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(a) Energy-status analyses of ATP, ADP, AMP, NAD and NADH with the related metabolite ratios ATP/AMP, ATP/ADP, ADP/AMP, and NADH/NAD. *p ≤ 0.05; **p ≤ 0.01. P-values were determined by Student’s t-test. Error bars represent s.e.m. N = 5 per group. (b) Western Blotting analyses of markers for GR and OR and proteins involved in the cellular energy metabolism. IMR90 cells were subjected to OR, GR and OGR for 24 h. After that cells were harvested and total cell lysate was analyzed using Western blotting and immunodetected with indicated antibodies. Tubulin was used as a loading control. For the densitometric quantification of the immunoreactive bands the absolute values measured were first normalized to tubulin and the resulting values to the control, which was set as 1. # ≤ 0.10, *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001. P-values were determined by one-way analysis of variance (ANOVA) with post-hoc Tukey honestly significant difference (HSD) test. Error bars represent s.e.m. N = 3 per group. (c) Autophagic degradation activity analyses upon OR, GR and OGR compared to control measured by Western blotting analyzing LC3 and LC3-II protein levels and LC3-II protein turnover with and without BafA. The autophagic degradation activity (autophagic flux) was determined by the following calculation: ΔLC3-II = ‘LC3-II + BafA’ - ‘LC3-II - BafA’. Tubulin was used as a loading control. # ≤ 0.10, *p ≤ 0.05, **p ≤ 0.01. P-values were determined by one-way ANOVA with post-hoc Tukey HSD test. Error bars represent s.e.m. N = 3 per group.
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The ability of cells to rearrange their metabolism plays an important role in compensating the energy shortage and may provide cell survival. Our study focuses on identifing the important adaptational changes under the conditions of oxygen and glucose reduction. Employing mass spectrometry-based metabolomics in combination with biochemistry and mic...
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... 52 In eukaryotic cells, autophagy consists of a highly conserved pathway for lysosomal degradation of long-lived proteins, lipids, and organelles, which is tightly regulated by the cellular metabolism. 53 Autophagy-inducing compounds, such as spermidine, selective AKT1 inhibitor MK-2206, and BECN1-stabilizing anthelmintic niclosamide (Fig. 6a), can reduce SARS-CoV-2 replication and hold potential for treating COVID-19. For instance, targeting autophagic pathways through the exogenous administration of these compounds inhibits SARS-CoV-2 propagation in vitro, with the compounds exhibiting IC 50 values of 7.67, 0.11, and 0.13 μM, respectively. ...
... Metabolic reprogramming is one of the host cell responses induced by viruses to intercept host cell machinery (Thaker et al. 2019). Viruses rapidly compensate for the lack of energy for survival by altering host cell metabolism, which consists primarily of OXPHOS and glycolysis (Weckmann et al. 2018). CVB3 also evolves molecular mechanisms that utilize the various compartments of the host cell (Garmaroudi et al. 2015). ...
Coxsackievirus B3 (CVB3) is a single-stranded RNA virus that belongs to the Enterovirus genus. CVB3 is a human pathogen associated with serious conditions such as myocarditis, dilated cardiomyopathy, and pancreatitis. However, there are no therapeutic interventions to treat CVB3 infections. In this study, we found that CVB3 induced metabolic alteration in host cells through increasing glycolysis level, as indicated by an increase in the extracellular acidification rate (ECAR). CVB3-mediated metabolic alteration was confirmed by metabolite change analysis using gas chromatography-mass spectrometry (GC-MS). Based on findings, a strategy to inhibit glycolysis has been proposed to treat CVB3 infection. Indeed, glycolysis inhibitors (2-Deoxy-D-glucose, sodium oxide) significantly reduced CVB3 titers after CVB3 infection, indicating that glycolysis inhibitors can be used as effective antiviral agents. Taken together, our results reveal a novel mechanism by which CVB3 infection is controlled by regulation of host cell metabolism.
