Oroboros Oxygraph Research Articles

7086 Disrupted Mitochondrial Function in Alström Syndrome- A Monogenic Model of Insulin Resistance and Obesity

Abstract Disclosure: S. Ali: None. S. Heising: None. V. Veeranna: None. A. Vincent: None. G.S. Xavier: None. T. Geberhiwot: None. Alström syndrome (ALMS) is a rare monogenic disease typified by severe insulin resistance (IR) and obesity. IR and obesity are cardinal features of metabolic syndrome. The metabolic effects of insulin at key target tissues leads to metabolic haemostasis by promoting glucose and lipid uptake and metabolism to generate energy in cells. Although insulin is a major regulator of fuel metabolism, its effects on mitochondrial ATP production in severe insulin resistance are unclear. RNASeq data from adipose tissue (AT) from patients with ALMS show downregulation of the expression of genes associated with mitochondrial respiratory chain function, pointing towards possible altered mitochondrial function in ALMS. We therefore undertook a detailed assessment of mitochondrial oxidative phosphorylation capacity (OXPHOS) in skeletal muscle (SM) in ALMS. We performed SM biopsy in 10 ALMS and 10 healthy control volunteers that were age, gender and Body Mass Index matched. The SM was subjected to high resolution respirometry, the gold standard method to quantify mitochondrial function ex vivo, using OROBOROS Oxygraph -2k (O2k) system. Overall OXPHOS was higher in control SM compared to ALMS, with maximal respiration of 62.96±19.2 and 44.24 ± 12.53 pmol/(s*mg), respectively (p<0.05).Reduced oxygen influx was observed in ALMS SM in comparison to controls for fatty acid ß oxidation (6.7±5.96 vs 7.87±2.7 pmol/(s*mg);p<0.05, at complex I (19.4 ± 8.98 vs 29.7 ±5.3 pmol/(s*mg);p<0.05, and at complex II (34.98 ±11.75 vs 53.52± 13.2 pmol/(s*mg) ;p<0.05. Mitochondrial membrane integrity was unaffected during these experiments. Therefore, our analysis unveils a consistent reduction in mitochondrial respiration across all the electron transport pathway. This noteworthy finding suggests impairment in respiratory function and the electron transport chain in mitochondria, a crucial aspect of cellular energy metabolism. Several studies have previously linked IR with mitochondrial dysfunction, where insulin-resistant rodents and humans have shown blunted mitochondrial response to ATP generation. Our results align with this body of evidence emphasizing the significance of investigating the underlying mechanism contributing to this phenomenon. These findings could pave the way to exploring potential mitochondrial-targeted therapies to restore mitochondrial function and therefore, improve insulin sensitivity to tackle IR in ALMS. Presentation: 6/2/2024

A Novel Approach for Assessing Mitochondrial Respiration in Freshly Isolated Glomerular and Tubular Fractions from Whole Mouse Kidney

Intro: Understanding mitochondrial function within specific renal compartments is crucial for unraveling the intricate metabolic processes underlying kidney physiology and pathophysiology. In this study, we present a novel method for accurately measuring mitochondrial respiration in freshly isolated glomerular and tubular fractions derived from dissected kidneys. Methods: Employing a differential adhesive sieving technique, we isolated glomerular and tubular fractions from freshly harvested whole kidneys (n=2) and subsequently utilized a standard substrate-inhibitor titration protocol to assess mitochondrial respiration in the Oroboros Oxygraph O2K (Oroboros Instruments, Innsbruck AT). Results: Notably, our findings revealed distinct state 3 respiration rates in various fractions, with the 70 μm tubule fraction exhibiting a rate of 2133.029 pmol/sec/mgprotein, the 40 μm tubule fraction displaying a rate of 522.4116 pmol/sec/mgprotein, the first glomerular fraction demonstrating a rate of 407.7911 pmol/sec/mgprotein, and the second tubular fraction manifesting a rate of 362.3286 pmol/sec/mgprotein. Moreover, these fragments exhibited differential substrate preferences, as the addition of succinate increased state 3 respiration by ~90% in the 70 μm tubule fraction. In contrast, succinate increased respiration by ~30% in the 40 μm tubule and glomerular fractions. Discussion: These ongoing experiments provide compelling evidence supporting the feasibility and reliability of our approach in quantifying mitochondrial respiration within isolated renal fractions. Our methodology not only enables precise measurements of mitochondrial function in distinct nephron segments but also lays the groundwork for further investigations into the physiological and pathological implications of mitochondrial dynamics in kidney health and disease. This research is supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award 5T32DK091317 from the National Institute of Diabetes and Digestive and Kidney Diseases and the National Kidney Foundation of Utah and Idaho. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A practical perspective on how to develop, implement, execute, and reproduce high-resolution respirometry experiments: The physiologist's guide to an Oroboros O2k.

The development of high-resolution respirometry (HRR) has greatly expanded the analytical scope to study mitochondrial respiratory control relative to specific tissue/cell types across various metabolic states. Specifically, the Oroboros Oxygraph 2000 (O2k) is a common tool for measuring rates of mitochondrial respiration and is the focus of this perspective. The O2k platform is amenable for answering numerous bioenergetic questions. However, inherent variability with HRR-derived data, both within and amongst users, can impede progress in bioenergetics research. Therefore, we advocate for several vital considerations when planning and conducting O2k experiments to ultimately enhance transparency and reproducibility across laboratories. In this perspective, we offer guidance for best practices of mitochondrial preparation, protocol selection, and measures to increase reproducibility. The goal of this perspective is to propagate the use of the O2k, enhance reliability and validity for both new and experienced O2k users, and provide a reference for peer reviewers.

