Mitochondrial efficiency : focus on dietary nitrate, hypoxia and exercise

Metabolic efficiency affects weight control, generation of heat, exercise performance and health. The tiny radical nitric oxide (NO) targets several cellular components that can influence metabolic efficiency. NO is produced endogenously from L-arginine and molecular oxygen by specific NO-synthases. In addition, extensive research during the past two decades shows that the inorganic anions nitrate and nitrite, which are oxidation products from endogenous NO generation, can be reduced back to NO and other nitrogen oxides. Apart from reflecting endogenous NOS-activity, circulating nitrate and nitrite are dependent on dietary intake, where green leafy vegetables in particular contain high amounts of nitrate. Circulating nitrate is actively taken up by the salivary glands and excreted in saliva. Oral commensal bacteria then reduce salivary nitrate into nitrite, which after swallowing and effective uptake in the gut, reaches the systemic circulation. In blood and tissues several enzymatic and non-enzymatic pathways are able to reduce nitrite to NO. These pathways are potentiated under acidic and hypoxic conditions. The nitratenitrite- NO pathway is considered a back-up system during conditions when NO-synthases are failing. Prior to the inception of this thesis, our group had shown that dietary nitrate was able to decrease oxygen cost during exercise and we wanted to further explore its metabolic effects With this background, we investigated the underlying mechanisms behind the oxygen sparing effect of dietary nitrate during exercise (Paper I). Moreover, we explored the effects of dietary nitrate on muscular function in mice (Paper II), oxygen consumption in a human model of global hypoxia (Paper III) and on resting metabolic rate in humans (Paper IV). In the last two papers we wanted to investigate if metabolic efficiency can affect exercise tolerance in hypoxia (Paper V) and how cytochrome c oxidase (COX) subunit IV isoform composition affects resting metabolic rate (Paper VI). Respirometric analysis of isolated mitochondria from healthy humans revealed that dietary nitrate improves mitochondrial efficiency (P/O ratio) and this effect correlated strongly with the reduction in oxygen consumption during cycling ergometry. In addition we found respirometric support for less uncoupling which was supported by reduced expression of adenine nucleotide transporter (ANT), a major determinant of proton conductance (Paper I). In muscle from mice fed for 7 days with nitrate, electric stimulation led to increased contractile force and speed of force development in fast twitch muscle compared to controls. This was accompanied by higher Ca2+ levels and increased expression of the Ca2+- handling proteins dihydropyridine receptor and calsequestrin-1 (Paper II). In the human model of global hypoxia a reduction in arterial oxygen saturation was achieved during prolonged breath-holding by experienced free divers after nitrate or placebo. In contrast to our hypothesis, nitrate during resting apnea increased pulmonary oxygen uptake, reduced arterial oxygen saturation and shortened maximal breathholding time. This was probably related to a NO-mediated attenuation of the oxygen conserving diving response as showed by less bradycardia and indications of an attenuation of the increase in blood pressure after nitrate (Paper III). In healthy humans we could demonstrate that dietary nitrate reduces resting metabolic rate by 4% and that acute administration of nitrite in vitro reduces respiration by 40% in primary myotubes from the same individuals (Paper IV). We found that healthy subjects with a high metabolic efficiency in normoxia had higher tolerance to exercise in hypoxia. Interestingly, these subjects acutely reduced their metabolic efficiency during hypoxia in order to maintain power output. On the other hand, isolated mitochondria, which work in the lower efficiency range, acutely increased their efficiency during a steady state hypoxic challenge in order to maintain ATP production (Paper V). There is a largely unexplained variation in resting metabolic rate between seemingly similar individuals. We found that the inter-individual variation in resting metabolic rate seems to depend on the composition of COX subunit IV isoforms. We could show that COX IV-2 isoform is present in human skeletal muscle and that a high COX IV-2/COX IV-1 ratio showed a strong negative correlation to resting metabolic rate. Moreover, concurrent overexpression of COX IV-2 and knock down of COX IV-1 in primary human myotubes significantly reduced basal cell respiration and ROS generation without affecting the COX activity (Paper VI). In conclusion, this thesis demonstrates profound effects of dietary nitrate on mitochondrial efficiency, muscle function and metabolism. In addition, metabolic efficiency plays a role in exercise tolerance during hypoxia and adapts to obtain optimal power. Finally, mitochondrial COX subunit IV isoform composition seems to affect resting metabolic rate. The physiological, therapeutic and nutritional aspects of these findings create a platform for further studies on dietary nitrate, mitochondrial function and metabolism

Diversity of respiratory parameters and metabolic adaptation to low oxygen tension in mesenchymal stromal cells