... BCL2L13 (BCL2 like 13), BNIP [NIP3 (BCL2/adenovirus E1B 19 KDa protein-interacting protein 3) and NIX (NIP3-like protein X)/BNIP3L, are located on OMM induce mitochondrial fragmentation and recruit autophagic machinery through their interaction with LC3 protein via their LIR (LC3 interacting) motif (Novak et al., 2010). BNIP and NIX proteins trigger mitophagy in response to hypoxia conditions (Zhang and Ney, 2009;Weckmann et al., 2018). In the MALM (Mieap-induced accumulation of lysosome-like organelles within mitochondria) pathway, BNIP3 or BNIP3L localized on OMM interacts with Mieap (mitochondria-eating protein) leading to pore formation in the mitochondrial membrane thus enabling lysosomes to enter into the mitochondria (Kamino et al., 2016). ...
... Mitochondrial biogenetic failure is a critical activator of inflammatory response pathways, indicated by an increase in oxidative stress, cytoplasmic calcium and reduction of mtDNA (Yan et al., 2020). Various mechanisms, particularly the Parkin/PINK1 pathway, are used to eliminate defective mitochondria (Whitworth and Pallanck, 2017). ...
Mitochondria provide neurons not only energy as ATP to keep them growing, proliferating and developing, but they also control apoptosis. Due to their high bioenergetic demand, neurons which are highly specific terminally differentiated cells, essentially depend on mitochondria. Defective mitochondrial function is thus related to numerous age-linked neurodegenerative ailments like Alzheimer’s disease (AD), in which the build-up of impaired and malfunctioning mitochondria has been identified as a primary sign, paying to disease development. Mitophagy, selective autophagy, is a key mitochondrial quality control system that helps neurons to stay healthy and functional by removing undesired and damaged mitochondria. Dysfunctional mitochondria and dysregulated mitophagy have been closely associated with the onset of ADs. Various proteins associated with mitophagy were found to be altered in AD. Therapeutic strategies focusing on the restoration of mitophagy capabilities could be utilized to strike the development of AD pathogenesis. We summarize the mechanism and role of mitophagy in the onset and advancement of AD, in the quality control mechanism of mitochondria, the consequences of dysfunctional mitophagy in AD, and potential therapeutic approaches involving mitophagy modulation in AD. To develop new therapeutic methods, a better knowledge of the function of mitophagy in the pathophysiology of AD is required.
... The observed upregulation of the "aspartate metabolism" under OS may also affect related pathways including "glycolysis", "gluconeogenesis", "citric acid cycle", "amino acids biosynthesis", and "pyrimidine and purine biosynthesis" (Bielarczyk et al., 1986;Darvey, 2000;Weckmann et al., 2018). Interestingly, upon 48-h recovery of OS, we observed an upregulation of the glycolysis metabolites such as Glucose-6phosphate (2.01-fold), Glycerol-3-phosphate (0.8-fold), and Lactate (1.59-fold) ( Figure 4B), which indicated that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in cardiomyocytes H9c2. ...
... HIF-1α upregulates the expression of Glut1 and glycolytic enzymes increasing the anaerobic glycolysis pathway (Fulda and Debatin, 2007). Moreover, HIF-1α has shown to limit the conversion of pyruvic acid to acetyl-CoA in the mitochondria, impairing the citric acid cycle activity as a strategy to couple to oxidative stress (Kim et al., 2006;Papandreou et al., 2006;Weckmann et al., 2018). The metabolite L-carnitine was also upregulated (1.9-fold) in cardiomyocytes under oxidative stress. ...
Cardiovascular diseases (CVDs) are noncommunicable diseases known for their complex etiology and high mortality rate. Oxidative stress (OS), a condition in which the release of free radical exceeds endogenous antioxidant capacity, is pivotal in CVC, such as myocardial infarction, ischemia/reperfusion, and heart failure. Due to the lack of information about the implications of OS on cardiovascular conditions, several methodologies have been applied to investigate the causes and consequences, and to find new ways of diagnosis and treatment as well. In the present study, cardiac dysfunction was evaluated by analyzing cells’ alterations with untargeted metabolomics, after simulation of an oxidative stress condition using hydrogen peroxide (H2O2) in H9c2 myocytes. Optimizations of H2O2 concentration, cell exposure, and cell recovery times were performed through MTT assays. Intracellular metabolites were analyzed right after the oxidative stress (oxidative stress group) and after 48 h of cell recovery (recovery group) by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in positive and negative ESI ionization mode. Significant alterations were found in pathways such as “alanine, aspartate and glutamate metabolism”, “glycolysis”, and “glutathione metabolism”, mostly with increased metabolites (upregulated). Furthermore, our results indicated that the LC-MS method is effective for studying metabolism in cardiomyocytes and generated excellent fit (R²Y > 0.987) and predictability (Q² > 0.84) values.