SIRT4 is a regulator of human skeletal muscle fatty acid metabolism influencing inner and outer mitochondrial membrane-mediated fusion

ObjectiveThe mitochondrial phenotype, governed by the balance of fusion-fission, is a key determinant of energy metabolism. The inner and outer mitochondrial membrane (IMM) fusion proteins optic atrophy 1 (OPA1) and Mitofusin 1 and 2 (Mfn1/2) play an important role in this process. Recent evidence also shows that Sirtuin 4 (SIRT4), located within the mitochondria, is involved in the regulation of fatty acid oxidation. The purpose of this study was to determine if SIRT4 expression regulates inner and outer mitochondrial-mediated fusion and substrate utilization within differentiated human skeletal muscle cells (HSkMC). Material and methodsSIRT4 expression was knocked down using small interfering RNA (siRNA) transfection in differentiated HSkMC. Following knockdown, mitochondrial respiration was determined by high-resolution respirometry (HRR) using the Oroboros Oxygraph O2k. Live cell confocal microscopy, quantified using the Mitochondrial Network Analysis (MiNA) toolset, was used to examine mitochondrial morphological change. This was further examined through the measurement of key metabolic and mitochondrial morphological regulators (mRNA and protein) induced by knockdown. ResultsSIRT4 knockdown resulted in a significant decrease in LEAK respiration, potentially explained by a decrease in ANT1 protein expression. Knockdown further increased oxidative phosphorylation and protein expression of key regulators of fatty acid metabolism. Quantitative analysis of live confocal imaging of fluorescently labelled mitochondria following SIRT4 knockdown supported the role SIRT4 plays in the regulation of mitochondrial morphology, as emphasized by an increase in mitochondrial network branches and junctions. Measurement of key regulators of mitochondrial dynamics illustrated a significant increase in mitochondrial fusion proteins Mfn1, OPA1 respectively, indicative of an increase in mitochondrial size. ConclusionsThis study provides evidence of a direct relationship between the mitochondrial phenotype and substrate oxidation in HSkMC. We identify SIRT4 as a key protagonist of energy metabolism via its regulation of IMM and OMM fusion proteins, OPA1 and Mfn1. SIRT4 knockdown increases mitochondrial capacity to oxidize fatty acids, decreasing LEAK respiration and further increasing mitochondrial elongation via its regulation of mitochondrial fusion.

Analysis of oxygen consumption rates in zebrafish reveals differences based on sex, age and physical activity recovery.

Introduction: Mitochondrial dysfunction is linked to a variety of human diseases. Understanding the dynamic alterations in mitochondrial respiration at various stages of development is important to our understanding of disease progression. Zebrafish provide a system for investigating mitochondrial function and alterations during different life stages. The purpose of this study was to investigate our ability to measure mitochondrial oxygen consumption rates in zebrafish embryos, larvae, and adults as an indicator of mitochondrial function. Methods: Basal respiration of entire zebrafish embryos (5dpf), larvae (0.6-0.9cm), young adults (3-month-old), and old adults (12-month-old) was measured using an Oroboros Oxygraph, with a stirrer speed of 26rpm. For embryos and larvae, "leak" respiration (plus oligomycin), maximum respiration (plus uncoupler), non-mitochondrial respiration (plus inhibitors), and complex IV activity were also measured. To induce physical activity in adult fish, the stirrer speed was increased to 200rpm. Results and Discussion: We demonstrate the ability to accurately measure respiration rates in zebrafish at various ages using the Oroboros Oxygraph. When comparing zebrafish embryos to larvae, embryos have a higher maximum respiration. Three-month-old zebrafish males have higher basal respiration than females, while 12-month-old zebrafish females exhibit greater rates of respiration than males and younger females. When the stirrer speed was increased, respiration rates decrease, but with differences depending on sex. This study demonstrates a simple and accessible method to assess zebrafish physiology by mitochondrial oxygen consumption measurements in an unmodified Oroboros Oxygraph. The method should facilitate studies to understand the intricate interplay between mitochondrial function, development, and aging.

Lysine acetylation regulates mitochondrial pyruvate carrier activity and cardiac pyruvate oxidation

Background/Objective: The normal heart can acutely switch fuel sources based on delivery. While this is beneficial in physiological scenarios, this flexibility is often lost in cardiac pathologies. During fasting, blood glucose levels drop and increased free fatty acid delivery causes the heart to shut off glucose/pyruvate oxidation and increase fat oxidation. This balance, known as the Randle cycle, has mainly been attributed to regulation of pyruvate dehydrogenase (PDH) activity via PDH phosphorylation. Hypothesis: Decreased pyruvate transport into the mitochondria via posttranslational modifications of the mitochondrial pyruvate carrier (MPC) plays a role in regulating cardiac pyruvate oxidation. Methods: Wildtype mice were allowed to consume normal chow ad libitum or were fasted for 24 hours. After euthanasia, plasma and hearts were collected and left-ventricular muscle fibers were saponin permeabilized and mitochondrial respiration measured in an Oroboros Oxygraph O2k. Cardiac protein lysates were immunoprecipitated with anti-acetyl-lysine beads, and western blotted using standard procedures. A bioluminescent resonance transfer (BRET) assay to detect conformational changes caused by pyruvate/inhibitor binding the MPC was used, and lysine mutant MPC2 constructs were generated by site-directed mutagenesis. Lastly, MPC2-/- H9C2 cardiomyocytes were generated by CRISPR, and respiration measured by Seahorse bioanalyzer in these cells after transfection with WT or lysine mutant MPC2 constructs. Results: 24h fasting reduced blood glucose and insulin concentrations, and increased circulating free fatty acids. Cardiac mitochondrial oxidation of pyruvate/malate was decreased, while oxidation of fatty acids (palmitoyl-CoA+carnitine) was increased in fasted hearts. Western blotting of cardiac lysates showed the expected increase in phosphorylation of PDH-E1α in fasted hearts, known to decrease PDH activity. Immunoprecipitating cardiac lysates with anti-acetyl-lysine beads and blotting for MPC2 suggested increased acetylation of MPC2 in fasted hearts. Using a model structure of the MPC, we have recently proposed and validated lysine 49 (K49) of MPC2 as a critical pyruvate binding site within the MPC. To assess the possible importance of K49 acetylation we mutated K49 to glutamine to mimic acetylation and observed that this K49Q-MPC2 mutant was no longer able to bind pyruvate or competitive inhibitors in a MPC BRET assay. We next created MPC2 knockout H9C2 cardiomyocytes by CRISPR-Cas9 and observed complete loss of MPC2 and MPC1 expression in these cells which decreased pyruvate respiration compared to wildtype H9C2s. Overexpression of wildtype MPC1 and MPC2 constructs increased pyruvate respiration in these MPC-/- cells, while overexpression of the MPC2-K49Q mutant was unable to improve pyruvate respiration. Conclusion: Fasting results in decreased cardiac oxidation of pyruvate in part by acetylation of the MPC and decreased mitochondrial pyruvate transport. National Institutes of Health and SLU institutional funding. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract TP235: Temporal Change Of Brain Mitochondrial Function In A Rodent Model Of Subarachnoid Hemorrhage

