Lower Mitochondrial Mass Research Articles

Energy Metabolic Profile in Oral Potentially Malignant Disorders and Oral Squamous Cell Carcinoma: A Preliminary Landscape of Warburg Effect in Oral Cancer.

We hypothesized that cell energy metabolic profiles correlate with normal, dysplastic, and tumor cell/tissue statuses and may be indicators of aggressiveness in oral squamous cell carcinoma (OSCC) cells. The energy-related proteins that were differentially expressed in human OSCC fragments (n = 3) and their adjacent epithelial tissue (TAE) were verified using mass spectrometry (MS). Immunohistochemistry for 4-hydroxynonenal (4-HNE) was performed to evaluate the oxidative stress patterns in OSCC (n = 10), epithelial dysplasia (n = 9), and normal epithelial (n = 4) biopsies. The metabolic energy profile of OSCC aggressiveness was investigated in human OSCC cell lines with different levels of epithelial-mesenchymal transition proteins. The genes associated with the proteins found by MS in this study were analyzed using survival analysis (OS), whereas the genes associated with a poorer prognosis were analyzed using context-specific expression, Gene Ontology (GO) and Cancer Hallmarks for function enrichment analysis. The rationale for all experimental approach was to investigate whether the variation in energy metabolism profile accompanies the different phenotypes (from epithelial to mesenchymal) during the epithelial-mesenchymal transition. All OSCC fragments exhibited an increase in glycolysis-related proteins and a decrease in mitochondrial activity compared to the TAE region (p < 0.05), probably due to the downregulation of pyruvate dehydrogenase and antioxidant proteins. Additionally, the OSCC cell lines with a mesenchymal profile (SCC4, SCC9, and SCC25) had a lower mitochondrial mass and membrane potential and generated lower levels of reactive oxygen and nitrogen species than the TAE region. When we analyzed 4-HNE, the reactive species levels were increased in the epithelial regions of OSCC and potentially malignant lesions. A decrease in the levels of 4-HNE/reactive species was observed in the connective tissue underlying the dysplastic regions and the OSCC invasion zone. Based on this scenario, aggressive OSCC is associated with high glycolytic and oxidative metabolism and low mitochondrial and antioxidant activities, which vary according to the differentiation level of the tumor cells and the stage of carcinogenesis.

Galectin-3-ITGB1 Signaling Mediates Interleukin 10 Production of Hepatic Conventional Natural Killer Cells in Hepatitis B Virus Transgenic Mice and Correlates with Hepatocellular Carcinoma Progression in Patients.

The outcomes of HBV infections are related to complex immune imbalances; however, the precise mechanisms by which HBV induces immune dysfunction are not well understood. HBV transgenic (HBs-Tg) mice were used to investigate intrahepatic NK cells in two distinct subsets: conventional NK (cNK) and liver-resident NK (LrNK) cells during a chronic HBV infection. The cNK cells, but not the LrNK cells, were primarily responsible for the increase in the number of bulk NK cells in the livers of ageing HBs-Tg mice. The hepatic cNK cells showed a stronger ability to produce IL-10, coupled with a higher expression of CD69, TIGIT and PD-L1, and lower NKG2D expression in ageing HBs-Tg mice. A lower mitochondrial mass and membrane potential, and less polarized localization were observed in the hepatic cNK cells compared with the splenic cNK cells in the HBs-Tg mice. The enhanced galectin-3 (Gal-3) secreted from HBsAg+ hepatocytes accounted for the IL-10 production of hepatic cNK cells via ITGB1 signaling. For humans, LGALS3 and ITGB1 expression is positively correlated with IL-10 expression, and negatively correlated with the poor clinical progression of HCC. Gal-3-ITGB1 signaling shapes hepatic cNK cells but not LrNK cells during a chronic HBV infection, which may correlate with HCC progression.

