allowed us to understand their involvement in cell death and hypertrophy. The simultaneous addition of PUFAs prevented the effects of palmitate and protected H9c2 cells. These results may have implications for the control of cardiac metabolism and dysfunction, particularly in relation to dietary habits and the quality of fatty acid intake.

Molecular Mechanisms Contributing to Mesenchymal Stromal Cell Aging.

Mesenchymal stem/stromal cells (MSCs) are a reservoir for tissue homeostasis and repair that age during organismal aging. Beside the fundamental in vivo role of MSCs, they have also emerged in the last years as extremely promising therapeutic agents for a wide variety of clinical conditions. MSC use frequently requires in vitro expansion, thus exposing cells to replicative senescence. Aging of MSCs (both in vivo and in vitro) can affect not only their replicative potential, but also their properties, like immunomodulation and secretory profile, thus possibly compromising their therapeutic effect. It is therefore of critical importance to unveil the underlying mechanisms of MSC senescence and to define shared methods to assess MSC aging status. The present review will focus on current scientific knowledge about MSC aging mechanisms, control and effects, including possible anti-aging treatments.

Polyamine supplementation reduces DNA damage in adipose stem cells cultured in 3-D.

According to previous research, natural polyamines exert a role in regulating cell committment and differentiation from stemness during skeletal development. In order to assess whether distinct polyamine patterns are associated with different skeletal cell types, primary cultures of stem cells, chondrocytes or osteoblasts were dedicated for HPLC analysis of intracellular polyamines. Spermine (SPM) and Spermidine (SPD) levels were higher in adipose derived stem cells (ASC) compared to mature skeletal cells, i.e. chondrocytes and osteoblasts, confirming the connection of polyamine content with stemness. To establish whether polyamines can protect ASC against oxidative DNA damage in a 3-D differentiation model, the level of γH2AX was measured by western blot, and found to correlate with age and BMI of patients. Addition of either polyamine to ASC was able to hinder DNA damage in the low micromolecular range, with marked reduction of γH2AX level at 10 µM SPM and 5 µM SPD. Molecular analysis of the mechanisms that might underlie the protective effect of polyamine supplementation evidences a possible involvement of autophagy. Altogether, these results support the idea that polyamines are able to manage both stem cell differentiation and cell oxidative damage, and therefore represent appealing tools for regenerative and cell based applications.

The N-Acetyl Phenylalanine Glucosamine Derivative Attenuates the Inflammatory/Catabolic Environment in a Chondrocyte-Synoviocyte Co-Culture System.

Osteoarthritis (OA), the most prevalent degenerative joint disease, still lacks a true disease-modifying therapy. The involvement of the NF-κB pathway and its upstream activating kinases in OA pathogenesis has been recognized for many years. The ability of the N-acetyl phenylalanine glucosamine derivative (NAPA) to increase anabolism and reduce catabolism via inhibition of IKKα kinase has been previously observed in vitro and in vivo. The present study aims to confirm the chondroprotective effects of NAPA in an in vitro model of joint OA established with primary cells, respecting both the crosstalk between chondrocytes and synoviocytes and their phenotypes. This model satisfactorily reproduces some features of the previously investigated DMM model, such as the prominent induction of ADAMTS-5 upon inflammatory stimulation. Both gene and protein expression analysis indicated the ability of NAPA to counteract key cartilage catabolic enzymes (ADAMTS-5) and effectors (MCP-1). Molecular analysis showed the ability of NAPA to reduce IKKα nuclear translocation and H3Ser10 phosphorylation, thus inhibiting IKKα transactivation of NF-κB signalling, a pivotal step in the NF-κB-dependent gene expression of some of its targets. In conclusion, our data confirm that NAPA could truly act as a disease-modifying drug in OA.

Biomaterials: Foreign Bodies or Tuners for the Immune Response?

The perspectives of regenerative medicine are still severely hampered by the host response to biomaterial implantation, despite the robustness of technologies that hold the promise to recover the functionality of damaged organs and tissues. In this scenario, the cellular and molecular events that decide on implant success and tissue regeneration are played at the interface between the foreign body and the host inflammation, determined by innate and adaptive immune responses. To avoid adverse events, rather than the use of inert scaffolds, current state of the art points to the use of immunomodulatory biomaterials and their knowledge-based use to reduce neutrophil activation, and optimize M1 to M2 macrophage polarization, Th1 to Th2 lymphocyte switch, and Treg induction. Despite the fact that the field is still evolving and much remains to be accomplished, recent research breakthroughs have provided a broader insight on the correct choice of biomaterial physicochemical modifications to tune the reaction of the host immune system to implanted biomaterial and to favor integration and healing.

Emerging Players at the Intersection of Chondrocyte Loss of Maturational Arrest, Oxidative Stress, Senescence and Low-Grade Inflammation in Osteoarthritis.

