Basal Mitochondrial Respiration Research Articles

Activation of the hypoxia-inducible factor pathway by roxadustat improves glucose metabolism in human primary myotubes from men.

Hypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) enzymes regulate adaptive cellular responses to low oxygen concentrations. Inhibition of HIF-P4Hs leads to stabilisation of hypoxia-inducible factors (HIFs) and activation of the HIF pathway affecting multiple biological processes to rescue cells from hypoxia. As evidence from animal models suggests that HIF-P4H inhibitors could be used to treat metabolic disorders associated with insulin resistance, we examined whether roxadustat, an HIF-P4H inhibitor approved for the treatment of renal anaemia, would have an effect on glucose metabolism in primary human myotubes. Primary skeletal muscle cell cultures, established from biopsies of vastus lateralis muscle from men with normal glucose tolerance (NGT) (n=5) or type 2 diabetes (n=8), were treated with roxadustat. Induction of HIF target gene expression was detected with quantitative real-time PCR. Glucose uptake and glycogen synthesis were investigated with radioactive tracers. Glycolysis and mitochondrial respiration rates were measured with a Seahorse analyser. Exposure to roxadustat stabilised nuclear HIF1α protein expression in human myotubes. Treatment with roxadustat led to induction of HIF target gene mRNAs for GLUT1 (also known as SLC2A1), HK2, MCT4 (also known as SLC16A4) and HIF-P4H-2 (also known as PHD2 or EGLN1) in myotubes from donors with NGT, with a blunted response in myotubes from donors with type 2 diabetes. mRNAs for LDHA, PDK1 and GBE1 were induced to a similar degree in myotubes from donors with NGT or type 2 diabetes. Exposure of myotubes to roxadustat led to a 1.4-fold increase in glycolytic rate in myotubes from men with NGT (p=0.0370) and a 1.7-fold increase in myotubes from donors with type 2 diabetes (p=0.0044), with no difference between the groups (p=0.1391). Exposure to roxadustat led to a reduction in basal mitochondrial respiration in both groups (p<0.01). Basal glucose uptake rates were similar in myotubes from donors with NGT (20.2 ± 2.7 pmol mg-1 min-1) and type 2 diabetes (25.3 ± 4.4 pmol mg-1 min-1, p=0.4205). Treatment with roxadustat enhanced insulin-stimulated glucose uptake in myotubes from donors with NGT (1.4-fold vs insulin-only condition, p=0.0023). The basal rate of glucose incorporation into glycogen was lower in myotubes from donors with NGT (233 ± 12.4 nmol g-1 h-1) than in myotubes from donors with type 2 diabetes (360 ± 40.3 nmol g-1 h-1, p=0.0344). Insulin increased glycogen synthesis by 1.9-fold (p=0.0025) in myotubes from donors with NGT, whereas roxadustat did not affect their basal or insulin-stimulated glycogen synthesis. Insulin increased glycogen synthesis by 1.7-fold (p=0.0031) in myotubes from donors with type 2 diabetes. While basal glycogen synthesis was unaffected by roxadustat, pretreatment with roxadustat enhanced insulin-stimulated glycogen synthesis in myotubes from donors with type 2 diabetes (p=0.0345). Roxadustat increases glycolysis and inhibits mitochondrial respiration in primary human myotubes regardless of diabetes status. Roxadustat may also improve insulin action on glycogen synthesis in myotubes from donors with type 2 diabetes.

Transport activity regulates mitochondrial bioenergetics and biogenesis in renal tubules.

Renal tubules are featured with copious mitochondria and robust transport activity. Mutations in mitochondrial genes cause congenital renal tubulopathies, and changes in transport activity affect mitochondrial morphology, suggesting mitochondrial function and transport activity are tightly coupled. Current methods of using bulk kidney tissues or cultured cells to study mitochondrial bioenergetics are limited. Here, we optimized an extracellular flux analysis (EFA) to study mitochondrial respiration and energy metabolism using microdissected mouse renal tubule segments. EFA detects mitochondrial respiration and glycolysis by measuring oxygen consumption and extracellular acidification rates, respectively. We show that both measurements positively correlate with sample sizes of a few centimeter-length renal tubules. The thick ascending limbs (TALs) and distal convoluted tubules (DCTs) critically utilize glucose/pyruvate as energy substrates, whereas proximal tubules (PTs) are significantly much less so. Acute inhibition of TALs' transport activity by ouabain treatment reduces basal and ATP-linked mitochondrial respiration. Chronic inhibition of transport activity by 2-week furosemide treatment or deletion of with-no-lysine kinase 4 (Wnk4) decreases maximal mitochondrial capacity. In addition, chronic inhibition downregulates mitochondrial DNA mass and mitochondrial length/density in TALs and DCTs. Conversely, gain-of-function Wnk4 mutation increases maximal mitochondrial capacity and mitochondrial length/density without increasing mitochondrial DNA mass. In conclusion, EFA is a sensitive and reliable method to investigate mitochondrial functions in isolated renal tubules. Transport activity tightly regulates mitochondrial bioenergetics and biogenesis to meet the energy demand in renal tubules. The system allows future investigation into whether and how mitochondria contribute to tubular remodeling adapted to changes in transport activity.