Relationship between serum β-hydroxybutyrate and hepatic fatty acid oxidation in individuals with obesity and NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excess lipid accumulation that can progress to inflammation (nonalcoholic steatohepatitis, NASH), and fibrosis. Serum β-hydroxybutyrate (β-HB), a product of the ketogenic pathway, is commonly used as a surrogate marker for hepatic fatty acid oxidation (FAO). However, it remains uncertain whether this relationship holds true in the context of NAFLD in humans. We compared fasting serum β-HB levels with direct measurement of liver mitochondrial palmitate oxidation in humans stratified based on NAFLD severity (n = 142). Patients were stratified based on NAFLD activity score (NAS): NAS = 0 (no disease), NAS = 1-2 (mild), NAS = 3-4 (moderate), and NAS ≥ 5 (advanced). Moderate and advanced NAFLD is associated with reductions in liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), serum β-HB, but not 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) mRNA, relative to no disease. Worsening liver mitochondrial complete palmitate oxidation corresponded with lower HMGCS2 mRNA but not total (complete + incomplete) palmitate oxidation. Interestingly, we found that liver HMGCS2 mRNA and serum β-HB correlated with liver mitochondrial β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity and CPT1A mRNA. Also, lower mitochondrial mass and markers of mitochondrial turnover positively correlated with lower HMGCS2 in the liver. These data suggest that liver ketogenesis and FAO occur at comparable rates in individuals with NAFLD. Our findings support the utility of serum β-HB to serve as a marker of liver injury and hepatic FAO in the context of NAFLD.NEW & NOTEWORTHY Serum β-hydroxybutyrate (β-HB) is frequently utilized as a surrogate marker for hepatic fatty acid oxidation; however, few studies have investigated this relationship during states of liver disease. We found that the progression of nonalcoholic fatty liver disease (NAFLD) is associated with reductions in circulating β-HB and liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). As well, decreased rates of hepatic fatty acid oxidation correlated with liver HMGCS2 mRNA and serum β-HB. Our work supports serum β-HB as a potential marker for hepatic fatty acid oxidation and liver injury during NAFLD.

Ketogenic Diets and Mitochondrial Function: Benefits for Aging But Not for Athletes.

As humans age, we lose skeletal muscle mass, even in the absence of disease (sarcopenia), increasing the risk of death. Low mitochondrial mass and activity contributes to sarcopenia. It is our hypothesis that a ketogenic diet improves skeletal muscle mitochondrial mass and function when they have declined because of aging or disease, but not in athletes where mitochondrial quality is high.

Zeb1 sustains hematopoietic stem cell functions by suppressing mitofusin-2-mediated mitochondrial fusion

Metabolic status is essential in maintaining normal functions of hematopoietic stem cells (HSCs). However, how the dynamic of the mitochondrion, as a central organelle in metabolism, is molecularly regulated to orchestrate metabolism and HSC stemness remains to be elucidated. Here, we focus on the role of Zeb1, a well-characterized epithelial-to-mesenchymal transition (EMT) inducer which has been demonstrated to confer stem-cell-like characteristics in multiple cancer types in stemness regulation of HSCs. Using a Zeb1-tdTomato reporter mouse model, we find that Zeb1+Lin−Sca-1+c-Kit+ cells (Zeb1+-LSKs) represent a subset of functional long-term HSCs. Zeb1+LSKs exhibit a reduced reactive oxygen species (ROS) level, low mitochondrial mass, low mitochondrial membrane potential (MMP), and particularly small, round fragmented mitochondria. Of note, ectopic expression of Zeb1 leads to a fragmented mitochondrial morphology with a low mitochondrial metabolic status in EML cells. In addition, Zeb1-knockout (Zeb1-KO) LSKs from fetal liver display an exhausted stem-cell activity. Zeb1 deficiency results in elongated and tubulated mitochondria with increased mitochondrial mass, elevated MMP, and higher ROS production. Mechanistically, Zeb1 acts as a transcriptional suppressor on the key mitochondrial-fusion protein Mitofusin-2 (encoded by Mfn2). We highlight an important role of Zeb1 in the regulation of mitochondrial morphology in HSC and the metabolic control of HSC stemness by repressing Mfn2-mediated mitochondrial fusion.