... In order to ensure robustness in our feature selection process, we further developed three different pairwise models to identify metabolite markers for each group: A significance analysis of microarray (SAM) [14], PLS-DA variable importance in projection (VIP) [15], and a random forest (RF) [16] classification model. SAM identified 73 out of 234 metabolites in the Tet-On Mfn2 induced fusion group, 71 out of 245 metabolites in the indirect fusion group, and 74 out of 233 metabolites in the leflunomide-treated group as significantly altered based on an FDR < 0.05 and a corresponding delta of 0.39, 0.38, and 0.32 for the Tet-On Mfn2, sgDrp1, and Leflunomide groups, respectively ( Figure 5B). ...
... Using our PLS-DA model, a VIP score greater than 1.0 [14] across all five principal components was used as a cutoff to identify discriminant metabolites after induction of mitochondrial fusion. From our original filtered metabolite set, we detected 83, 72, and 59 potential metabolites of interest in our Tet-On Mfn2, sgDrp1, and Leflunomide-treated groups accordingly ( Figure 5C and Table S4A-C). ...
Mitochondria are dynamic organelles that constantly alter their shape through the recruitment of specialized proteins, like mitofusin-2 (Mfn2) and dynamin-related protein 1 (Drp1). Mfn2 induces the fusion of nearby mitochondria, while Drp1 mediates mitochondrial fission. We previously found that the genetic or pharmacological activation of mitochondrial fusion was tumor suppressive against pancreatic ductal adenocarcinoma (PDAC) in several model systems. The mechanisms of how these different inducers of mitochondrial fusion reduce pancreatic cancer growth are still unknown. Here, we characterized and compared the metabolic reprogramming of these three independent methods of inducing mitochondrial fusion in KPC cells: overexpression of Mfn2, genetic editing of Drp1, or treatment with leflunomide. We identified significantly altered metabolites via robust, orthogonal statistical analyses and found that mitochondrial fusion consistently produces alterations in the metabolism of amino acids. Our unbiased methodology revealed that metabolic perturbations were similar across all these methods of inducing mitochondrial fusion, proposing a common pathway for metabolic targeting with other drugs.
... The inhibition of PDE4 enzymes is in testosterone production by the Leydig cells have been reported to cause a signi cant increase. In rats, it has been asserted that autophagy participates in the regulation of steroid synthesis in Leydig cells (Weckmann et al. 2018). It has been reported that PDE4 and PDE8 play important roles in the physiology of steroidogenic tissues and there is synergism between signaling compartments regulated by PDE4 and PDE8 to facilitate maximum steroid output ). ...
Roflumilast (ROF) (3-cyclo-propylmethoxy-4-difuorome-thoxy-N-[3,5-di-chloropyrid-4-yl] benzamide) is a second generation and forcible phosphodiesterase-4 (PDE4) inhibitor. This study aims to investigate the effects of chronic Roflumilast in different doses on testicular tissue and testosterone levels in healthy Sprague-Dawley rats.
During the research, the 6 weeks old (180–200 gr), 36 male rats were divided into 4 groups. Roflumilast (ROF) was administered as 0.5 mg/kg and 1 mg/kg by oral gavage for four weeks, once each day. Hematoxylin-Eosin (H&E) staining for histopathological examinations in testicular tissue, immunohistochemical and immunofluorescence examinations for Caspase-3, Apoptosis Inducing Factor (AIF) and Light Chain 3β (LC3B) expression levels, and ELISA method used to determine serum testosterone levels. Data were analyzed using SPSS v.22 with Kruskal-Wallis, Mann Whitney-U, and Wilcoxon tests.