An E115A missense variant in CERS2 is associated with increased sleeping energy expenditure and hepatic insulin resistance in American Indians.

Genetic determinants of inter-individual differences in energy expenditure (EE) are largely unknown. Sphingolipids, such as ceramides, have been implicated in the regulation of human EE via mitochondrial uncoupling. In this study, we investigated whether genetic variants within enzymes involved in sphingolipid synthesis and degradation affect EE and insulin-related traits in a cohort of American Indians informative for 24-hour EE and glucose disposal rates during a hyperinsulinemic-euglycemic clamp. Association analysis of 10,084 genetic variants within 28 genes involved in sphingolipid pathways identified a missense variant (rs267738, A>C, E115A) in exon 4 of CERS2 that was associated with higher sleeping EE (+116 kcal/day) and increased rates of endogenous glucose production during basal (+5%) and insulin-stimulated (+43%) conditions, both indicators of hepatic insulin resistance. The rs267738 variant did not affect ceramide synthesis in HepG2 cells but resulted in a 30% decrease in basal mitochondrial respiration. In conclusion, we provide evidence that the CERS2 rs267738 missense variant may influence hepatic glucose production and post-absorptive sleeping metabolic rate.

NLRX1: a key regulator in mitochondrial respiration and colorectal cancer progression.

Colorectal cancer (CRC) is a prevalent and aggressive malignancy with high mortality rates and significant risks to human well-being. Population-wide screening for tumor suppressor genes and oncogenes shows promise for reducing the incidence and fatality of CRC. Recent studies have suggested that NLRX1, an innate immunity suppressor, may play a role in regulating chronic inflammation and tumorigenesis. However, further investigation is needed to understand the specific role of NLRX1 in CRC. To evaluate the impact of NLRX1 on migration, invasion, and metastasis, two human colon cancer cell lines were studied in vitro. Additionally, a knockout mouse tumor-bearing model was used to validate the inhibitory effect of NLRX1 on tumor emergence and progression. The Seahorse XF96 technology was employed to assess mitochondrial function and glycolysis in colorectal cancer cells overexpressing NLRX1. Moreover, public databases were consulted to analyze gene and protein expression levels of NLRX1. Finally, the results were validated using a series of CRC patient samples. Our findings demonstrate that downregulation of NLRX1 enhances proliferation, colony formation, and tumor-forming capacity in HCT116 and LoVo cells. Conversely, overexpression of NLRX1 negatively impacts basal respiration and mitochondrial ATP-linked respiration in both cell lines, resulting in a notable decrease in maximal respiration during the standard mitochondrial stress test. Furthermore, analysis of data from the TCGA database reveals a significant reduction in NLRX1 expression in colon and rectal cancer tissues compared to normal tissues. This result was validated using clinical samples, where immunohistochemistry staining and western blotting demonstrated a notable reduction in NLRX1 protein levels in CRC compared to adjacent normal tissues. The decreased expression of NLRX1 may serve as a significant prognostic indicator and diagnostic biomarker for CRC patients.

Unique features of β-cell metabolism are lost in type 2 diabetes.

Pancreatic β cells play an essential role in the control of systemic glucose homeostasis as they sense blood glucose levels and respond by secreting insulin. Upon stimulating glucose uptake in insulin-sensitive tissues post-prandially, this anabolic hormone restores blood glucose levels to pre-prandial levels. Maintaining physiological glucose levels thus relies on proper β-cell function. To fulfill this highly specialized nutrient sensor role, β cells have evolved a unique genetic program that shapes its distinct cellular metabolism. In this review, the unique genetic and metabolic features of β cells will be outlined, including their alterations in type 2 diabetes (T2D). β cells selectively express a set of genes in a cell type-specific manner; for instance, the glucose activating hexokinase IV enzyme or Glucokinase (GCK), whereas other genes are selectively "disallowed", including lactate dehydrogenase A (LDHA) and monocarboxylate transporter 1 (MCT1). This selective gene program equips β cells with a unique metabolic apparatus to ensure that nutrient metabolism is coupled to appropriate insulin secretion, thereby avoiding hyperglycemia, as well as life-threatening hypoglycemia. Unlike most cell types, β cells exhibit specialized bioenergetic features, including supply-driven rather than demand-driven metabolism and a high basal mitochondrial proton leak respiration. The understanding of these unique genetically programmed metabolic features and their alterations that lead to β-cell dysfunction is crucial for a comprehensive understanding of T2D pathophysiology and the development of innovative therapeutic approaches for T2D patients.