Introduction: Subarachnoid hemorrhage (SAH) is a devastating disease. A third of the patients die and a large percentage of the survivors require support in daily living and suffers from reduced quality of life. As part of clinical practice for these patients, a cerebral microdialysis catheter may be placed to monitor cerebral metabolic function. Using this monitoring technique, the patients often demonstrate an increase in the lactate-pyruvate ratio, despite adequate supply of glucose and oxygen, which may be an indirect marker of mitochondrial dysfunction. We wanted to identify a possible mitochondrial dysfunction in a rat SAH model at different time points by directly measuring mitochondrial function in brain tissue ex vivo . Method: The pre-chiasmatic single injection model of SAH was used. Oroboros Oxygraph high resolution respirometry was used to evaluate the electron transport chain of the mitochondria. We also analyzed ROS generation simultaneously at the same time points using the Oroboros LED2 module. We measured tissue from the cortex and the hippocampus. Results: 45 rats at four different timepoints, 6-, 24-, 48- and 96 hours after surgery, were included. We identified a significant reduction in respiration through complexes I and II of the electron transport chain in hippocampal tissue at 96 hours after SAH compared to sham. The respiration of cortical tissue at the same time point was reduced compared to sham but did not reach significance. No differences were identified at other time points and no differences in ROS generation were identified. Conclusion: We conclude that the SAH model can be used as a suitable model to identify mitochondrial dysfunction and that the respiratory capacity of the electron transport chain is reduced in the hippocampus at 96 h post insult in this rat model of SAH.

Donor variation in stored platelets: Higher metabolic rates of platelets are associated with mean platelet volume, activation and donor health.

Platelets (PLTs) differ in glycolytic activity, resulting in rapid acidification of 'poor' storing PLT concentrates (PCs) in plasma, or depletion of glucose when stored in PLT additive solution (PAS). We aimed to understand why PLT glycolysis rates vary between donors and how this affects storage performance. Buffy coats from donors <45, 45-70 and >70 years were selected and single-donor PCs in plasma or PAS-E were prepared. PCs were stored for 8 days at 22 ± 2°C and sampled regularly for analysis. Mitochondrial activity was analyzed with an Oroboros oxygraph. Age groups, or subgroups divided into quartiles based on glucose consumption, were analyzed with ANOVA. In each comparison, PCs of the different groups were not different in volume and cellular composition. PLTs with the highest glucose consumption had a higher initial mean platelet volume (MPV) and developed higher CD62P expression and Annexin A5 binding during storage. Higher glycolytic activity in these PLTs was not a compensation for lower mitochondrial ATP production, because mitochondrial ATP-linked respiration of fresh PLTs correlated positively with MPV (R2 = 0.71). Donors of high glucose-consuming PLTs had more health-related issues. Storage properties of PCs from donors over 70 were not significantly different compared to PCs from donors younger than 45 years. High glucose-consuming PCs developing higher activation levels, not only displayed enhanced mitochondrial activity but were also found to contain larger PLTs, as determined by MPV. Storage performance of PLTs was found to be associated with donor health, but not with donor age.

Diurnal Differences in Mitochondrial Bioenergetics is Lost in Bmal1 Knockout Rats

In the kidney, 95% of O2 consumed through mitochondrial respiration is used for Na+ transport. The circadian clock regulates Na+ transport with a necessary diurnal variation for optimal health. The circadian clock transcription factor BMAL1 is proposed to regulate mitochondrial bioenergetic function; however, this has not been extensively investigated in the kidney. Therefore, we tested the hypothesis that renal mitochondrial respiration may be impaired or altered in a novel Bmal1 clock gene knock‐out rat model and is associated with loss of diurnal control of Na+ excretion. Male and female global Bmal1+/+ and Bmal1‐/‐ rats at 12‐14 weeks of age maintained in a regular 12‐hour light/dark cycle was used to measure renal mitochondrial O2consumption. Outer renal medullary tissue was dissected and prepared for respiration measurements at either day (ZT2‐4) or night (ZT14‐16) periods to correspond with minimum and maximum whole‐body energy consumption as well as peak and trough BMAL1 protein expression, respectively (n = 10, Bmal1+/+; n = 22, Bmal1‐/‐). Respiration was assessed using permeabilized tissue in the Oroboros Oxygraph and analyzed using Data Lab 2 software (O2K, Oroboros Instruments GmbH). Mitochondrial gene expression analysis was assessed using digital drop PCR. Data from both sexes were combined for each genotype at the two time points and analyzed by two‐way ANOVA. Mitochondrial state 3 O2 consumption was significantly higher (main effect of genotype p = 0.0310) in Bmal1‐/‐ (118 ± 156 pmol/s*mg) compared to BMAL1+/+ rats (75 ± 15 pmol/s*mg; Tukey post‐hoc p = 0.0303). We also observed a significant increase in complex IV activity in Bmal1‐/‐ rats during the dark vs. light period (164 ± 40 vs. 306 ± 40 pmol/s*mg; p=0.0031, t‐test). Mitochondrial‐associated genes, optic atrophy 1 (Opal1) and mitofusion 1 (Mfn1) show as significant day‐night difference in mRNA expression in kidneys from BMAL1+/+ control rats that is lost in Bmal1‐/‐ rats consistent with disrupted mitochondrial fusion and fission processes. Our lab previously reported that male Bmal1‐/‐ rats do not have the typical night‐day difference in Na+ excretion, which is consistent with our new data showing an alteration in mitochondrial function in kidneys from Bmal1‐/‐ rats. Therefore, our findings demonstrate that BMAL1 may play a role in maintaining the coupling of mitochondrial respiration and ATP generation in the kidney and support the hypothesis that diurnal Na+handling is regulated in part by BMAL1‐dependent mitochondrial function.

