Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and ...neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy-
d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.
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► SH-SY5Y cells adopts a more oxidative phenotype on differentiation. ► Differentiated SH-SY5Y cells haves increased cytochrome c oxidase and MnSOD. ► Bioenergetic reserve capacity is increased in the differentiated SH-SY5Y cells. ► Differentiated SH-SY5Y cells are more resistant to oxidative stress.
Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic ...fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression in neurodegeneration, ...diabetes, cancer and cardiovascular disease. A key concept is that the mitochondrion can act as the 'canary in the coal mine' by serving as an early warning of bioenergetic crisis in patient populations. We propose that new clinical tests to monitor changes in bioenergetics in patient populations are needed to take advantage of the early and sensitive ability of bioenergetics to determine severity and progression in complex and multifactorial diseases. With the recent development of high-throughput assays to measure cellular energetic function in the small number of cells that can be isolated from human blood these clinical tests are now feasible. We have shown that the sequential addition of well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic profile to be measured in cells isolated from normal or pathological samples. From these data we propose that a single value-the Bioenergetic Health Index (BHI)-can be calculated to represent the patient's composite mitochondrial profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI could serve as a dynamic index of bioenergetic health and how it can be measured in platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a new biomarker for assessing patient health with both prognostic and diagnostic value.
Understanding metabolic pathways that regulate Th17 development is important to broaden therapeutic options for Th17-mediated autoimmunity. Here, we report a pivotal role of mitochondrial oxidative ...phosphorylation (OXPHOS) for lineage specification toward pathogenic Th17 differentiation. Th17 cells rapidly increase mitochondrial respiration during development, and this is necessary for metabolic reprogramming following T cell activation. Surprisingly, specific inhibition of mitochondrial ATP synthase ablates Th17 pathogenicity in a mouse model of autoimmunity by preventing Th17 pathogenic signature gene expression. Notably, cells activated under OXPHOS-inhibited Th17 conditions preferentially express Foxp3, rather than Th17 genes, and become suppressive Treg cells. Mechanistically, OXPHOS promotes the Th17 pioneer transcription factor, BATF, and facilitates T cell receptor (TCR) and mTOR signaling. Correspondingly, overexpression of BATF rescues Th17 development when ATP synthase activity is restricted. Together, our data reveal a regulatory role of mitochondrial OXPHOS in dictating the fate decision between Th17 and Treg cells by supporting early molecular events necessary for Th17 commitment.
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•CD4 T cells rapidly increase mitochondrial respiration during Th17 differentiation•OXPHOS is essential for Th17 cell pathogenicity in a mouse model of MS•Mitochondrial respiration shapes the Th17 and Treg cell fate decision•OXPHOS facilitates TCR and mTOR signaling, which in turn support BATF induction
Shin et al. report that ATP-linked mitochondrial respiration controls the Th17 and Treg cell fate decision by supporting TCR signaling and Th17-associated molecular events. Inhibition of mitochondrial OXPHOS ablates Th17 pathogenicity in a mouse model of MS and results in generation of functionally suppressive Treg cells under Th17 conditions.
Myofibroblasts participate in physiological wound healing and pathological fibrosis. Myofibroblast differentiation is characterized by the expression of α-smooth muscle actin and extracellular matrix ...proteins and is dependent on metabolic reprogramming. In this study, we explored the role of glutaminolysis and metabolites of TCA in supporting myofibroblast differentiation. Glutaminolysis converts Gln into α-ketoglutarate (α-KG), a critical intermediate in the TCA cycle. Increases in the steady-state concentrations of TCA cycle metabolites including α-KG, succinate, fumarate, malate, and citrate were observed in TGF-β1–differentiated myofibroblasts. The concentration of glutamate was also increased in TGF-β1–differentiated myofibroblasts compared with controls, whereas glutamine levels were decreased, suggesting enhanced glutaminolysis. This was associated with TGF-β1–induced expression of the glutaminase (GLS) isoform, GLS1, which converts Gln into glutamate, at both the mRNA and protein levels. The stimulation of GLS1 expression by TGF-β1 was dependent on both SMAD3 and p38 mitogen-activated protein kinase activation. Depletion of extracellular Gln prevented TGF-β1–induced myofibroblast differentiation. The removal of extracellular Gln postmyofibroblast differentiation decreased the expression of the profibrotic markers fibronectin and hypoxia-inducible factor-1α and reversed TGF-β1–induced metabolic reprogramming. Silencing of GLS1 expression, in the presence of Gln, abrogated TGF-β1–induced expression of profibrotic markers. Treatment of GLS1-deficient myofibroblasts with exogenous glutamate or α-KG restored TGF-β1–induced expression of profibrotic markers in GLS1-deficient myofibroblasts. Together, these data demonstrate that glutaminolysis is a critical component of myofibroblast metabolic reprogramming that regulates myofibroblast differentiation.
