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Journal of Animal Science Aug 2022Beta-adrenergic agonists (β-AAs) are widely used supplements in beef and pork production to improve feed efficiency and increase lean muscle mass, yet little is known...
Beta-adrenergic agonists (β-AAs) are widely used supplements in beef and pork production to improve feed efficiency and increase lean muscle mass, yet little is known about the molecular mechanism by which β-AAs achieve this outcome. Our objective was to identify the influence of ractopamine HCl and zilpaterol HCl on mitochondrial respiratory activity in muscle satellite cells isolated from crossbred beef steers (N = 5), crossbred barrows (N = 2), Yorkshire-cross gilts (N = 3), and commercial weather lambs (N = 5). Real-time measurements of oxygen consumption rates (OCRs) were recorded using extracellular flux analyses with a Seahorse XFe24 analyzer. After basal OCR measurements were recorded, zilpaterol HCl, ractopamine HCl, or no β-AA was injected into the assay plate in three technical replicates for each cell isolate. Then, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, and rotenone were injected into the assay plate sequentially, each inducing a different cellular state. This allowed for the measurement of OCR at these states and for the calculation of the following measures of mitochondrial function: basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, adenosine triphosphate (ATP)-linked respiration, and spare respiratory capacity. Incubation of bovine cells with either zilpaterol HCl or ractopamine HCl increased maximal respiration (P = 0.046) and spare respiratory capacity (P = 0.035) compared with non-supplemented counterparts. No difference (P > 0.05) was observed between zilpaterol HCl and ractopamine HCl for maximal respiration and spare respiratory capacity in bovine cell isolates. No measures of mitochondrial function (basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, ATP-linked respiration, and spare respiratory capacity) were altered by β-AA treatment in ovine or porcine cells. These findings indicate that β-AAs in cattle may improve the efficiency of oxidative metabolism in muscle satellite cells by modifying mitochondrial respiratory activity. The lack of response by ovine and porcine cells to β-AA incubation also demonstrates differing physiological responses to β-AA across species, which helps to explain the variation in its effectiveness as a growth supplement.
Topics: Adenosine Triphosphate; Adrenergic beta-Agonists; Animals; Cattle; Female; Myoblasts; Oxidative Phosphorylation; Phenethylamines; Protons; Sheep; Sheep, Domestic; Swine
PubMed: 35908785
DOI: 10.1093/jas/skac208 -
Antibiotics (Basel, Switzerland) Jul 2022PAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from . Its mutation...
PAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from . Its mutation drastically abates filipin, but also oligomycin production, a macrolide ATP-synthase inhibitor, and delays sporulation; thus, it has been considered a transcriptional activator. Transcriptomic analyses were performed in D and its parental strain. Both strains were grown in a YEME medium without sucrose, and the samples were taken at exponential and stationary growth phases. A total of 257 genes showed an altered expression in the mutant, most of them at the exponential growth phase. Surprisingly, despite PteF being considered an activator, most of the genes affected showed overexpression, thereby suggesting a negative modulation. The affected genes were related to various metabolic processes, including genetic information processing; DNA, energy, carbohydrate, and lipid metabolism; morphological differentiation; and transcriptional regulation, among others, but were particularly related to secondary metabolite biosynthesis. Notably, 10 secondary metabolite gene clusters out of the 38 encoded by the genome showed altered expression profiles in the mutant, suggesting a regulatory role for PteF that is wider than expected. The transcriptomic results were validated by quantitative reverse-transcription polymerase chain reaction. These findings provide important clues to understanding the intertwined regulatory machinery that modulates antibiotic biosynthesis in .
PubMed: 35892384
DOI: 10.3390/antibiotics11080994 -
Biochimica Et Biophysica Acta.... Oct 2022Most macrophages generate energy to mount an inflammatory cytokine response by increased glucose metabolism through intracellular glycolysis. Previous studies have...