P387: UNRAVELING LEUKEMIC STEM CELLS MITOCHONDRIAL DEPENDENCY IN PEDIATRIC ACUTE MYELOID LEUKEMIA

Background: Relapse still represents an unsolved problem for children with acute myeloid leukemia (AML), therefore more effective therapeutic strategies are needed for a complete blasts eradication. Leukemic stem cells (LSCs) have been proposed as the therapy-resistant reservoir of cells responsible for failure of antiproliferative chemotherapy and disease recurrence in AML due to their self-renewal capacity and quiescence allowing evasion from chemotherapy. Thus, the identification of LSCs-specific vulnerabilities is needed for more efficacious treatments. Aims: We aim to dissect novel LSCs characteristics to unravel mechanisms of chemoresistance and disease recurrence, and characterize biological properties of LSCs that can be targeted to completely eradicate AML. Methods: To study LSCs derived from primary pediatric AML samples, we based on reactive oxygen species (ROS) content since it was shown that functionally defined LSCs have relatively low levels of ROS. We isolated the lowest 20% of cells fluorescing with CellROX probe (ROS low) and the highest 20% (ROS high) by cell sorting, and analyzed cell cycle, engraftment in mice and RNA sequencing to identify deregulated pathways. Results: After isolating ROS low and ROS high cells, we first assessed classical stemness features. By colony-forming assay, we observed that ROS low and ROS high cells have similar colony-forming capacity, but only ROS low cells preserve this capacity after a second replating whereas the ROS high population does not (p=0.002). By BrdU assay, we found that ROS low cells are relatively dormant, being more in G0/G1 cell cycle phase (41.9%) than the ROS high counterpart (7.9%, p=0.001), also confirmed by CFSE staining, revealing that ROS low cells proliferate more slowly (at day 4, 31% undivided cells in ROS low vs 8% in ROS high). According to our previously published data, we confirmed that ROS low cells express higher levels of CDK6-AS1 (RQ=1.38) and CDK6 genes (RQ=2.69; RQ=1 for ROS high) and that, being more quiescent, are characterized by lower levels of mitochondrial membrane potential by TMRE and lower content of mitochondrial ROS, byproducts of mitochondrial activity, by MitoSOX staining. We also verified that ROS low cells have a lower mitochondrial mass by MitoTracker and TOMM20 staining (mitochondria to nucleus ratio 1.18 vs 1.35 for ROS high, p=0.0005), suggesting that mitochondrial mass and functions might be involved in quiescent cells properties. However, engraftment experiments did not reveal different capacity of ROS low or ROS high cells in recreating leukemia in NSG mice, neither in terms of human CD45+ cells in mice peripheral blood (23.3% vs 41.9% respectively, p=0.12) nor bone marrow infiltration (73% vs 84.8%, p=0.44) at 8 weeks post-engraftment, opening for further dissection of the presence of LSCs within ROS high cells. By RNA sequencing, we found that the top 150 differentially expressed genes between ROS low and ROS high cells are involved in focal adhesion (logP=-13.51), regulation of hemopoiesis (logP=-12.31), cell response to stress (logP=-8.73) and cytokine production (logP=-8.67). Further analyses will confirm the role of these pathways in maintaining ROS low LSCs. Moreover, we identified 5 differentially expressed surface antigens that are under investigation as putative ROS low LSCs markers. Summary/Conclusion: In conclusion, the ROS low strategy permits to identify a quiescent AML subpopulation with peculiar mitochondrial features and transcriptome allowing to pinpoint novel molecular pathways that might be targeted to enhance LSCs clearance.

Deciphering the Heterogeneity of Mitochondrial Functions During Hematopoietic Lineage Differentiation.

The heterogeneity of mitochondrial function is an important feature of hematopoietic cell lineage differentiation, but its stage wise contribution is not adequately studied. To establish a model to compare the lineage differentiation of hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and differentiated blood cells, the mitochondrial mass (MM), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitophagy level were analyzed. HSCs had lower mitochondrial metabolic activity than committed progenitor populations, indicated by lower MM, MMP, and ROS and higher mitophagy. HPC1s shared more stem cell characteristics than HPC2s and committed progenitor populations in terms of mitochondrial number and function. The mitochondrial metabolism of mature blood cells had greater heterogeneity than hematopoietic stem and progenitor cells, with granulocytes being similar to monocytes. Moreover, HSCs exhibited heterogeneity in the selection of mitophagy-related PINK1/PARK2, BNIP3/NIX, and FUNDC1 pathways. Myeloid differentiation had greater morphological and functional heterogeneity of hematopoietic cells than lymphoid differentiation. Additionally, leukemia stem cells had higher aerobic metabolism and better stem cell function through elevated mitophagy than normal hematopoietic cells. ROS and MMP levels in differentiated leukemia cells were higher, but the level of mitophagy was lower than in differentiated hematopoietic cells. This study provides a complete set of methods and basic reference values for the systematic study of the mitochondrial metabolic function of different types of hematopoietic cells under physiological and pathological conditions. The findings contribute to the future research of tumor and aging based on mitochondrial metabolism.