Mitochondrial respiration of a primary mixed culture of neurons from hippocampus at various stages of differentiation

BACKGROUND: Primary mixed culture of neurons is often used to evaluate the molecular mechanisms underlying neurodegenerative disorders. However, isolating cells from the brain is accompanied by profound changes in cell morphology, behavior, and metabolic rate due to enzymatic disintegration and microenvironmental changes in cells. In this regard, the mitochondrial basal respiration of neurons should be considered as an indicator of the formation of functional activity of the cell.&#x0D; AIM: To evaluate mitochondrial respiration in a primary mixed culture of hippocampal neurons at various stages of differentiation.&#x0D; METHODS: This study included mice of line CD-1 and was conducted in two series: the first studied the primary mixed embryonic culture on the 18th day of gestation (E18) and the second used the postnatal culture on the 2nd day after birth (P2). A Seahorse XF HS Mini (Agilent, USA) was used to measure the functional parameters of cell metabolism. The oxygen consumption rate was calculated based on the results of the mitochondrial respiration profile of the primary mixed culture built during its differentiation.&#x0D; RESULTS: On the 5th day of neuronal differentiation in the culture, maximum mitochondrial respiration was established both in the hippocampal culture obtained from embryos on the 18th day of gestation and in the culture taken from mice on the 2nd day after birth. As cells differentiated in culture from days 2 to 11, the substrate oxidation rate increased almost twofold in the culture of hippocampal neurons obtained from embryos, which increased the metabolic potential.&#x0D; CONCLUSION: The study results prove that the hippocampus can be used to study the role of mitochondria in neurogenesis.

Aerobic capacity and mitochondrial function in bipolar disorder: a longitudinal study during acute phases and after clinical remission.

Aerobic capacity has shown to predict physical and mental health-related quality of life in bipolar disorder (BD). However, the correlation between exercise respiratory capacity and mitochondrial function remains understudied. We aimed to assess longitudinally intra-individual differences in these factors during mood episodes and remission in BD. This study included eight BD patients admitted to an acute psychiatric unit. Incremental cardiopulmonary exercise test (CPET) was conducted during acute episodes (T0), followed by constant work rate cycle ergometry (CWRCE) to evaluate endurance time, oxygen uptake at peak exercise (VO2peak) and at the anaerobic threshold. The second test was repeated during remission (T1). Mitochondrial respiration rates were assessed at T0 and T1 in peripheral blood mononuclear cells. Endurance time, VO2peak, and anaerobic threshold oxygen consumption showed no significant variations between T0 and T1. Basal oxygen consumption at T1 tended to inversely correlate with maximal mitochondrial respiratory capacity (r=-0.690, p=0.058), and VO2peak during exercise at T1 inversely correlated with basal and minimum mitochondrial respiration (r=-0.810, p=0.015; r=-0.786, p=0.021, respectively). Our preliminary data showed that lower basal oxygen consumption may be linked to greater mitochondrial respiratory capacity, and maximum oxygen uptake during the exercise task was associated with lower basal mitochondrial respiration, suggesting that lower oxygen requirements could be associated with greater mitochondrial capacity. These findings should be replicated in larger samples stratified for manic and depressive states.