Most macrophages generate energy to mount an inflammatory cytokine response by increased glucose metabolism through intracellular glycolysis. Previous studies have suggested that alveolar macrophages (AMs), which reside in a glucose-poor natural environment, are less capable to utilize glycolysis and instead rely on other substrates to fuel oxidative phosphorylation (OXPHOS) for energy supply. At present, it is not known whether AMs are capable to use glucose metabolism to produce cytokines when other metabolic options are blocked. Here, we studied human AMs retrieved by bronchoalveolar lavage from healthy subjects, and examined their glucose metabolism in response to activation by the gram-negative bacterial component lipopolysaccharide (LPS) ex vivo. The immunological and metabolic responses of AMs were compared to those of cultured blood monocyte-derived macrophages (MDMs) from the same subjects. LPS stimulation enhanced cytokine release by both AMs and MDMs, which was associated with increased lactate release by MDMs (reflecting glycolysis), but not by AMs. In agreement, LPS induced higher mRNA expression of multiple glycolytic regulators in MDMs, but not in AMs. Flux analyses of [C]-glucose revealed no differences in [C]-incorporation in glucose metabolism intermediates in AMs. Inhibition of OXPHOS by oligomycin strongly reduced LPS-induced cytokine production by AMs, but not by MDMs. Collectively, these results indicate that human AMs, in contrast to MDMs, do not use glucose metabolism during LPS-induced activation and fully rely on OXPHOS for cytokine production.
Topics: Cytokines; Glucose; Humans; Lipopolysaccharides; Macrophages; Macrophages, Alveolar
PubMed: 35835414
DOI: 10.1016/j.bbadis.2022.166488 -
International Journal of Molecular... Jun 2022The presence of liver cancer stem cells (LCSCs) is one of the reasons for the treatment failure of hepatocellular carcinoma (HCC). For LCSCs, one of their prominent...
The presence of liver cancer stem cells (LCSCs) is one of the reasons for the treatment failure of hepatocellular carcinoma (HCC). For LCSCs, one of their prominent features is metabolism plasticity, which depends on transporters and ion channels to exchange metabolites and ions. The K channel protein (Potassium Calcium-Activated Channel Subfamily N Member 4) has been reported to promote cell metabolism and malignant progression of HCCs, but its influence on LCSC stemness has remained unclear. Here, we demonstrated that was highly expressed in L-CSCs by RT-PCR and Western blot. Then, we illustrated that promoted the stemness of HC-C cells by CD133CD44 LCSC subpopulation ratio analysis, in vitro stemness transcription factor detection, and sphere formation assay, as well as in vivo orthotopic liver tumor formation and limiting dilution tumorigenesis assays. We also showed that enhanced the glucose metabolism in LCSCs by metabolic enzyme detections and seahorse analysis, and the -promoted increase in LCSC ratios was abolished by glycolysis inhibitor 2-DG or OXPHOS inhibitor oligomycin. Collectively, our results suggested that promoted LCSC stemness via enhancing glucose metabolism, and that would be a potential molecular target for eliminating LCSCs in HCC.
Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Glucose; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Liver Neoplasms; Neoplastic Stem Cells
PubMed: 35805963
DOI: 10.3390/ijms23136958 -
Biochemistry and Biophysics Reports Sep 2022Acute lymphoblastic leukemia (ALL) is still incurable hematologic neoplasia in an important percentage of patients. Therefore, new therapeutic approaches need to be...
BACKGROUND
Acute lymphoblastic leukemia (ALL) is still incurable hematologic neoplasia in an important percentage of patients. Therefore, new therapeutic approaches need to be developed.
METHODS
To evaluate the cellular effect of cell-penetrating peptides C-PP on leukemia cells, Jurkat cells -a model of ALL were exposed to increasing concentration (50-500 μM) Aβ, R-G-Aβ and Aβ-G-R peptide for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry (FC), and fluorescent microscopy (FM) analysis were used to assess metabolic viability, cell cycle and proliferation, mitochondria functionality, oxidative stress, and cell death markers.