Abstract 11214: Ibrutinib Decreases Mitochondrial Oxidative Phosphorylation and Metabolic Gene Expression in Atrial-Like Engineered Heart Tissue

Introduction: Ibrutinib is a Bruton kinase inhibitor which treats many hematological malignancies, but also precipitates atrial fibrillation (AF). The precise mechanism(s) remain to be elucidated. Hypothesis: We hypothesized ibrutinib treatment would decrease mitochondrial function and metabolic gene expression in human inducible pluripotent stem cell-derived atrial cardiomyocytes grown as atrial-like engineered heart tissues (aEHTs). Methods: aEHTs were treated with vehicle or 1 μM ibrutinib daily, for 72 hours. Oxidative phosphorylation was assessed (n=5/group) using an Oroboros Oxygraph. Gene expression was evaluated by RNAseq (n=3/group). Results: Oxidative phosphorylation in the presence of complex I and complex II substrates, and maximal oxidative capacity, was decreased in ibrutinib-treated aEHTs compared to vehicle (Panel A). Subunit expression of complex I (NDUFA6, NDUFA9, NDUFS1, NDUFS2, NDUFS4, ND3, ND4, ND5, ND4L), complex II (SDHA, SDHB, SDHD), complex III (UQCR10, UQCRB, UQCRC1, UQCRC2, UQCRFS1), and complex IV (CO2, COX10, COX7A2, CYCS) was decreased. Ingenuity Pathway Analysis of genes differentially expressed identified TCA Cycle and Glycolysis I (Panel B), pathways that provide reducing equivalents to the electron transport chain, as pathways that were decreased by ibrutinib. These changes were accompanied by decreased AMPK Signaling, including key metabolic regulators (PRKAA2, PRKAB2, PRKAG2, SIRT1, and PPARGC1A), and Calcium Signaling. Tumor protein (TP53), nuclear protein 1 (NUPR1), and erb-B2 receptor tyrosine kinase (ERBB2) were identified as top upstream regulators. Conclusions: Ibrutinib treatment decreased oxidative phosphorylation and gene expression of electron transport chain subunits and key modulators of atrial metabolism. Together, these data identify metabolic pathways that are altered by ibrutinib in aEHTs and metabolic regulators that could be targeted for AF therapeutic intervention.

Abstract 13104: L-carnitine Supplementation Improves Right Ventricular Function in Two Models of Right Ventricular Failure

Introduction: Previous studies have shown that right ventricular (RV) failure in pulmonary arterial hypertension is characterized by diminished fatty acid oxidation and ultimate lipotoxicity due to accumulation of toxic lipid intermediates. Whether or not augmenting fatty acid oxidation can reverse lipotoxic RV failure in PAH is not known. Hypothesis: In this study, we tested the hypothesis that augmentation of fatty acid oxidation through L-carnitine supplementation reverses RV failure in two models of RV failure. Methods: BMPR2 (R899X) mice were allowed to develop PAH for 10 weeks prior to supplementation of L-carnitine or control in their water for 8 weeks. In a second model, wild type C57/BL6 mice underwent pulmonary artery banding and were treated with L-carnitine or control 2 weeks after banding surgery. Finally, L-carnitine supplementation was also tested on H9C2 cardiomyocyte-like cells transfected with mutant BMPR2. Lipid accumulation was measured by Oil Red O or BODIPY stain. Fatty acid-mediated mitochondrial respiration was measured by Oroboros Oxygraph or Seahorse analysis. Results: In vivo , L-carnitine in BMPR2 mice and pulmonary artery banded mice increased RV cardiac output, RV ejection fraction, and mitochondrial respiration of long-chain fatty acids. Hemodynamic improvement in RV cardiac output was confirmed in pulmonary artery banded mice. In vitro , L-carnitine reduced lipid accumulation and increased fatty acid oxidation in BMPR2 mutant H9C2 cells through a Cpt1-dependent mechanism. Conclusions: In summary, augmentation of fatty acid oxidation in right ventricular failure through L-carnitine supplementation improves RV function, increases fatty acid oxidation, and decreases lipid accumulation.

Importance of Mitochondrial Metabolism As a Selective Therapeutic Approach in Chronic Lymphocytic Leukemia Patients