Abstract 259: Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) patients who experience relapse have a dismal prognosis, with 5-year overall survival rates of 10% for adults and 36% for children. Relapse is attributed to cytotoxic chemotherapy’s inability to target Leukemia Initiating Cells (LICs) that have the potential to re-populate the cancer. There is a need for novel therapeutic strategies to target LIC self-renewal in T-ALL with improved safety profiles and better chances of transition to clinical development. This project utilized a transgenic zebrafish model that accurately recapitulates the most aggressive form of the human T-ALL. This model offered several advantages, including a high frequency of LICs and suitability for drug testing. Over 770 FDA-approved drugs were screened, in vivo using &amp;gt;2,500 syngeneic CG1 zebrafish. Further secondary screening in human T-ALL cells narrowed down the potential hits into 4 compounds that specifically interfere with self-renewal. Finally, the hit drug Amiloride was confirmed by a limiting dilation assay (LDA), resulting in a more than 4-fold decrease in the LIC frequency (p=0.0026). The top hit, Amiloride, which reduced the frequency of LICs in zebrafish and human T-ALL cells, is an inhibitor of the Sodium Hydrogen Exachanger-1 (NHE1). NHE1 has been previously linked to the proliferation, migration, and drug resistance of different cancers. In addition, Amiloride treatment was found to impair mitochondrial function in cancer cells, offering a possible mechanism for the inhibition of LICs. Research has not yet investigated how NHE1, and the altered mitochondrial dynamics related to its inhibition affect the development and persistence of LICs in T-ALL. In our human T-ALL cells, we found that Amiloride significantly reduced mitochondrial basal respiration (p=0.0074), ATP production (p=0.0008) and increased the expression of genes associated with mitochondrial stress such as TFAM, PINK1, PARKIN and MT-CO1. Experiments using shRNA mediated knock down of the NHE1 in human T-ALL cells are underway and will be followed by investigating the impact of NHE1 inhibition on primary patient-derived T-ALL cells in a mouse model. Overall, this project showcases of the power of the zebrafish in cancer research in an efficient and accurate large-scale screen for drugs that impact LIC self-renewal. We have identified Amiloride as a top hit that reduces the frequency of LIC in vivo and in vitro. Finally, we are continuing to investigate the role of the NHE1 in self-renewal in human T-ALL and the associated mitochondrial alterations. Citation Format: Majd Al-Hamaly, Yelena Chernyavskaya, Emma Arvin, Logan Turner, Meghan Haney, Jessica Blackburn. Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 259.

Upregulated GIRK2 Counteracts Ethanol-Induced Changes in Excitability and Respiration in Human Neurons.

Genome-wide association studies (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified noncoding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G-protein-coupled inwardly rectifying potassium channel that regulates neuronal excitability. We studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multielectrode arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21 d of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-induced changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons.

Non-lethal sampling for assessment of mitochondrial function does not affect metabolic rate and swimming performance.

A fundamental issue in the metabolic field is whether it is possible to understand underlying mechanisms that characterize individual variation. Whole-animal performance relies on mitochondrial function as it produces energy for cellular processes. However, our lack of longitudinal measures to evaluate how mitochondrial function can change within and among individuals and with environmental context makes it difficult to assess individual variation in mitochondrial traits. The aims of this study were to test the repeatability of muscle mitochondrial metabolism by performing two biopsies of red muscle, and to evaluate the effects of biopsies on whole-animal performance in goldfish Carassius auratus. Our results show that basal mitochondrial respiration and net phosphorylation efficiency are repeatable at 14-day intervals. We also show that swimming performance (optimal cost of transport and critical swimming speed) was repeatable in biopsied fish, whereas the repeatability of individual oxygen consumption (standard and maximal metabolic rates) seemed unstable over time. However, we noted that the means of individual and mitochondrial traits did not change over time in biopsied fish. This study shows that muscle biopsies allow the measurement of mitochondrial metabolism without sacrificing animals and that two muscle biopsies 14 days apart affect the intraspecific variation in fish performance without affecting average performance of individuals. This article is part of the theme issue 'The evolutionary significance of variation in metabolic rates'.

Bile acids promote endothelial cell homeostasis via the regulation of cell metabolism and quiescence