Roflumilast group lost weight compared to the control and sham. Shedding in the seminiferous epithelium, degenerations in the interstitial area, separation between cells, desquamation, interstitial edema and degenerative changes in the testicular tissue was observed. While apoptosis and autophagy determined by Caspase-3, AIF and LC3B were close and statistically insignificant in control and sham, there was significantly increased apoptotic and autophagic changes and immunopositivity in the ROF groups. The 1 mg/kg Roflumilast group’s serum testosterone level was lower than control, sham and 0.5 mg/kg Roflumilast groups.
When the research data was evaluated, it was determined that the chronic use of the active ingredient Roflumilast, which has a broad-spectrum area such as COPD, arthritis, neurodegenerative diseases, liver and dermatology, had negative effects on the testicular tissue and testosterone level of rats.
... The UPS facilitates ubiquitintargeted rapid degradation of proteins via the proteasome 8 . Autophagy, which is tightly controlled by metabolism, is a highly conserved lysosomal degradation process of long-lived proteins, lipids, and organelles in eukaryotic cells 9,10 . The mechanistic target of rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) regulate autophagy by responding to metabolic alterations via continuous crosstalk with glucose and protein homeostasis 11 . ...
Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC 50 values of 136.7, 7.67, 0.11, and 0.13 μM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.
... Fusion and fission mechanisms are characterized by the condensation of two smaller mitochondria or the fragmentation of a single big mitochondrion, respectively [19]. It is now well-established that mitochondria modulate their morphology when cells are subjected to physical [20][21][22], chemical [23][24][25], and biological [26][27][28][29] cues. ...
... The impact of autophagy on energy metabolism was also described under hypoxia and glucose restriction, which significantly induce autophagic response [161,162]. The authors demonstrated that glucose or oxygen restriction deactivated anabolic pathways, increased hypoxia inducible factor-1α (HIF-1α), and autophagic machinery in IMR90 fibroblasts [25]. In particular, they observed increased levels of energy pathway intermediates belonging to the pentose phosphate pathway, glycolysis, TCA cycle, and amino acid metabolism, together with mitophagy activation. ...
... In particular, they observed increased levels of energy pathway intermediates belonging to the pentose phosphate pathway, glycolysis, TCA cycle, and amino acid metabolism, together with mitophagy activation. As stated by the authors, the results collected from this study were in line with previous observations obtained in cancer cells and contribute to understanding the relationship between autophagy and metabolism in tumoral settings [25]. ...
Mitochondria represent the energy hub of cells and their function is under the constant influence of their tethering with other subcellular organelles. Mitochondria interact with the endoplasmic reticulum, lysosomes, cytoskeleton, peroxisomes, and nucleus in several ways, ranging from signal transduction, vesicle transport, and membrane contact sites, to regulate energy metabolism, biosynthetic processes, apoptosis, and cell turnover. Tumorigenesis is often associated with mitochondrial dysfunction, which could likely be the result of an altered interaction with different cell organelles or structures. The purpose of the present review is to provide an updated overview of the links between inter-organellar communications and interactions and metabolism in cancer cells, with a focus on mitochondria. The very recent publication of several reviews on these aspects testifies the great interest in the area. Here, we aim at (1) summarizing recent evidence supporting that the metabolic rewiring and adaptation observed in tumors deeply affect organelle dynamics and cellular functions and vice versa; (2) discussing insights on the underlying mechanisms, when available; and (3) critically presenting the gaps in the field that need to be filled, for a comprehensive understanding of tumor cells’ biology. Chemo-resistance and druggable vulnerabilities of cancer cells related to the aspects mentioned above is also outlined.
... The degraded products like amino acids or fatty acids can further be metabolized to generate energy, or they can be recycled to protein or membrane biogenesis. Autophagy is particularly important and upregulated during metabolic stress, including glucose reduction and other bioenergetic deficiencies [3]. Impaired autophagy plays a major role in aging and various human disorders, including many neurodegenerative disorders [4,5]. ...