The prevalence of Osteoarthritis (OA) is increasing because of the progressive aging and unhealthy lifestyle. These risk factors trigger OA by removing constraints that keep the tightly regulated low turnover of the extracellular matrix (ECM) of articular cartilage, the correct chondrocyte phenotype, and the functionality of major homeostatic mechanisms, such as mitophagy, that allows for the clearance of dysfunctional mitochondria, preventing increased production of reactive oxygen species, oxidative stress, and senescence. After OA onset, the presence of ECM degradation products is perceived as a "danger" signal by the chondrocytes and the synovial macrophages that release alarmins with autocrine/paracrine effects on the same cells. Alarmins trigger innate immunity in the joint, with important systemic crosstalks that explain the beneficial effects of dietary interventions and improved lifestyle. Alarmins also boost low-grade inflammation: the release of inflammatory molecules and chemokines sustained by continuous triggering of NF-κB within an altered cellular setting that allows its higher transcriptional activity. Chemokines exert pleiotropic functions in OA, including the recruitment of inflammatory cells and the induction of ECM remodeling. Some chemokines have been successfully targeted to attenuate structural damage or pain in OA animal models. This represents a promising strategy for the future management of human OA.

MicroRNAs and Autophagy: Fine Players in the Control of Chondrocyte Homeostatic Activities in Osteoarthritis.

Osteoarthritis (OA) is a debilitating degenerative disease of the articular cartilage with a multifactorial etiology. Aging, the main risk factor for OA development, is associated with a systemic oxidative and inflammatory phenotype. Autophagy is a central housekeeping system that plays an antiaging role by supporting the clearance of senescence-associated alterations of macromolecules and organelles. Autophagy deficiency has been related to OA pathogenesis because of the accumulation of cellular defects in chondrocytes. Microribonucleic acids (microRNAs or miRs) are a well-established class of posttranscriptional modulators belonging to the family of noncoding RNAs that have been identified as key players in the regulation of cellular processes, such as autophagy, by targeting their own cognate mRNAs. Here, we present a state-of-the-art literature review on the role of miRs and autophagy in the scenario of OA pathogenesis. In addition, a comprehensive survey has been performed on the functional connections of the miR network and the autophagy pathway in OA by using "microRNA," "autophagy," and "osteoarthritis" as key words. Discussion of available evidence sheds light on some aspects that need further investigation in order to reach a more comprehensive view of the potential of this topic in OA.

Hydroxytyrosol prevents chondrocyte death under oxidative stress by inducing autophagy through sirtuin 1-dependent and -independent mechanisms.

BACKGROUND: Hydroxytyrosol (HT), a major phenolic antioxidant found in olive oil, can afford protection from oxidative stress in several types of non-tumoral cells, including chondrocytes. Autophagy was recently identified as a protective process during osteoarthritis (OA) development and critical for survival of chondrocytes. Therefore we have investigated the possibility to modulate chondrocyte autophagy by HT treatment. METHODS: DNA damage and cell death were estimated in human C-28/I2 and primary OA chondrocytes exposed to hydrogen peroxide. Autophagic flux and mitophagy were monitored by measuring levels and location of autophagy markers through western blot, immunostaining and confocal laser microscopy. Late autophagic vacuoles were stained with monodansylcadaverine. The involvement of sirtuin 1 (SIRT-1) was evaluated by immunohistochemistry, western blot and gene silencing with specific siRNA. RESULTS: HT increases markers of autophagy and protects chondrocytes from DNA damage and cell death induced by oxidative stress. The protective effect requires the deacetylase SIRT-1, which accumulated in the nucleus following HT treatment. In fact silencing of this enzyme prevented HT from promoting the autophagic process and cell survival. Furthermore HT supports autophagy even in a SIRT-1-independent manner, by increasing p62 transcription, required for autophagic degradation of polyubiquitin-containing bodies. CONCLUSIONS: These results support the potential of HT as a chondroprotective nutraceutical compound against OA, not merely for its antioxidant ability, but as an autophagy and SIRT-1 inducer as well. GENERAL SIGNIFICANCE: HT may exert a cytoprotective action by promoting autophagy in cell types that may be damaged in degenerative diseases by oxidative and other stress stimuli.

Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients.

INTRODUCTION: Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3ß inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3ß inactivation in vitro to assess their contribution to cell senescence and hypertrophy. METHODS: In vivo level of phosphorylated GSK3ß was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3ß inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45ß and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated ß galactosidase activity, and PAS staining. RESULTS: In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3ß, oxidative damage and expression of GADD45ß and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3ß inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45ß and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10. CONCLUSIONS: In articular chondrocytes, GSK3ß activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3ß inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3ß inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response.

mTOR, AMPK, and Sirt1: Key Players in Metabolic Stress Management.

Cells adapt their metabolism and activities in response to signals from their surroundings, and this ability is essential for their survival in the face of environmental changes. In mammalian tissues a deficit of these mechanisms is commonly associated with cellular aging and degenerative diseases related to aging, such as cardiovascular disease, cancer, immune system decline, and neurological pathologies. Several proteins have been identified as able to respond directly to energy, nutrient, and growth factor levels and stress stimuli in order to mediate adaptations in the cell. Many of these proteins are enzymes that positively or negatively modulate the autophagic process. This review focuses on biochemical mechanisms involving enzymes--specifically, mTOR, AMPK, and Sirt1--that are currently considered important for these adaptive responses, providing an overview of the interactions of the main players in this process.