Objective: Chronic lymphocytic leukemia (CLL) is the most common blood cancer among adults in Western countries. Impairments in mitochondrial physiology play a role in CLL. Selectively targeting mitochondrial bioenergetics in leukemia cells may be an effective therapeutic approach for CLL. However, mitochondrial metabolism in CLL remains relatively unexplored. Nicotinamidephospho-robosyltransferase (NAMPT) is a key enzyme in the nitoinamide adenine dinucleotide (NAD) salvage pathway. FK866, a chemical inhibitor of NAMPT depletes cellular NAD and ATP levels and triggers apoptosis, suggesting NAMPT contributes to the prolonged survival of CLL cells. Idelalisib, an inhibitor of phosphatidylinositol-3-kinase-delta (PI3Kδ) is an approved therapy for B-cell malignancies including CLL. Herein, bioenergetics profile in 20 CLL patients have been defined and the effects of these drugs on this profile, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and protein levels of mitochondrial complexes and pPI3Kδ were analyzed. We hypothesize that both inhibitors will alter CLL mitochondrial bioenergetics profiles and function, cell viability, ROS levels and enable lower doses and profound effects for combination therapies rather than single agent therapy.Methods: A high resolution Oroboros Oxygraph 2K (Oroboros Instruments, Innsbruck, Austria), a Clarke-type oxygen electrode is used to measure mitochondrial respiration rates CLL cells at 370C. Freshly isolated CLL cells (5-10 mil./ml) were added to the chamber in 2 ml of RPMI. After the measurement of basal respiration rates (BR), the following chemicals were added: oligomycin (2μM), FCCP: carbonyl cyanide p-trifluoromethoxy phenyhydrazone (2.5-12.5 μM), and antimycin A (2 μM). Oxygen consumption rate is expressed in pmol oxygen/s/mil. cells. Protein levels in CLL cell lysates were determined by Western blotting. Cell viability, MMP, and ROS were assessed by Novocyte flow cytometer.Results: Bioenergetics profile (BR: 14.45 ± 3.40; FCCP-induced MR: 57.48 ± 14.94; reserve respiratory capacity, RRC: 43.03 ± 12.80; respiratory control ratio, RCR: 9.36 ± 3.99; proton leak: 6.77 ± 2.45 and residual respiration: 6.03 ± 1.49; Mean ± SD) of 20 CLL patients was individually illustrated and summarized. Our data show that CLL cells have a high mitochondrial RRC and require a higher concentration of uncoupler, namely FCCP to achieve MR than commonly used. BR was not significantly decreased by 24 hours of treatment with FK866 at 1 nM (FK1) nor idelalisib at 6.25 μM (ID6.25) or the combination. After 48 hours, basal respiration of ID6.25 or ID6.25 + FK1 - treated cells was significantly decreased compared to DMSO vehicle control. However, after 24 or 48 hours MR, RRC, and RCR were significantly decreased by each individual drug or its combination compared to DMSO vehicle control. After 24 hours, combination therapy did not significantly decrease BR, RRC, and RCR compared to the treatment of FK1 (except MR), but MR, RRC, and RCR were decreased significantly compared to ID6.25 (except BR). The combination treatment of these two drugs significantly decreased all bioenergetics parameters compared to each individual drug after 48 hours. Preliminary data with another NAMPT-inhibitor, GMX-1778 at 1nM also showed similar effect to FK866 and in combination with ID6.25. MMP was not affected by these drug treatments, cell viability corresponded to the effect of mitochondrial bioenergetics profile, however, ROS levels were increased. Protein expression of certain subunits of mitochondrial complexes I, III and V and pPI3Kδ were decreased after the combined treatment.Conclusion:Bioenergetics profile can be directly evaluated by high resolution Oroboros oxygraph in CLL cells without any excess manipulation. CLL cells have immense mitochondrial RRC and highly sensitive to an uncoupler. Both FK866 and idelalisib affected bioenergetics profile in CLL cells at doses as low as 1 nM or 6.25 µM, respectively. The combined effect of these drugs is more profound than each agent alone on mitochondrial bioenergetics profile, cell viability, ROS and protein levels of mitochondrial complexes and pPI3Kδ. These novel data derived from the combined treatment with lower doses targeting mitochondrial metabolism provide rational therapeutic combination that may lead to reduce toxicity and increased drug efficacy in CLL. DisclosuresNo relevant conflicts of interest to declare.

283-OR: Inadequate Hepatic Mitochondrial Adaptation in Patients with NASH Is Related to the Severity of Steatohepatitis and Fibrosis Stage

Dysfunctional mitochondrial function plays an important role in the progression of nonalcoholic steatohepatitis (NASH). Mitochondrial (mt) respiratory function assessed by oxidative phosphorylation/ OXPHOS (activated respiration with mt complex I [OXPHOS CI] and mt complex I + II [OXPHOS CI+II] substrates) and electron transport system capacity [ETC] were measured using high-resolution respirometry (Oroboros Oxygraph 2k). The aim of this study was to assess mitochondrial function in patients a) with vs. without NASH, and b) NASH with vs. without fibrosis. Liver tissue was obtained from 40 patients (age: 52 ± 12 yrs; BMI: 39.4 ± 8.4 kg/m2; HbA1c: 6.9 ± 1.5%) with NAFLD and obesity or T2DM either by percutaneous liver biopsy (n=28) or during bariatric surgery (n=12). Patients with vs. without NASH (n=20 in each group) were more insulin resistant/ IR (HOMA-IR: 8.1 ± 4.2 vs. 4.5 ± 2.7 mg/dl x μU/ml; Adipo-IR: 10.4 ± 7.2 vs. 6.2 ± 3.8 mmol/L x µU/mL) and had higher OXPHOS CI+II (all p&amp;lt;0.05), likely an adaptation to IR and higher FFA flux to the liver. We then compared those with worse steatohepatitis (necroinflammation score/ NIS), divided as mild (n=16), moderate (n=15) or severe (n=9). Patients with moderate vs. mild NIS had increased mitochondrial respiration, expressed as pmol/(s x mg wet weight) (OXPHOS CI: 20.8 ± 4.8 vs. 16.4 ± 4.6; OXPHOS CI+II: 46.0 ± 11.4 vs. 31.3 ± 9.8; ETC: 60.5 ± 17.1 vs. 46.4 ± 15.2; all p&amp;lt;0.05). There was a trend for these parameters to decline with the most severe disease activity, that was significant when patients with NASH also had significant fibrosis vs. mild or no fibrosis (OXPHOS CI+II: 38.6 ± 7.8 vs. 49.8 ± 12.5, p=0.04; ETC: 50.3 ± 12.9 vs. 67.5 ± 16.1; p=0.02). Conclusion: In patients with NASH vs. simple steatosis, there is an early hepatic mitochondrial adaptation to severe insulin resistance. This adaptation is impaired when disease activity worsens, being most evident once patients develop steatohepatitis with significant fibrosis. Disclosure S. Kalavalapalli: None. D. Poulton: None. L. Mansour: None. S. Shrestha: None. F. Bril: None. S. K. Subbarayan: None. S. Kadiyala: None. K. Cusi: None. D. Barb: None. E. Godinez: None. R. Lomonaco: None. J. Friedman: Consultant; Self; Ethicon, Inc. S. Wohlgemuth: None. N. Fanous: None. R. E. Dillard: None. T. M. Cowan: None.

Ex Vivo Mitochondrial Respiration Parallels Biochemical Response to Ibrutinib in CLL Cells.