Objective Cell metabolism has been shown to play an active role in regulation of stemness and fate decision. In order to identify favorable culture conditions for mesenchymal stromal cells (MSCs) prior to transplantation, this study aimed to characterize the metabolic function of MSCs from different developmental stages in response to different oxygen tension during expansion. Materials and methods We cultured human fetal cardiac MSCs and human adult bone-marrow MSCs for a week under hypoxia (3% O2) and normoxia (20% O2). We performed mitochondrial characterization and assessed oxygen consumption- and extracellular acidification-rates (OCR and ECAR) in addition to oxygen-sensitive respiration and mitochondrial complex activities, using both the Seahorse and Oroboros systems. Results Adult and fetal MSCs displayed similar basal respiration and mitochondrial amount, however fetal MSCs had lower spare respiratory capacity and apparent coupling efficiency. Fetal MSCs expanded in either hypoxia or normoxia demonstrated similar acidification rates, while adult MSCs downregulated their aerobic glycolysis in normoxia. Acute decrease in oxygen tension caused a higher respiratory inhibition in adult compared to fetal MSCs. In both sources of MSCs, minor changes in complex activities in normoxic and hypoxic cultures were found. Conclusions In contrast to adult MSCs, fetal MSCs displayed similar respiration and aerobic glycolysis at different O2 culture concentrations during expansion. Adult MSCs adjusted their respiration to glycolytic activities, depending on the culture conditions thus displaying a more mature metabolic function. These findings are relevant for establishing optimal in vitro culturing conditions, with the aim to maximize engraftment and therapeutic outcome.CC BY-NC-ND 4.0Corresponding author: Department of Surgical Sciences, Uppsala University, 751 85, Uppsala, Sweden. E-mail address: [email protected] (K.-H. Grinnemo).Available online 3 February 2022, Version of Record 5 February 2022The project was funded by Karolinska Institute-Mayo Clinic Collaborative Grant 2013; The Swedish Research Council young investigator: 2013–3590; Stockholm county; The Swedish Research Council; The Family Erling-Persson Foundation; ERC-2018-AdG (834860 EYELETS); Uppsala county; Uppsala County Association against Heart and Lung Diseases; and Higher Education of the Russian Federation (agreement no. 075-15-2020-899).</p

Repression of hypoxia-inducible factor-1 contributes to increased mitochondrial reactive oxygen species production in diabetes

Background: Excessive production of mitochondrial reactive oxygen species (ROS) is a central mechanism for the development of diabetes complications. Recently, hypoxia has been identified to play an additional pathogenic role in diabetes. In this study, we hypothesized that ROS overproduction was secondary to the impaired responses to hypoxia due to the inhibition of hypoxia-inducible factor-1 (HIF-1) by hyperglycemia. Methods: The ROS levels were analyzed in the blood of healthy subjects and individuals with type 1 diabetes after exposure to hypoxia. The relation between HIF-1, glucose levels, ROS production and its functional consequences were analyzed in renal mIMCD-3 cells and in kidneys of mouse models of diabetes. Results: Exposure to hypoxia increased circulating ROS in subjects with diabetes, but not in subjects without diabetes. High glucose concentrations repressed HIF-1 both in hypoxic cells and in kidneys of animals with diabetes, through a HIF prolyl-hydroxylase (PHD)-dependent mechanism. The impaired HIF-1 signaling contributed to excess production of mitochondrial ROS through increased mitochondrial respiration that was mediated by Pyruvate dehydrogenase kinase 1 (PDK1). The restoration of HIF-1 function attenuated ROS overproduction despite persistent hyperglycemia, and conferred protection against apoptosis and renal injury in diabetes. Conclusions: We conclude that the repression of HIF-1 plays a central role in mitochondrial ROS overproduction in diabetes and is a potential therapeutic target for diabetic complications. These findings are timely since the first PHD inhibitor that can activate HIF-1 has been newly approved for clinical use. Funding: This work was supported by grants from the Swedish Research Council, Stockholm County Research Council, Stockholm Regional Research Foundation, Bert von Kantzows Foundation, Swedish Society of Medicine, Kung Gustaf V:s och Drottning Victorias Frimurarestifelse, Karolinska Institute's Research Foundations, Strategic Research Programme in Diabetes, and Erling-Persson Family Foundation for S-B.C.; grants from the Swedish Research Council and Swedish Heart and Lung Foundation for T.A.S.; and ERC consolidator grant for M.M.Peer reviewe

KCNMA1 Encoded Cardiac BK Channels Afford Protection against Ischemia-Reperfusion Injury

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+-and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioninglike effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and reoxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP),but differed upon IP. While the area of infarction comprised 28 +/- 3% of the area at risk in wild-type, it was increased to 58 +/- 5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations

Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic β cells

Male mice lacking the androgen receptor (AR) in pancreatic β cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in β cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male β cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of CO2, activating the HCO3--sensitive soluble adenylate cyclase; and (2) increased Gαs recruitment to GLP-1 receptor and AR complexes, activating transmembrane adenylate cyclase. Additionally, testosterone enhances GSIS in human islets via a focal adhesion kinase/SRC/phosphatidylinositol 3-kinase/mammalian target of rapamycin complex 2 actin remodeling cascade. We describe the testosterone-stimulated AR interactome, transcriptome, proteome, and metabolome that contribute to these effects. This study identifies AR genomic and non-genomic actions that enhance GLP-1-stimulated insulin exocytosis in male β cells

Architecture of Androgen Receptor Pathways Amplifying Glucagon-Like Peptide-1 Insulinotropic Action in Male Pancreatic β Cells

Male mice lacking the androgen receptor (AR) in pancreatic β cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in β cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male β cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of C

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p&lt;0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (&lt;1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (&lt;1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Report of the National Institutes of Health SARS-CoV-2 Antiviral Therapeutics Summit

The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development