Chronic Manganese Exposure and the Enteric Nervous System: An in Vitro and Mouse in Vivo Study.

Background:Chronic environmental exposure to manganese (Mn) can cause debilitating damage to the central nervous system. However, its potential toxic effects on the enteric nervous system (ENS) have yet to be assessed.Objective:We examined the effect of Mn on the ENS using both cell and animal models.Method:Rat enteric glial cells (EGCs) and mouse primary enteric cultures were exposed to increasing concentrations of Mn and cell viability and mitochondrial health were assessed using various morphological and functional assays. C57BL/6 mice were exposed daily to a sublethal dose of Mn () for 30 d. Gut peristalsis, enteric inflammation, gut microbiome profile, and fecal metabolite composition were assessed at the end of exposure.Results:EGC mitochondria were highly susceptible to Mn neurotoxicity, as evidenced by lower mitochondrial mass, adenosine triphosphate–linked respiration, and aconitase activity as well as higher mitochondrial superoxide, upon Mn exposure. Minor differences were seen in the mouse model: specifically, longer intestinal transit times and higher levels of colonic inflammation.Conclusion:Based on our findings from this study, Mn preferentially induced mitochondrial dysfunction in a rat EGC line and in vivo resulted in inflammation in the ENS. https://doi.org/10.1289/EHP7877

Connecting the dots between mitochondrial dysfunction and Parkinson's disorder: focus mitochondria-targeting therapeutic paradigm in mitigating the disease severity.

Mitochondria are unique cell organelles, which exhibit multifactorialroles in numerous cell physiological processes, significantly preserving the integrity of neural synaptic interconnections, mediating ATP production, and regulating apoptotic signaling pathways and calcium homeostasis. Multiple neurological disorders occur as a consequence of impaired mitochondrial functioning, with greater sensitivity of dopaminergic (DA) neurons to mitochondrial dysfunction, due to oxidative nature and low mitochondrial mass, thus supporting the contribution of mitochondrial impairment in Parkinson's disorder (neuronal damage due to curbed dopamine levels). The pathophysiology of the second most common disorder, PD, is potentiated by various mitochondrial homeostasis regulating genes, as discussed in the review. The PD symptoms are known to be aggravated by multiple mitochondria-linked alterations, like reactive oxygen species (ROS) production, Ca2+ buffering, imbalanced mitochondrial dynamics (fission, fusion, mitophagy), biogenetic dysfunctions, disrupted mitochondrial membrane potential (MMP), protein aggregation, neurotoxins, and genetic mutations, which manifest the central involvement of unhealthy mitochondria in neurodegeneration, resulting in retarded DA neurons in region of substantia nigra pars compacta (SNpc), causing PD. Furthermore, the review tends to target altered mitochondrial components, like oxidative stress, inflammation, biogenetic alterations, impaired dynamics, uncontrolled homeostasis, and genetic mutations, to provide a sustainable and reliable alternative in PD therapeutics and to overcome the pitfalls of conventional therapeutic agents. Therefore, the authors elaborate the relationship between PD pathogenesis and mitochondrial dysfunctions, followed by a suitable mitochondria-targeting therapeutic portfolio, as well as future considerations, aiding the researchers to investigate novel strategies to mitigate the severity of the disease.

Mitochondrial state determines functionally divergent stem cell population in planaria.

SummaryMitochondrial state changes were shown to be critical for stem cell function. However, variation in the mitochondrial content in stem cells and the implication, if any, on differentiation is poorly understood. Here, using cellular and molecular studies, we show that the planarian pluripotent stem cells (PSCs) have low mitochondrial mass compared with their progenitors. Transplantation experiments provided functional validation that neoblasts with low mitochondrial mass are the true PSCs. Further, the mitochondrial mass correlated with OxPhos and inhibiting the transition to OxPhos dependent metabolism in cultured cells resulted in higher PSCs. In summary, we show that low mitochondrial mass is a hallmark of PSCs in planaria and provide a mechanism to isolate live, functionally active, PSCs from different cell cycle stages (G0/G1 and S, G2/M). Our study demonstrates that the change in mitochondrial metabolism, a feature of PSCs is conserved in planaria and highlights its role in organismal regeneration.