Simple SummaryChronic lymphocytic leukemia (CLL) is a cancer that is characterized by dysfunctional mitochondria. This results in a deranged mitochondrial metabolism. Many drugs are tested using simple live or dead cell assays and as such the impact on the altered mitochondrial function is not evaluated. One such drug is ibrutinib. The use of ibrutinib at full doses can lead to significant side effects resulting in dose reduction or even discontinuation of the drug in clinical practice. In this study, we reviewed the effect of the dose of ibrutinib on mitochondrial function of the CLL cells from patients treated with ibrutinib and did not observe any difference in the mitochondrial respiration. We also evaluated the effect of progression of CLL cells from patients on ibrutinib treatment and the impact on mitochondrial respiration. We believe our findings are novel and suggest that evaluation of mitochondrial function of CLL cells is of importance to help design safe treatments in the preclinical setting.Mitochondrial respiration is becoming more commonly used as a preclinical tool and potential biomarker for chronic lymphocytic leukemia (CLL) and activated B-cell receptor (BCR) signaling. However, respiration parameters have not been evaluated with respect to dose of ibrutinib given in clinical practice or the effect of progression on ibrutinib treatment on respiration of CLL cells. We evaluated the impact of low and standard dose ibrutinib on CLL cells from patients treated in vivo on mitochondrial respiration using Oroboros oxygraph. Cytokines CCL3 and CCL4 were evaluated using the Mesoscale. Western blot analysis was used to evaluate the BCR and apoptotic pathways. We observed no difference in the mitochondrial respiration rates or levels of plasma chemokine (C-C motif) ligands 3 and 4 (CCL3/CCL4), β-2 microglobulin (β-2 M) and lactate dehydrogenase (LDH) between low and standard doses of ibrutinib. This may confirm why clinical observations of the safety and efficacy of low dose ibrutinib are observed in practice. Of interest, we also observed that the mitochondrial respiration of CLL cells paralleled the increase in β-2 M and LDH at progression. Our study further supports mitochondrial respiration as a biomarker for response and progression on ibrutinib in CLL cells and a valuable pre-clinical tool.

A Protocol to Study Mitochondrial Function in Human Neural Progenitors and iPSC-Derived Astrocytes.

Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfunctioning of astrocytes is related to many neuropathologies. Here we describe protocols for generating induced pluripotent stem cell (iPSC)-derived astrocytes and evaluating multiple aspects of mitochondrial function. We use a high-resolution respirometry assay that measures real-time variations in mitochondrial oxygen flow, allowing the evaluation of cellular respiration in the context of an intact intracellular microenvironment, something not possible with permeabilized cells or isolated mitochondria, where the cellular microenvironment is disrupted. Given that an impairment in the mitochondrial regulation of intracellular calcium homeostasis is involved in many pathologic stresses, we also describe a protocol to evaluate mitochondrial calcium dynamics in human neural cells, by fluorimetry. Lastly, we outline a mitochondrial function assay that allows for the measurement of the enzymatic activity of mitochondrial hexokinase (mt-HK), an enzyme that is functionally coupled to oxidative phosphorylation and is involved in redox homeostasis, particularly in the brain. In all, these protocols allow a detailed characterization of mitochondrial function in human neural cells. High-resolution respirometry, calcium dynamics, and mt-HK activity assays provide data regarding the functional status of mitochondria, which may reflect mitochondrial stress or dysfunction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of iPSC-derived human astrocytes Basic Protocol 2: Measuring real-time oxygen flux in human iPSC-derived astrocytes using a high-resolution OROBOROS Oxygraph 2k (O2k) Basic Protocol 3: Measuring mitochondrial calcium dynamics fluorometrically in permeabilized human neural cells Basic Protocol 4: Measuring OXPHOS-dependent activity of mitochondrial hexokinase in permeabilized human neural cells using a spectrophotometer.

High-fat Diet Induces Nr4a3-dependent Decrease In Respiratory Capacity Of Mouse Soleus Muscle

The Nuclear Hormone Receptor 4A family of genes have been observed to play a role in proper metabolic function in various tissues, including skeletal muscle. PURPOSE: To analyze the effect of the Nr4a3 gene on respiratory capacity of mitochondria in skeletal muscle of mice on a normal or high fat diet. METHODS: Nr4a3-/- and WT mice were fed a normal chow (NC) or high fat diet (HF) for at least 20 weeks. After euthanasia, soleus muscle was harvested and wet weight was measured. Muscle fibers were teased apart and permeabilized with saponin in preparation for respirometry. Mitochondrial respiration was evaluated using an Oroboros Oxygraph Respirometer. Respiratory capacity comparisons were made with a two-way ANOVA and Tukey multiple comparison test. RESULTS: Oxygen consumption is reported as pmol/(s*mg wet tissue) and statistics are represented as mean ± SEM. In the WT male mice there was a decrease in coupled complex I supported respiration in HF vs. NC diet (25.9 ± 7.3 vs. 64.5 ± 5.0, p=0.004). In the HF WT group there was also a decrease in coupled complex I and II supported respiration (57.2 ± 13.4 vs. 102.5 ± 7.0, p=0.0005) and uncoupled respiration (61.4 ± 15.0 vs. 107.8 ± 7.1, p=0.0004) compared to NC WT. In female mice there was also a decrease between HF WT and NC WT in complex I (28.2 ± 3.7 vs. 57.4 ± 5.7, p=0.0005) and complex I and II (78.2 ± 6.1 vs. 108.8 ± 6.7, p=0.0003) supported respiration as well as in uncoupled respiration (87.1 ± 7.1 vs. 119.4 ± 8.9, p=0.0001). However, there was no significant difference between the WT NC mice and either of the Nr4a3-/- groups. Coupled complex I, complex I and II and uncoupled respiration states in both Nr4a3-/- groups were not significantly different from WT. CONCLUSIONS: The Nr4a3 gene plays a role in mitochondrial function in mouse skeletal muscle. Feeding mice a high fat diet impairs proper mitochondrial function in muscle when compared to a normal chow diet. The decrease in respiration from the HF diet is dependent upon the function of the Nr4a3 gene, as no decrease was observed in Nr4a3-/- mice. A limitation of this study is that this effect could be due to the lack of Nr4a3 in the skeletal muscle, or a secondary effect of lacking the gene in other parts of the body.