Abstract Background Endothelial cell (EC) homeostasis is characterized by a quiescent and low-metabolic state. Obesity leads to EC dysfunction and CVD. We demonstrated that bariatric surgery improved obesity-induced EC dysfunction and increased fasting and post-prandial plasma levels of bile acids (BAs)(in particular, cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)). BAs are emerging as metabolic signaling molecules in several tissues, but their effect on ECs has not been clearly established yet. Purpose We investigate the effect of BA on the maintenance of EC quiescence. Methods Human Aortic Endothelial Cells (HAECs) were treated for 24h with vehicle (0.1% DMSO), CA, CDCA and DCA (50uM each) in 2%FBS EGM-2 media. Percentage of apoptotic cells was assessed with AnnexinV/PI. Seahorse Mitostress test, Mitoplate and glucose uptake with 0.5µCi/ml of 14C-2-deoxy-D-glucose were used to evaluate changes of HAEC metabolism. Immunostaining for Ki67, Crystal Violet staining and scratch assay were performed to assess HAEC proliferation and migration. NO production and endothelial NO synthase (eNOS) activity were measured via DAF-2 probe and Western Blot, respectively. By qPCR genes involved in Notch pathway were evaluated. Mesoscale vascular injury panel, scratch assay, Ki67 immunostaining and glucose uptake were also performed in HAECs pre-treated with 50uM of BAs and stimulated with 0.5ng/mL TNF-α for 24h. Results HAECs treated with BAs increased NO production compared to vehicle and a reduction in both basal and maximal mitochondrial respiration and extracellular acidification rate was shown by Seahorse Mitostress test. Mitoplate assay displayed an overall reduction in the metabolic rate of HAECs treated with CA and DCA and a peculiar metabolic pattern in cells treated with CDCA. Reduction in glucose uptake was also observed after BA treatment, suggesting that BAs are able to decrease EC metabolism. Immunostaining for Ki67 and Crystal Violet highlighted that BAs reduced cell proliferation. The scratch assay also displayed a reduction in EC migration after BA treatment. BAs also reduced EC apoptosis, as significant differences in the percentage of AnnexinV/PI positive cells was observed in BA-treated cells compared to vehicle. qPCR showed increased expression of genes involved in Notch pathway promoting quiescence after BA treatment. Glucose uptake, scratch assay and Ki67 immunostaining performed in TNF-α stimulated HAECs suggested a BA-dependent endothelial protection in conditions of EC pro-inflammatory activation. We did not observed a reduction in pro-inflammatory biomarkers released in the supernatant of HAECs pre-treated with BA and stimulated with TNF-α. Conclusions Our results suggest that BAs may be involved in the maintenance of EC quiescence by regulating EC metabolism and gene expression profile. These findings highlight a potential role of BAs as endothelial-protective molecules.

Increased platelet mitochondrial function correlates with clot strength in a rodent fracture model.

Thromboelastographic measures of clot strength increase early after injury, portending higher risks for thromboembolic complications during recovery. Understanding the specific role of platelets is challenging because of a lack of clinically relevant measures of platelet function. Platelet mitochondrial respirometry may provide insight to global platelet function but has not yet been correlated with functional coagulation studies. Wistar rats underwent anesthesia and either immediate sacrifice for baseline values (n = 6) or (1) bilateral hindlimb orthopedic injury (n = 12), versus (2) sham anesthesia (n = 12) with terminal phlebotomy/hepatectomy after 24 hours. High-resolution respirometry was used to measure basal respiration, mitochondrial leak, maximal oxidative phosphorylation, and Complex IV activity in intact platelets; Complex I- and Complex II-driven respiration was measured in isolated liver mitochondria. Results were normalized to platelet number and protein mass, respectively. Citrated native thromboelastography (TEG) was performed in triplicate. Citrated native TEG maximal amplitude was significantly higher (81.0 ± 3.0 vs. 73.3 ± 3.5 mm, p < 0.001) in trauma compared with sham rats 24 hours after injury. Intact platelets from injured rats had higher basal oxygen consumption (17.7 ± 2.5 vs. 15.1 ± 3.2 pmol O 2 /[s × 10 8 cells], p = 0.045), with similar trends in mitochondrial leak rate ( p = 0.19) when compared with sham animals. Overall, platelet basal respiration significantly correlated with TEG maximal amplitude ( r = 0.44, p = 0.034). As a control for sex-dependent systemic mitochondrial differences, females displayed higher liver mitochondria Complex I-driven respiration (895.6 ± 123.7 vs. 622.1 ± 48.7 mmol e - /min/mg protein, p = 0.02); as a control for systemic mitochondrial effects of injury, no liver mitochondrial respiration differences were seen. Platelet mitochondrial basal respiration is increased after injury and correlates with clot strength in this rodent hindlimb fracture model. Several mitochondrial-targeted therapeutics exist in common use that are underexplored but hold promise as potential antithrombotic adjuncts that can be sensitively evaluated in this preclinical model.

Brain-derived neurotrophic factor protects neurons by stimulating mitochondrial function through protein kinase A.