Sepsis is associated with mitochondrial DNA damage and a reduced mitochondrial mass in the kidney of patients with sepsis-AKI

BackgroundSepsis is a life-threatening condition accompanied by organ dysfunction subsequent to a dysregulated host response to infection. Up to 60% of patients with sepsis develop acute kidney injury (AKI), which is associated with a poor clinical outcome. The pathophysiology of sepsis-associated AKI (sepsis-AKI) remains incompletely understood, but mitochondria have emerged as key players in the pathogenesis. Therefore, our aim was to identify mitochondrial damage in patients with sepsis-AKI.MethodsWe conducted a clinical laboratory study using “warm” postmortem biopsies from sepsis-associated AKI patients from a university teaching hospital. Biopsies were taken from adult patients (n = 14) who died of sepsis with AKI at the intensive care unit (ICU) and control patients (n = 12) undergoing tumor nephrectomy. To define the mechanisms of the mitochondrial contribution to the pathogenesis of sepsis-AKI, we explored mRNA and DNA expression of mitochondrial quality mechanism pathways, DNA oxidation and mitochondrial DNA (mtDNA) integrity in renal biopsies from sepsis-AKI patients and control subjects. Next, we induced human umbilical vein endothelial cells (HUVECs) with lipopolysaccharide (LPS) for 48 h to mimic sepsis and validate our results in vitro.ResultsCompared to control subjects, sepsis-AKI patients had upregulated mRNA expression of oxidative damage markers, excess mitochondrial DNA damage and lower mitochondrial mass. Sepsis-AKI patients had lower mRNA expression of mitochondrial quality markers TFAM, PINK1 and PARKIN, but not of MFN2 and DRP1. Oxidative DNA damage was present in the cytosol of tubular epithelial cells in the kidney of sepsis-AKI patients, whereas it was almost absent in biopsies from control subjects. Oxidative DNA damage co-localized with both the nuclei and mitochondria. Accordingly, HUVECs induced with LPS for 48 h showed an increased mnSOD expression, a decreased TFAM expression and higher mtDNA damage levels.ConclusionSepsis-AKI induces mitochondrial DNA damage in the human kidney, without upregulation of mitochondrial quality control mechanisms, which likely resulted in a reduction in mitochondrial mass.

Abstract PO-025: Mitochondrial elongation through Drp1 deletion increases CD8 T cell oxidative phosphorylation and memory formation

Abstract Despite the positive effects of tumor infiltrating lymphocytes (TIL) seen in patients with melanoma and other tumor types, TIL often fail to completely control disease progression even with immune responses enhanced by checkpoint blockade or other immunotherapy. These TIL exhibit characteristics of exhaustion such as poor proliferation, poor effector function, and an increase in inhibitory receptor expression. By flow cytometry, we find that TIL have lower mitochondrial mass as well as lower mitochondrial membrane potential when compared to functional effector T-cells. During the activation and differentiation of T cells, the metabolic demands fluctuate between the necessity for glycolysis or oxidative phosphorylation (OXPHOS), which is supported by shifting of the mitochondrial network state. Using mice lacking Drp1, a mitochondrial fission protein, in T cells as a model of elongated mitochondria, we see increased spare respiratory capacity and OXPHOS as compared to WT T cells with fission capability. We find that these mice are more likely to generate memory precursor CD8 T cells as represented by KLRG1 lo and CD127 hi during a primary response to anti-CD40, PolyI:C, and ovalbumin protein. As indicated by the increase in memory precursors, we find that Drp1 −/− T cells form more memory than WT animals after a 28-day rest period. At this time, mice were challenged with an adenovirus expressing ovalbumin to elicit a recall response. This recall response of CD8 T cells is greater in animals lacking Drp1 in the T cells. In addition to the increase in T cell numbers, we anticipate an increase in the metabolic and possibly cytokine function of these cells, which is still under investigation. We aim to exploit mitochondrial dynamics to enforce elongation of the mitochondrial network of TIL in order to improve TIL functions and have begun investigations into mitochondrial elongation in T cells in mice bearing B16OVA melanoma tumors. Citation Format: Marissa A. Gonzales, Timothy NJ Bullock. Mitochondrial elongation through Drp1 deletion increases CD8 T cell oxidative phosphorylation and memory formation [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-025.

Mitochondrial Potentiation Ameliorates Age-Related Heterogeneity in Hematopoietic Stem Cell Function.

Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs invivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function.

The feasibility of muscle mitochondrial respiratory chain phenotyping across the cognitive spectrum in Parkinson's disease.