Donor brain stem death and cardiac transplantation causes mitochondrial dyscoupling and oxidative stress

Introduction: Mitochondria are essential for adequate cardiovascular function and are important in the generation of reactive oxygen species (ROS). However, mitochondrial function and ROS post donor brain stem death (BD) and heart transplantation HTx is poorly understood and may contribute to donor heart dysfunction (DHD) and post HTx graft failure (GF). Objectives∖Aims: To examine mitochondrial function and ROS production in hearts both after 24hrs of BSD and post HTx. Methods: Donor sheep underwent BD (BD n=13) or sham instrumentation (SH, n=8) and monitored for 24hrs followed by heart procurement or cold static storage. Orthotopic HTx followed, in 7∖13 BD (BD-Tx) and 4/8 SH donors (SH-Tx). Mitochondrial respirometry was assessed using the Oroboros Oxygraph using carbohydrate (CHO) or fatty acid (FAO) substrates for both ventricles. O2 flux control ratios (FCR) were calculated in LEAK (no ADP) and Complex- I (CI) & II (CII) OXPHOS (with ADP) respiration states. Tissue levels of 3-nitrotyrosine and glutathione:reduced glutathione (GSH:GSSG) ratio were assessed using plate based assays. Results: BD caused a significant increase in LEAK RV proton slip. Both ventricles showed a trend toward higher O2 consumption at CI (known driver of ROS) and a significant decrease in CII respiration for both substrates. Post HTx showed mitochondrial dyscoupling evidenced by a trend toward higher CI O2 consumption, significantly higher RV proton slip, and depressed LV and RV CII respiration. For the LV, GSH:GSSG ratios were significantly lower for BD, BD-Tx and SH-Tx. For the RV, GSH:GSSG ratios were non-significantly depressed post BD both pre and post Tx. Conclusions: We have shown that donor BD and Tx caused significant mitochondrial dyscoupling and higher oxidative stress. Mitochondrial function, particularly CI ROS production may then contribute to DHD and consequently post-HTx GF. Therapeutically targeting mitochondrial ROS could improve patient prognosis.

Ibrutinib in Combination with Venetoclax Decreases Mitochondrial Bioenergetics through the Impaired BTK, AKT and AMPK/SIRT/PGC-1α Signaling Pathway in CLL

Objective: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Altered mitochondrial metabolism has been shown to be involved in the pathogenesis of CLL. Ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK), and venetoclax, an inhibitor of B-cell lymphoma 2 (Bcl-2) protein are approved therapies for CLL. The adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling axis senses the metabolic demands of cells and regulates mitochondrial function. Silent information regulator T1 (SIRT1) is a cytoplasmic enzyme that mediates NAD+-dependent deacetylation of target substrates. The importance of BTK, AKT, and AMPK/SIRT/PGC-1α signaling pathway have to be explored yet in CLL. Ibrutinib and venetoclax may trigger a switching off of AMPK and /or SIRT1 signaling leading to impaired PGC-1α expression/activity and diminished mitochondrial activity. The effects of these drugs on mitochondrial bioenergetics profile, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and protein levels of p-BTK, p-AKT, Bcl-2, p-AMPK, SIRT1, and PGC-1α were analyzed. We hypothesize that ibrutinib, along with venetoclax, will alter CLL mitochondrial bioenergetics profiles and function, cell viability, and ROS levels through the involvement of BTK, AKT and AMPK/SIRT/PGC-1α signaling pathway and enable lower doses and profound effects for combination therapies rather than single agent therapy. Methods: The high resolution Oroboros Oxygraph 2K (Oroboros Instruments, Austria), a Clarke-type oxygen electrode, is used to measure mitochondrial respiration rates of CLL cells at 370C. Freshly isolated CLL cells (10 mil. cells/ml) were added to the chamber in 2 ml of RPMI. After the measurement of basal respiration rates, the following chemicals were added: oligomycin (2uM), FCCP: carbonylcyanide p-trifluoromethoxyphenylhydrazone (2-12 uM), and antimycin A (2 uM). Oxygen consumption rate is expressed in pmol oxygen/s /mil. cells. Protein levels in CLL cell lysates were determined by Western blotting. Cell viability, MMP, and ROS were assessed by the flow cytometer (NovoCyte). Results: CLL cells obtained ex vivo from ibrutinib treated patients had decreased mitochondrial bioenergetics compared to pre-treatment samples. Primary CLL cells treated in vitro with sub-lethal doses of ibrutinib for 24 or 48h showed significantly decreased basal respiration rates, maximal respiration rates, and spare respiratory capacity compared to DMSO vehicle control. These parameters were also significantly affected by low doses (0.5 - 2 nM) of venetoclax. The combination treatment of ibrutinib with venetoclax for 24 or 48h significantly decreased these bioenergetics parameters compared to each individual drug alone except for the spare respiratory capacity vs venetoclax alone at 24 h. MMP was significantly decreased by these drug treatments and its combinations compared to DMSO. Cell viability was used to confirm a sub-lethal phenotype. ROS levels were significantly increased by venetoclax compared to DMSO, and in combination with ibrutinib vs ibrutinib alone. However, ROS levels by ibrutinib were not significantly changed compared to DMSO or venetoclax. Protein levels of p-BTK, p-AKT, p-AMPK, SIRT1, and PGC-1α were decreased by ibrutinib or venetoclax alone compared to DMSO and further decreased by the combination of these two drugs compared to single agent. Conclusion: CLL cells from ibrutinib treated patients, a clinically approved BTK inhibitor, demonstrated decreased bioenergetics similar to normal B-lymphocytes suggesting ibrutinib treatment normalizes the mitochondrial bioenergetics in CLL. Ibrutinib and venetoclax affected bioenergetics profiles in CLL cells at low doses. The combined effect of these drugs on the mitochondrial bioenergetics profiles and cell viability is more profound than each BTK or Bcl-2 inhibitor agent alone. Protein levels of p-BTK, p-AKT, p-AMPK, SIRT1, and PGC-1α in ibrutinib and venetoclax treated samples were reduced compared to DMSO and each single agent inhibitor. These novel data suggest the involvement of BTK, AKT and AMPK/SIRT/PGC-1α signaling pathway to target mitochondrial metabolism and provide rational therapeutic combinations that may lead to reduced toxicity and increased drug efficacy in CLL. Disclosures Johnston: Janssen: Research Funding. Banerji:Janssen: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Licensing fee, Research Funding; Abbvie: Consultancy, Honoraria; CIHR: Research Funding; LLSC: Research Funding; Research Manitoba: Research Funding; Astra-Zeneca: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Dana-Farber Cancer Institute: Other: Licencing fee; CCMF: Research Funding; CancerCare Manitoba/University of Manitoba: Employment; CAPhO: Honoraria; BIOGEN: Other: Licensing fee.