Brain-derived neurotrophic factor (BDNF) stimulates dendrite outgrowth and synaptic plasticity by activating downstream protein kinase A (PKA) signaling. Recently, BDNF has been shown to modulate mitochondrial respiration in isolated brain mitochondria, suggesting that BDNF can modulate mitochondrial physiology. However, the molecular mechanisms by which BDNF stimulates mitochondrial function in neurons remain to be elucidated. In this study, we surmised that BDNF binds to the TrkB receptor and translocates to mitochondria to govern mitochondrial physiology in a PKA-dependent manner. Confocal microscopy and biochemical subcellular fractionation assays confirm the localization of the TrkB receptor in mitochondria. The translocation of the TrkB receptor to mitochondria was significantly enhanced upon treating primary cortical neurons with exogenous BDNF, leading to rapid PKA activation. Showing a direct role of BDNF in regulating mitochondrial structure/function, time-lapse confocal microscopy in primary cortical neurons showed that exogenous BDNF enhances mitochondrial fusion, anterograde mitochondrial trafficking, and mitochondrial content within dendrites, which led to increased basal and ATP-linked mitochondrial respiration and glycolysis as assessed by an XF24e metabolic analyzer. BDNF-mediated regulation of mitochondrial structure/function requires PKA activity as treating primary cortical neurons with a pharmacological inhibitor of PKA or transiently expressing constructs that target an inhibitor peptide of PKA (PKI) to the mitochondrion abrogated BDNF-mediated mitochondrial fusion and trafficking. Mechanistically, western/Phos-tag blots show that BDNF stimulates PKA-mediated phosphorylation of Drp1 and Miro-2 to promote mitochondrial fusion and elevate mitochondrial content in dendrites, respectively. Effects of BDNF on mitochondrial function were associated with increased resistance of neurons to oxidative stress and dendrite retraction induced by rotenone. Overall, this study revealed new mechanisms of BDNF-mediated neuroprotection, which entails enhancing mitochondrial health and function of neurons.

P10.36.B ROLE OF AMPK IN GLIOBLASTOMA BIOENERGETICS

Abstract BACKGROUND Glioblastoma is the most prevalent and aggressive primary brain tumor. AMP-activated kinase (AMPK), the main energy sensor of the cell, has been previously described by our group as a key factor in glioblastoma oncogenesis and proliferation and is known for taking part in tumoral metabolic transformation. Our group has also formerly reported how AMPK regulates the expression of lipoprotein receptors and alters lipid reliance of glioblastoma cells, suggesting AMPK orchestrates nutrient dependency and flexibility on glioblastoma. Overall, this evidence positions AMPK inhibition as a promising antitumoral therapy for glioblastoma. MATERIAL AND METHODS To further study the role of AMPK in glioblastoma bioenergetics, we used the human glioblastoma cell lines U87 and U373 and silenced AMPK by transfecting the cells with a siRNA for AMPKα1/2. To assess mitochondrial function (OCR), fuel oxidation, and glycolytic parameters (ECAR), we carried out Seahorse metabolic analyses. This technology provides non-invasive and real-time monitoring of glycolytic and mitochondrial metabolic profiles. The expression of genes related to mitochondrial energetics (CPT1C, PPARGC1A, and PPARA) in these cells was studied using RT-qPCR. We are also currently studying lactate dehydrogenase (LDH) function and expression, as well as glutamine metabolism. RESULTS Our data show that silencing of AMPKα1/2 reduces basal and maximum mitochondrial respiration, as well as decreases the expression of CPT1C, PPARGC1A, and PPARA. Altogether, these results suggest alterations in mitochondrial energetic pathways. Our results do not reveal a significant effect of AMPK on the glycolytic activity of these glioblastoma cell lines. Finally, we found a great capacity of these cells to use glucose, but also fatty acids and glutamine as energy substrates, although with a very high dependency on glucose. CONCLUSION Even though in glioblastoma cells there is an increment in aerobic glycolysis (Warburg effect), these tumoral cells still maintain an elevated mitochondrial activity and a great capacity to use lipids as a fuel. AMPK plays an important role in regulating glioblastoma mitochondrial activity and bioenergetics. The authors would like to acknowledge the Funding for the Consolidation of Competitive Groups of Research (ED431B 2020/26, Xunta de Galicia, 2020-2022).

Oxygen-consumption based quantification of chemogenetic H2O2 production in live human cells.

Reactive Oxygen Species (ROS) in the form of H2O2 can act both as physiological signaling molecules as well as damaging agents, depending on their concentration and localization. The downstream biological effects of H2O2 were often studied making use of exogenously added H2O2, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H2O2 production by for instance mitochondrial respiration. The enzyme d-Amino Acid Oxidase (DAAO) catalyzes H2O2 formation using d-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H2O2. However, a method to directly quantify the amount of H2O2 produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H2O2. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H2O2 production. The oxygen consumption rate (OCR) of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, allowing to estimate whether the ensuing level of H2O2 production is within the range of physiological mitochondrial ROS production. In the tested monoclonal RPE1-hTert cells, addition of 5 mM D-Ala to the culture media amounts to a DAAO-dependent OCR that surpasses ∼5% of the OCR that stems from basal mitochondrial respiration and hence produces supra-physiological levels of H2O2. We show that the assay can also be used to select clones that express differentially localized DAAO with the same absolute level of H2O2 production to be able to discriminate the effects of H2O2 production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward.