There has been little work on the relationship between sarcopenia, a progressive skeletal muscle disorder, and age-related neurodegenerative diseases such as Parkinson's disease (PD). We aimed to determine: 1) the feasibility of characterizing skeletal muscle across a range of cognitive function in PD; 2) if muscle mitochondrial respiratory chain (MRC) function and content are preserved in older adults with PD. Sarcopenia was defined using handgrip strength, chair rise and bioimpedance analysis. MRC function was assessed using phosphorous magnetic resonance spectroscopy (MRS) by estimating τ1/2 PCr (s) (phosphocreatine half-time recovery) in the calf muscles following a bout of aerobic exercise. Biopsy of the vastus lateralis muscle was performed, and MRC content assessed by fluorescent immunohistochemistry for porin and components of MRC Complexes I and IV. Nine participants (78% male; mean age 79.9; PD duration 3.3years) were recruited. Four had cognitive impairment. Six participants had probable sarcopenia. Eight participants completed MRS and had mean (SD) τ1/2 PCr of 37.8 (7.6) seconds, suggesting preserved mitochondrial function. Muscle biopsies were obtained in all and the procedure was well tolerated. Porin Z-score, a proxy for mitochondrial mass, was lower than expected compared to controls (0-89% of fibres with low porin). There was a small amount of Complex I (0.16-4.59%) and Complex IV (0-3.79%) deficiency. Detailed phenotyping, muscle biopsy and imaging was feasible and acceptable across a spectrum of cognitive function in PD. Sarcopenia was relatively common and may be associated with lower mitochondrial mass and low levels of MRC deficiency.

Dysregulation of glycerophospholipid metabolism during Behçet’s disease contributes to a pro-inflammatory phenotype of circulating monocytes

Behçet’s disease (BD) is a relapsing, multisystem and inflammatory condition characterized by systemic vasculitis of small and large vessels. Although the etiopathogenesis of BD remains unknown, immune-mediated mechanisms play a major role in the development of the disease. BD patients present leukocyte infiltration in the mucocutaneous lesions as well as neutrophil hyperactivation. In contrast to neutrophils, whose involvement in the pathogenesis of BD has been extensively studied, the biology of monocytes during BD is less well known. In this study, we analyzed the phenotype and function of circulating monocytes of 38 BD patients from Hospital of Braga. In addition, we evaluated the impact of inflammatory and metabolomic plasma environment on monocyte biology. We observed a worsening of mitochondrial function, with lower mitochondrial mass and increased ROS production, on circulating monocytes of BD patients. Incubation of monocytes from healthy donors with the plasma of BD patients mimicked the observed phenotype, strongly suggesting the involvement of serum mediators. BD patients, regardless of their symptoms, had higher serum pro-inflammatory TNF-α and IP-10 levels and IL-1β/IL-1RA ratio. Untargeted metabolomic analysis identified a dysregulation of glycerophospholipid metabolism on BD patients, where a significant reduction of phospholipids was observed concomitantly with an increase of lysophospholipids and fatty acids. These observations converged to an enhanced phospholipase A2 (PLA2) activation. Indeed, inhibition of PLA2 with dexamethasone or the downstream cyclooxygenase (COX) enzyme with ibuprofen was able to significantly revert the mitochondrial dysfunction observed on monocytes of BD patients. Our results show that the plasma inflammatory environment coupled with a dysregulation of glycerophospholipid metabolism in BD patients contribute to a dysfunction of circulating monocytes.

The effect of maternal high-fat/high-sugar diet on offspring oocytes and early embryo development.

Observational human data and several lines of animal experimental data indicate that maternal obesity impairs offspring health. Here, we comprehensively tested the model that maternal obesity causes defects in the next three generations of oocytes and embryos. We exposed female F0 mice to a high-fat/high-sugar (HF/HS) diet for 6weeks before conception until weaning. Sires, F1 offspring and all subsequent generations were fed control chow diet. Oocytes from F1, F2 and F3 offspring of obese mothers had lower mitochondrial mass and less ATP and citrate than oocytes from offspring of control mothers. F0 blastocysts from HF/HS-exposed mice, but not F1 and F2 blastocysts, had lower mitochondrial mass and membrane potential, less citrate and ATP and smaller total cell number than F0 blastocysts from control mothers. Finally, supplementation of IVF media with the anti-oxidant mito-esculetin partially prevented the oocyte mitochondrial effects caused by maternal HF/HS diet. Our results support the idea that maternal obesity impairs offspring oocyte quality and suggest that antioxidant supplementation should be tested as a means to improve IVF outcomes for obese women.