RESULTS
We report for the first time that the R-G-Aβ, but not Aβ peptide, induced selective cell death in Jurkat cells more efficiently than the Aβ-G-R peptide. Indeed, R-G-Aβ (200 μM) altered the metabolic activity (-25%), arrested the cell cycle in the G2/M-phase (15%), and induced a significant reduction of cellular proliferation (i.e., -74% reduction of Ki-67 nuclei reactivity). Moreover, R-G-Aβ induced the dissipation of mitochondrial membrane potential (ΔΨ 51%) and produced an important amount of reactive oxygen species (ROS, 75% at 8 h) in Jurkat cells. The exposure of cells to antioxidant/cytoprotectant N-acetylcysteine (NAC) did not prevent R-G-Aβ from a loss of ΔΨ in Jurkat cells. The peptide was also unable to activate the executer CASPASE-3, thereby preserving the integrity of the cellular DNA corroborated by the fact that the caspase-3 inhibitor NSCI was unable to protect cells from R-G-Aβ -induced cell damage. Further analysis showed that the R-G-Aβ peptide is specifically localized at the outer mitochondria membrane (OMM) according to colocalization with the protein translocase TOMM20. Additionally, the cytotoxic effect of the poly-R peptide resembles the toxic action of the uncoupler FCCP, mitocan oligomycin, and rotenone in Jurkat cells. Importantly, the R-G-Aβ peptide was innocuous to menstrual mesenchymal stromal cells (MenSC) -normal non-leukemia proliferative cells.
CONCLUSION
Our findings demonstrated that the cationic Aβ peptide possesses specific anti-leukemia activity against Jurkat cells through oxidative stress (OS)- and CASPASE-3-independent mechanism but fast mitochondria depolarization.
PubMed: 35755270
DOI: 10.1016/j.bbrep.2022.101300 -
Journal of Lipid and Atherosclerosis May 2022Impaired cardiac efficiency is a hallmark of diabetic cardiomyopathy in models of type 2 diabetes. Adiponectin receptor 1 (AdipoR1) deficiency impairs cardiac efficiency...
OBJECTIVE
Impaired cardiac efficiency is a hallmark of diabetic cardiomyopathy in models of type 2 diabetes. Adiponectin receptor 1 (AdipoR1) deficiency impairs cardiac efficiency in non-diabetic mice, suggesting that hypoadiponectinemia in type 2 diabetes may contribute to impaired cardiac efficiency due to compromised AdipoR1 signaling. Thus, we investigated whether targeting cardiac adiponectin receptors may improve cardiac function and energetics, and attenuate diabetic cardiomyopathy in type 2 diabetic mice.
METHODS
A non-selective adiponectin receptor agonist, AdipoRon, and vehicle were injected intraperitoneally into Eight-week-old or C57BLKS/J mice for 10 days. Cardiac morphology and function were evaluated by echocardiography and working heart perfusions.
RESULTS
Based on echocardiography, AdipoRon treatment did not alter ejection fraction, left ventricular diameters or left ventricular wall thickness in mice compared to vehicle-treated mice. In isolated working hearts, an impairment in cardiac output and efficiency in mice was not improved by AdipoRon. Mitochondrial respiratory capacity, respiration in the presence of oligomycin, and 4-hydroxynonenal levels were similar among all groups. However, AdipoRon induced a marked shift in the substrate oxidation pattern in mice towards increased reliance on glucose utilization. In parallel, the diabetes-associated increase in serum triglyceride levels in vehicle-treated mice was blunted by AdipoRon treatment, while an increase in myocardial triglycerides in vehicle-treated mice was not altered by AdipoRon treatment.
CONCLUSION
AdipoRon treatment shifts myocardial substrate preference towards increased glucose utilization, likely by decreasing fatty acid delivery to the heart, but was not sufficient to improve cardiac output and efficiency in mice.
PubMed: 35656151
DOI: 10.12997/jla.2022.11.2.161 -
Biochimica Et Biophysica Acta.... Sep 2022Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The FA proteins have functions in genome maintenance and in the cytoplasmic process of...
Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The FA proteins have functions in genome maintenance and in the cytoplasmic process of selective autophagy, beyond their canonical roles of repairing DNA interstrand cross-links. FA core complex proteins FANCC, FANCF, FANCL, FANCA, FANCD2, BRCA1 and BRCA2, which previously had no known direct functions outside the nucleus, have recently been implicated in mitophagy. Although mutations in FANCL account for only a very small number of cases in FA families, it plays a key role in the FA pathophysiology and might drive carcinogenesis. Here, we demonstrate that FANCL protein is present in mitochondria in the control and Oligomycin and Antimycin (OA)-treated cells and its ubiquitin ligase activity is not required for its localization to mitochondria. CRISPR/Cas9-mediated knockout of FANCL in HeLa cells overexpressing parkin results in increased sensitivity to mitochondrial stress and defective clearing of damaged mitochondria upon OA treatment. This defect was reversed by the reintroduction of either wild-type FANCL or FANCL(C307A), a mutant lacking ubiquitin ligase activity. To summarize, FANCL protects from mitochondrial stress and supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner.