Leucine-rich diet induces a shift in tumour metabolism from glycolytic towards oxidative phosphorylation, reducing glucose consumption and metastasis in Walker-256 tumour-bearing rats

Leucine can stimulate protein synthesis in skeletal muscle, and recent studies have shown an increase in leucine-related mitochondrial biogenesis and oxidative phosphorylation capacity in muscle cells. However, leucine-related effects in tumour tissues are still poorly understood. Thus, we described the effects of leucine in both in vivo and in vitro models of a Walker-256 tumour. Tumour-bearing Wistar rats were randomly distributed into a control group (W; normoprotein diet) and leucine group (LW; leucine-rich diet [normoprotein + 3% leucine]). After 20 days of tumour evolution, the animals underwent 18-fludeoxyglucose positron emission computed tomography (18F-FDG PET-CT) imaging, and after euthanasia, fresh tumour biopsy samples were taken for oxygen consumption rate measurements (Oroboros Oxygraph), electron microscopy analysis and RNA and protein extraction. Our main results from the LW group showed no tumour size change, lower tumour glucose (18F-FDG) uptake, and reduced metastatic sites. Furthermore, leucine stimulated a shift in tumour metabolism from glycolytic towards oxidative phosphorylation, higher mRNA and protein expression of oxidative phosphorylation components, and enhanced mitochondrial density/area even though the leucine-treated tumour had a higher number of apoptotic nuclei with increased oxidative stress. In summary, a leucine-rich diet directed Walker-256 tumour metabolism to a less glycolytic phenotype profile in which these metabolic alterations were associated with a decrease in tumour aggressiveness and reduction in the number of metastatic sites in rats fed a diet supplemented with this branched-chain amino acid.

Venetoclax in Combination with NAMPT Inhibitors Decreases Mitochondrial Bioenergetics through the Impaired AMPK/SIRT/PGC1α Signaling Pathway in CLL

Objective: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Altered mitochondrial metabolism has been shown to be involved in the pathogenesis of CLL. Nicotinamidephospho-ribosyltransferase (NAMPT) is a key enzyme in the nicotinamide adenine dinucleotide (NAD) salvage pathway. FK866 and GMX1778, chemical inhibitors of NAMPT, deplete cellular NAD and ATP levels and trigger apoptosis in CLL cells, suggesting NAMPT contributes to the prolonged survival of these cells. Venetoclax is an approved therapy for CLL and blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein, leading to programmed cell death of CLL cells. The adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signaling axis senses the metabolic demands of cells and regulates mitochondrial function. Silent information regulator T1 (SIRT1) is a cytoplasmic enzyme that mediates NAD+-dependent deacetylation of target substrates. The importance of the AMPK/SIRT/PGC1α signaling pathway has to be explored yet in CLL. Venetoclax may trigger a switching off of AMPK and /or SIRT1 signaling leading to impaired PGC-1α expression/activity and diminished mitochondrial activity. The effects of these drugs on mitochondrial bioenergetics profile, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and protein levels of Bcl-2, AMPK, SIRT1, and PGC1α were analyzed. We hypothesize that venetoclax, along with FK866 and GMX1778, will alter CLL mitochondrial bioenergetics profiles and function, cell viability, and ROS levels through the involvement of the AMPK/SIRT/PGC1α signaling pathway and enable lower doses and profound effects for combination therapies rather than single agent therapy.Methods: A high resolution Oroboros Oxygraph 2K (Oroboros Instruments, Innsbruck, Austria), a Clarke-type oxygen electrode is used to measure mitochondrial respiration rates of CLL cells at 370C. Freshly isolated CLL cells (10 mil./ml) were added to the chamber in 2 ml of RPMI. After the measurement of basal respiration rates, the following chemicals were added: oligomycin (2uM), FCCP: carbonyl cyanide p-trifluoromethoxy phenyhydrazone (2.5-12.5 uM), and antimycin A (2 uM). Oxygen consumption rate is expressed in pmol oxygen/s/mil. cells. Protein levels in CLL cell lysates were determined by Western blotting. Cell viability, MMP, and ROS were assessed by Novocyte flow cytometer.Results: Primary CLL cells treated with 1.25 nM of venetoclax for 24 or 48h showed significantly decreased basal respiration rates, maximal respiration rates, and spare respiratory capacity compared to DMSO vehicle control. These parameters were also significantly affected by 1nM of FK866 or 1 nM of GMX1778 except basal respiration rates. The combination treatment of venetoclax with FK866 or GMX1778 for 24 or 48h significantly decreased all these bioenergetics parameters compared to each individual drug. MMP was not affected by these drug treatments and cell viability corresponded to the effect of the mitochondrial bioenergetics profile. ROS levels after 24 hours in venetoclax treated CLL cells were significantly increased compared to DMSO, any single agent or in combination. However, after 48 hours of treatment, increased ROS levels were not statistically significant compared to DMSO. Protein levels of Bcl-2, P-AMPK, AMPK, SIRT1, and PGC1α were decreased by venetoclax alone or in combination with FK866 or GMX1778.Conclusion: Venetoclax, FK866 and GMX1778 affected bioenergetics profiles in CLL cells at doses as low as 1.25 nM, 1nM, and 1 nM, respectively. The combined effect of these drugs on the mitochondrial bioenergetics profiles and cell viability is more profound than each NAMPT inhibitor agent alone. Protein levels of Bcl-2, AMPK, SIRT1, and PGC1α in venetoclax treated samples were reduced compared to DMSO and each single agent NAMPT inhibitor. These novel data suggest the involvement of the AMPK/SIRT/PGC1α signaling pathway to target mitochondrial metabolism and provide rational therapeutic combinations that may lead to reduced toxicity and increased drug efficacy in CLL. DisclosuresBanerji:Roche: Other: Unrestricted grant received in the past; Janssen: Other: Unrestricted grant received in the past; Gilead: Other: Unrestricted grant received in the past; Abbvie: Other: Unrestricted grant received in the past; Teva: Other: Unrestricted grant received in the past.