Macroautophagy is a conserved degradative process for maintaining cellular homeostasis and plays a key role in aging and various human disorders. The microtubule-associated protein 1A/1B light chain 3B (MAP1LC3B or LC3B) is commonly analyzed as a key marker for autophagosomes and as a proxy for autophagic flux. Three paralogues of the LC3 gene exist in humans: LC3A, LC3B and LC3C. The molecular function, regulation and cellular localization of LC3A and LC3C have not been investigated frequently, even if a similar function to that described for LC3B appears likely. Here, we have selectively decapacitated LC3B by three separate strategies in primary human fibroblasts and analyzed the evoked effects on LC3A, LC3B and LC3C in terms of their cellular distribution and co-localization with p62, a ubiquitin and autophagy receptor. First, treatment with pharmacological sirtuin 1 (SIRT1) inhibitors to prevent the translocation of LC3B from the nucleus into the cytosol induced an increase in cytosolic LC3C, a heightened co-localization of LC3C with p62, and an increase LC3C-dependent autophagic flux as assessed by protein lipidation. Cytosolic LC3A, however, was moderately reduced, but also more co-localized with p62. Second, siRNA-based knock-down of SIRT1 broadly reproduced these findings and increased the co-localization of LC3A and particularly LC3C with p62 in presumed autophagosomes. These effects resembled the effects of pharmacological sirtuin inhibition under normal and starvation conditions. Third, siRNA-based knock-down of total LC3B in cytosol and nucleus also induced a redistribution of LC3C as if to replace LC3B in the nucleus, but only moderately affected LC3A. Total protein expression of LC3A, LC3B, LC3C, GABARAP and GABARAP-L1 following LC3B decapacitation was unaltered. Our data indicate that nuclear trapping and other causes of LC3B functional loss in the cytosol are buffered by LC3A and actively compensated by LC3C, but not by GABARAPs. The biological relevance of the potential functional compensation of LC3B decapacitation by LC3C and LC3A warrants further study.
... In ventricular cardiomyocytes, Roberts et al. (2014) have shown that glucose deprivation, through hexokinase-II (that catalyzes glycolysis) binding mTORC1, upregulates autophagy (Roberts et al., 2014). Recently, in glucose-starved fibroblasts, metabolomics analysis also demonstrates a downregulation of gluconeogenesis, pentose phosphate route, glycolysis, nucleotide sugars metabolism together with autophagy, correlating these catabolic pathways in mammalian cell (Weckmann et al., 2018). Besides, glucose starvation could also reprogram cell metabolism to a mitochondrial fatty acid oxidation and, in this condition, it could mobilizes lipid droplets and autophagosomes to deliver fatty acids and sustain mitochondria network in stress (Rambold et al., 2015). ...
Trypanosoma cruzi causes Chagas disease, a neglected illness that affects millions of people worldwide, especially in Latin America. The balance between biochemical pathways triggered by the parasite and host cells response will ultimately define the progression of a life-threatening disease, justifying the efforts to understand cellular mechanisms for infection restrain. In this interaction, parasite and host cells are affected by different physiological responses as autophagy modulation, which could be under intense cellular stress, such as nutrient deprivation, hormone depletion, or infection. Autophagy is a constitutive pathway that leads to degradation of macromolecules and cellular structures and may induce cell death. In Trypanosoma cruzi infection, the relevance of host autophagy is controversial regarding in vitro parasite intracellular life cycle. In the present study, we evaluated host cell autophagy during T. cruzi infection in phagocytic and non-professional phagocytic cells. We described that the presence of the parasite increased the number of LC3 puncta, a marker for autophagy, in cardiac cells and peritoneal macrophages in vitro. The induction of host autophagy decreased infection in macrophages in early and late time-periods. We suggest that starved phagocytic cells reduced internalization, also confirmed by inert particles and dead trypomastigotes. Whereas, in cardiac cells, starvation-induced autophagy decreased lipid droplets and infection in later time-point, by reducing parasite differentiation/proliferation. In ATG5 knockout MEF cells, we confirmed our hypothesis of autophagy machinery activation during parasite internalization, increasing infection. Our data suggest that host autophagy downregulates T. cruzi infection through impairing parasite intracellular life cycle, reducing the infection in primary culture cells.