Topics: Fanconi Anemia; Fanconi Anemia Complementation Group L Protein; Fanconi Anemia Complementation Group Proteins; HeLa Cells; Humans; Mitophagy; Ubiquitin; Ubiquitin-Protein Ligases
PubMed: 35644338
DOI: 10.1016/j.bbadis.2022.166453 -
Journal of Inherited Metabolic Disease Sep 2022Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet...
Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.
Topics: Brain Diseases; DNA, Complementary; Humans; Leigh Disease; Mitochondria; Mitochondrial Proton-Translocating ATPases; Mutation; Proteins
PubMed: 35621276
DOI: 10.1002/jimd.12526 -
Cell Death and Differentiation Dec 2022Binding of the mitochondrial chaperone TRAP1 to client proteins shapes bioenergetic and proteostatic adaptations of cells, but the panel of TRAP1 clients is only...
Binding of the mitochondrial chaperone TRAP1 to client proteins shapes bioenergetic and proteostatic adaptations of cells, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Electrophysiological measurements indicate that TRAP1 directly inhibits a channel activity of purified F-ATP synthase endowed with the features of the permeability transition pore (PTP) and that it reverses PTP induction by CyPD, antagonizing PTP-dependent mitochondrial depolarization and cell death. Conversely, CyPD outcompetes the TRAP1 inhibitory effect on the channel. Our data identify TRAP1 as an F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.
Topics: Humans; Mitochondrial Permeability Transition Pore; Mitochondrial Membrane Transport Proteins; Mitochondrial Proton-Translocating ATPases; Peptidyl-Prolyl Isomerase F; Mitochondria; Molecular Chaperones; Adenosine Triphosphate; HSP90 Heat-Shock Proteins
PubMed: 35614131
DOI: 10.1038/s41418-022-01020-0 -
Immune Network Apr 2022Dysfunction of mitochondrial metabolism is implicated in cellular injury and cell death. While mitochondrial dysfunction is associated with lung injury by lung...
Dysfunction of mitochondrial metabolism is implicated in cellular injury and cell death. While mitochondrial dysfunction is associated with lung injury by lung inflammation, the mechanism by which the impairment of mitochondrial ATP synthesis regulates necroptosis during acute lung injury (ALI) by lung inflammation is unclear. Here, we showed that the impairment of mitochondrial ATP synthesis induces receptor interacting serine/threonine kinase 3 (RIPK3)-dependent necroptosis during lung injury by lung inflammation. We found that the impairment of mitochondrial ATP synthesis by oligomycin, an inhibitor of ATP synthase, resulted in increased lung injury and RIPK3 levels in lung tissues during lung inflammation by LPS in mice. The elevated RIPK3 and RIPK3 phosphorylation levels by oligomycin resulted in high mixed lineage kinase domain-like (MLKL) phosphorylation, the terminal molecule in necroptotic cell death pathway, in lung epithelial cells during lung inflammation. Moreover, the levels of protein in bronchoalveolar lavage fluid (BALF) were increased by the activation of necroptosis via oligomycin during lung inflammation. Furthermore, the levels of ATP5A, a catalytic subunit of the mitochondrial ATP synthase complex for ATP synthesis, were reduced in lung epithelial cells of lung tissues from patients with acute respiratory distress syndrome (ARDS), the most severe form of ALI. The levels of RIPK3, RIPK3 phosphorylation and MLKL phosphorylation were elevated in lung epithelial cells in patients with ARDS. Our results suggest that the impairment of mitochondrial ATP synthesis induces RIPK3-dependent necroptosis in lung epithelial cells during lung injury by lung inflammation.
PubMed: 35573150
DOI: 10.4110/in.2022.22.e18