Amyloid-β Pathology-Specific Cytokine Secretion Suppresses Neuronal Mitochondrial Metabolism

IntroductionNeuroinflammation and metabolic dysfunction are early alterations in Alzheimer’s disease (AD) brain that are thought to contribute to disease onset and progression. Glial activation due to protein deposition results in cytokine secretion and shifts in brain metabolism, which have been observed in AD patients. However, the mechanism by which this immunometabolic feedback loop can injure neurons and cause neurodegeneration remains unclear.MethodsWe used Luminex XMAP technology to quantify hippocampal cytokine concentrations in the 5xFAD mouse model of AD at milestone timepoints in disease development. We used partial least squares regression to build cytokine signatures predictive of disease progression, as compared to healthy aging in wild-type littermates. We applied the disease-defining cytokine signature to wild-type primary neuron cultures and measured downstream changes in gene expression using the NanoString nCounter system and mitochondrial function using the Seahorse Extracellular Flux live-cell analyzer.ResultsWe identified a pattern of up-regulated IFNγ, IP-10/CXCL10, and IL-9 as predictive of advanced disease. When healthy neurons were exposed to these cytokines in proportions found in diseased brain, gene expression of mitochondrial electron transport chain complexes, including ATP synthase, was suppressed. In live cells, basal and maximal mitochondrial respiration were impaired following cytokine stimulation.ConclusionsWe identify a pattern of cytokine secretion predictive of progressing amyloid-β pathology in the 5xFAD mouse model of AD that reduces expression of mitochondrial electron transport complexes and impairs mitochondrial respiration in healthy neurons. We establish a mechanistic link between disease-specific immune cues and impaired neuronal metabolism, potentially causing neuronal vulnerability and susceptibility to degeneration in AD.

Novel Iron Chelator SK4 Drives Cytotoxicity through Inhibiting Mitochondrial Metabolism in Ovarian and Triple Negative Breast Cancer Cell Lines.

Anti-cancer therapy by iron chelation has been shown to inhibit many cellular processes including DNA replication, mitochondrial metabolism and oncogenic signalling pathways (e.g., EGFR). Iron chelator SK4 represents a double pronged approach towards treating cancer. SK4 enters through LAT1, a commonly overexpressed amino acid transporter in tumours, thus targeting iron addiction and LAT1 overexpression. The aim of this study was to characterise the mode of action of SK4 through proteomics, metabolomics, lipidomics and seahorse real-time analysis in ovarian cell line SKOV3 and triple negative breast cancer cell line MDA MB 231. Pathway enrichment of proteomics data showed an overrepresentation of metabolism related pathways. Metabolic change after SK4 exposure have been confirmed in investigations of changes in basal and maximal mitochondrial respiration using seahorse real-time analysis of mitochondrial metabolism. Metabolomics also showed an increase in AMP and glucose-1-phosphate. Interestingly, our lipidomics data show a decrease in phospholipid synthesis in the SKOV3 cells which is in contrast with previous data which showed an upregulation of ceramide driven apoptosis. In summary, our data highlight impairment of energy metabolism as a mechanism of action underlying SK4 apoptosis, but also suggest a potential role of ceramide induction in the phenotypic outcome of the cell model.

Mitochondrial Dysfunction in Peripheral Blood Mononuclear Cells as Novel Diagnostic Tools for Non-Alcoholic Fatty Liver Disease: Visualizing Relationships with Known and Potential Disease Biomarkers.

Non-alcoholic fatty liver disease (NAFLD) is a health emergency worldwide due to its high prevalence and the lack of specific therapies. Noninvasive biomarkers supporting NAFLD diagnosis are urgently needed. Liver mitochondrial dysfunction is a central NAFLD pathomechanism that changes throughout disease progression. Blood-cell bioenergetics reflecting mitochondrial organ dysfunction is emerging for its potential applications in diagnostics. We measured real-time mitochondrial respirometry in peripheral blood mononuclear cells (PBMCs), anthropometric parameters, routine blood analytes, and circulating cytokines from a cohort of NAFLD patients (N = 19) and non-NAFLD control subjects (N = 18). PBMC basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity were significantly reduced in NAFLD compared to non-NAFLD cases. Correlation plots were applied to visualize relationships between known or potential NAFLD-related biomarkers, while non-parametric methods were applied to identify which biomarkers are NAFLD predictors. Basal and ATP-linked mitochondrial respiration were negatively correlated with triglycerides and fasting insulin levels and HOMA index. Maximal and spare respiratory capacity were negatively correlated with IL-6 levels. All the mitochondrial respiratory parameters were positively correlated with HDL-cholesterol level and negatively correlated with fatty liver index. We propose including blood cell respirometry in panels of NAFLD diagnostic biomarkers to monitor disease progression and the response to current and novel therapies, including mitochondrial-targeted ones.