Sirt3 (sirtuin 3) is an NAD-dependent deacetylase localized to mitochondria. Sirt3 expression is increased in mouse muscle and liver by starvation, which could protect against the ...starvation-dependent increase in oxidative stress and protein damage. Damaged proteins and organelles depend on autophagy for removal and this is critical for cell survival, but the role of Sirt3 is unclear. To examine this, we used Sirt3-KO (knockout) mouse embryonic fibroblast cells, and found that, under basal conditions, Sirt3-KO cells exhibited increased autophagy flux compared with WT (wild-type) cells. In response to nutrient deprivation, both WT and KO cells exhibited increased basal and ATP-linked mitochondrial respiration, indicating an increased energy demand. Both cells exhibited lower levels of phosphorylated mTOR (mammalian target of rapamycin) and higher autophagy flux, with KO cells exhibiting lower maximal mitochondrial respiration and reserve capacity, and higher levels of autophagy than WT cells. KO cells exhibit higher phospho-JNK (c-Jun N-terminal kinase) and phospho-c-Jun than WT cells under starvation conditions. However, inhibition of JNK activity in Sirt3-KO cells did not affect LC3-I (light chain 3-I) and LC3-II levels, indicating that Sirt3-regulated autophagy is independent of the JNK pathway. Caspase 3 activation and cell death are significantly higher in Sirt3-KO cells compared with WT cells in response to nutrient deprivation. Inhibition of autophagy by chloroquine exacerbated cell death in both WT and Sirt3-KO cells, and by 3-methyadenine exacerbated cell death in Sirt3-KO cells. These data suggest that nutrient deprivation-induced autophagy plays a protective role in cell survival, and Sirt3 decreases the requirement for enhanced autophagy and improves cellular bioenergetics.
Autophagy is an important cell recycling program responsible for the clearance of damaged or long-lived proteins and organelles. Pharmacological modulators of this pathway have been extensively ...utilized in a wide range of basic research and pre-clinical studies. Bafilomycin A1 and chloroquine are commonly used compounds that inhibit autophagy by targeting the lysosomes but through distinct mechanisms. Since it is now clear that mitochondrial quality control, particularly in neurons, is dependent on autophagy, it is important to determine whether these compounds modify cellular bioenergetics. To address this, we cultured primary rat cortical neurons from E18 embryos and used the Seahorse XF96 analyzer and a targeted metabolomics approach to measure the effects of bafilomycin A1 and chloroquine on bioenergetics and metabolism. We found that both bafilomycin and chloroquine could significantly increase the autophagosome marker LC3-II and inhibit key parameters of mitochondrial function, and increase mtDNA damage. Furthermore, we observed significant alterations in TCA cycle intermediates, particularly those downstream of citrate synthase and those linked to glutaminolysis. Taken together, these data demonstrate a significant impact of bafilomycin and chloroquine on cellular bioenergetics and metabolism consistent with decreased mitochondrial quality associated with inhibition of autophagy.
•Autophagy inhibition decreased mitochondrial bioenergetics in intact neurons.•Autophagy inhibition decreased mitochondrial complexes I, II or IV substrate linked respiration.•Autophagy inhibition increased mitochondrial DNA damage.•Autophagy inhibition decreased major components of the Krebs cycle.•Autophagy inhibition resulted in decreased citrate synthase activities.