High mitochondrial mass is associated with reconstitution capacity and quiescence of hematopoietic stem cells

Key Points HSCs can be separated based on high or low mitochondrial mass. Higher mitochondrial mass is associated with quiescence and greater reconstitution capacity of HSCs.

Low VDAC1 Expression Is Associated with an Aggressive Phenotype and Reduced Overall Patient Survival in Cholangiocellular Carcinoma.

Cancer cells frequently exhibit dysfunctional oxidative phosphorylation (OXPHOS) and a concomitant increase in glycolytic flux. We investigated the expression of OXPHOS complex subunits and mitochondrial mass in 34 human cholangiocellular carcinomas (CCCs) and adjacent normal tissue by using tissue microarrays. In the tumor periphery, all OXPHOS complexes were reduced except complex I. In addition, significantly lower levels of complex IV were found at the tumor center (p < 0.0001). Mitochondrial mass, as indicated by VDAC1 expression, was significantly increased in CCCs compared to corresponding normal tissue (p < 0.0001). VDAC1 levels were inversely correlated with UICC (Union Internationale Contre le Cancer) cancer stage classification (p = 0.0065). Furthermore, significantly lower VDAC1 was present in patients with lymph node involvement (p = 0.02). Consistent with this, patients whose carcinomas expressed VDAC1 at low to moderate levels had significantly reduced survival compared to high expressors (p < 0.05). Therefore, low mitochondrial mass is associated with more aggressive CCC. These metabolic features are indicative of a Warburg phenotype in CCCs. This metabolic signature has potential therapeutic implications because tumors with low mitochondrial function may be targeted by metabolic therapies such as a high-fat, low-carbohydrate ketogenic diet.

Modulating T cell function through elongation of the mitochondrial network to improve cytotoxic CD8+ tumor infiltrating lymphocyte function

Abstract For a number of tumor types, including melanoma, positive clinical outcomes correlate with infiltration of T-cells into the tumor. However, failure to completely control disease progression often occurs, even with immune responses enhanced by checkpoint blockade or other immunotherapy. When T-cells fail to control tumor growth, the tumor infiltrating T lymphocytes (TIL) exhibit characteristics of exhaustion such as poor proliferation, poor effector function, and an increase in inhibitory receptor expression. Metabolic activity of T-cells contributes to differentiation and effector function, leading us to investigate the metabolic state of TIL. By flow cytometry, we find that CD8+ cytotoxic TIL have lower mitochondrial mass, as well as lower mitochondrial membrane potential, compared to effector CD8+ T-cells that respond to vaccination. We hypothesize that TIL are unable to effectively regulate their mitochondrial network, and it is likely that one or more fusion or fission machinery proteins has altered expression or can be manipulated to improve TIL function. We find that TIL have punctate mitochondria as compared to functional effector cells and hypothesize that elongation of the mitochondrial network will improve TIL function such as increasing the capacity for OXPHOS, which is seen in memory T cells with elongated mitochondrial networks. Using mice lacking the fission protein, Drp1, in T cells as a model of elongated mitochondria, we see increased mitochondrial membrane potential, spare respiratory capacity and OXPHOS as compared to WT cells with fission capability. We continue to investigate the effects of mitochondrial elongation of CD8+ cytotoxic TIL.

Dye-Independent Methods Reveal Elevated Mitochondrial Mass in Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) produce most cellular energy through glycolysis rather than through mitochondrial respiration. Consistent with this notion, mitochondrial mass has been reported to be low in HSCs. However, we found that staining with MitoTracker Green, a commonly used dye to measure mitochondrial content, leads to artefactually low fluorescence specifically in HSCs because of dye efflux. Using mtDNA quantification, enumeration of mitochondrial nucleoids, and fluorescence intensity of a genetically encoded mitochondrial reporter, we unequivocally show here that HSCs and multipotential progenitors (MPPs) have higher mitochondrial mass than lineage-committed progenitors and mature cells. Despite similar mitochondrial mass, respiratory capacity of MPPs exceeds that of HSCs. Furthermore, although elevated mitophagy has been invoked to explain low mitochondrial mass in HSCs, we observed that mitochondrial turnover capacity is comparatively low in HSCs. We propose that the role of mitochondria in HSC biology may have to be revisited in light of these findings.