Asiatic improves brain mitochondrial function, activates antioxidant response and attenuates cognitive deficits in beta amyloid overexpressing mice

AbstractBackgroundThe plant Centella asiatica can enhance mitochondrial function, promote antioxidant activity and improve cognitive deficits in mouse models of aging and Alzheimer’s disease. Asiatic acid (AA) is one of the constituent triterpene compounds present in the plant. In this study we explore the effects of increasing concentrations of AA on mitochondrial function and antioxidant response in healthy mice and investigate whether high doses of AA can elicit beneficial effects on mitochondrial function, antioxidant response and cognitive function in the 5xFAD mouse model of beta‐amyloid accumulation.MethodsNine month old female CF1 mice were treated with 0, 5mg, 50mg or 1000 mg of AA per 1kg of AIN‐93M diet for four weeks. Toxicity was also monitored. Hippocampal mitochondrial bioenergetics and the expression of mitochondrial and antioxidant response genes was determined. Results were compared to animals treated with 1000mg/kg of a water extract of Centella asiatica (CAW) incorporated into their diet. Additionally, six month old male and female 5xFAD mice were treated with 0 or 1000 mg/kg AA in their diet for four weeks. In the third week mice underwent cognitive testing. Hippocampal mitochondrial bioenergetics and expression of synaptic, mitochondrial and antioxidant response genes was assessed.ResultsIn the healthy mice increasing concentrations of AA resulted in a dose dependent increase in basal and maximal mitochondrial respiration, mitochondrial gene expression and antioxidant gene expression. Results from the 1000mg/kg AA dose were similar to what was observed with CAW. No evidence of toxicity was observed at any concentration of AA. In the 5xFAD mice 1000mg/kg AA attenuated deficits in cognitive and mitochondrial function and induced the expression of antioxidant genes. Interestingly, there was not a consistent effect of AA on mitochondrial or synaptic gene expression.ConclusionThese data show that high concentrations of AA can improve mitochondrial function and antioxidant response in the brains of treated animals. Consistent with the fact that mitochondrial dysfunction and oxidative stress are known to contribute to cognitive impairments this improvement was accompanied by improved cognitive function in the 5xFAD mice. Taken together these results suggest that AA is a promising cognitive enhancing therapy to develop for use in AD.

β-Hydroxy-β-methyl Butyrate Regulates the Lipid Metabolism, Mitochondrial Function, and Fat Browning of Adipocytes.

A growing number of in vivo studies demonstrated that β-hydroxy-β-methyl butyrate (HMB) can serve as a lipid-lowering nutrient. Despite this interesting observation, the use of adipocytes as a model for research is yet to be explored. To ascertain the effects of HMB on the lipid metabolism of adipocytes and elucidate the underlying mechanisms, the 3T3-L1 cell line was employed. Firstly, serial doses of HMB were added to 3T3-L1 preadipocytes to evaluate the effects of HMB on cell proliferation. HMB (50 µM) significantly promoted the proliferation of preadipocytes. Next, we investigated whether HMB could attenuate fat accumulation in adipocytes. The results show that HMB treatment (50 µM) reduced the triglyceride (TG) content. Furthermore, HMB was found to inhibit lipid accumulation by suppressing the expression of lipogenic proteins (C/EBPα and PPARγ) and increasing the expression of lipolysis-related proteins (p-AMPK, p-Sirt1, HSL, and UCP3). We also determined the concentrations of several lipid metabolism-related enzymes and fatty acid composition in adipocytes. The HMB-treated cells showed reduced G6PD, LPL, and ATGL concentrations. Moreover, HMB improved the fatty acid composition in adipocytes, manifested by increases in the contents of n6 and n3 PUFAs. The enhancement of the mitochondrial respiratory function of 3T3-L1 adipocytes was confirmed via Seahorse metabolic assay, which showed that HMB treatment elevated basal mitochondrial respiration, ATP production, H+ leak, maximal respiration, and non-mitochondrial respiration. In addition, HMB enhanced fat browning of adipocytes, and this effect might be associated with the activation of the PRDM16/PGC-1α/UCP1 pathway. Taken together, HMB-induced changes in the lipid metabolism and mitochondrial function may contribute to preventing fat deposition and improving insulin sensitivity.