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It is now clear that mitochondria are an important target for oxidative stress in a broad range of pathologies, including cardiovascular disease, diabetes, neurodegeneration, and cancer. Methods for ...assessing the impact of reactive species on isolated mitochondria are well established but constrained by the need for large amounts of material to prepare intact mitochondria for polarographic measurements. With the availability of high-resolution polarography and fluorescence techniques for the measurement of oxygen concentration in solution, measurements of mitochondrial function in intact cells can be made. Recently, the development of extracellular flux methods to monitor changes in oxygen concentration and pH in cultures of adherent cells in multiple-sample wells simultaneously has greatly enhanced the ability to measure bioenergetic function in response to oxidative stress. Here we describe these methods in detail using representative cell types from renal, cardiovascular, nervous, and tumorigenic model systems while illustrating the application of three protocols to analyze the bioenergetic response of cells to oxidative stress.
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► The impact of oxidative stress on bioenergetics is important to assess in a cellular setting. ► Protocols are reported to obtain the bioenergetic response to oxidative stress in adherent cells. ► Oxidative phosphorylation and glycolysis are integrated to make a metabolic profile. ► Reserve capacity is the most critical parameter in cellular bioenergetics.
Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for ...freshly isolated mitochondria hinders the feasibility of respirometry in multi‐site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90–95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.
Synopsis
Freeze‐thawing events cause mitochondrial membrane permeabilization and disrupt mitochondrial functionality in cells and tissues. Reconstitution of maximal mitochondrial respiration allows the analysis of mitochondrial bioenergetics in frozen and thawed crude samples, thus overcoming limitations associated with the current methods.
Following reconstitution, mitochondrial maximal respiration can be assessed in frozen isolated mitochondria or total tissue lysate.
Respiratory rate measurements from tissue lysates reduce by an order of magnitude the minimal mass of tissue required.
Maximal respiration rates in reconstituted frozen samples are comparable to those in fresh ones.
Respiratory rates can be normalized per cell, per total protein or per mitochondrial content in frozen specimens.
Respiratory rates can be measured in stored samples from clinical and animal studies, and drug toxicity assays.
Reconstitution of maximal mitochondrial respiration circumvents the limitations associated with current methods for assessing mitochondrial bioenergetics in frozen clinical samples.
Elevated infiltration of immunosuppressive alternatively polarized (M2) macrophages is associated with poor prognosis in patients with cancer. The tumor microenvironment remarkably orchestrates ...molecular mechanisms that program these macrophages. Here we identify a novel role for oncogenic Hedgehog (Hh) signaling in programming signature metabolic circuitries that regulate alternative polarization of tumor-associated macrophages. Two immunocompetent orthotopic mouse models of mammary tumors were used to test the effect of inhibiting Hh signaling on tumor-associated macrophages. Treatment with the pharmacologic Hh inhibitor vismodegib induced a significant shift in the profile of tumor-infiltrating macrophages. Mass spectrometry-based metabolomic analysis showed Hh inhibition induced significant alterations in metabolic processes, including metabolic sensing, mitochondrial adaptations, and lipid metabolism. In particular, inhibition of Hh in M2 macrophages reduced flux through the UDP-GlcNAc biosynthesis pathway. Consequently, O-GlcNAc-modification of STAT6 decreased, mitigating the immune-suppressive program of M2 macrophages, and the metabolically demanding M2 macrophages shifted their metabolism and bioenergetics from fatty acid oxidation to glycolysis. M2 macrophages enriched from vismodegib-treated mammary tumors showed characteristically decreased O-GlcNAcylation and altered mitochondrial dynamics. These Hh-inhibited macrophages are reminiscent of inflammatory (M1) macrophages, phenotypically characterized by fragmented mitochondria. This is the first report highlighting the relevance of Hh signaling in controlling a complex metabolic network in immune cells. These data describe a novel immunometabolic function of Hh signaling that can be clinically exploited. SIGNIFICANCE: These findings illustrate that Hh activity regulates a metabolic and bioenergetic regulatory program in tumor-associated macrophages that promotes their immune-suppressive polarization.
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