Low ADP Research Articles

Abstract Tu104: Mitochondrial Hydrogen Sulfide Regulates Skeletal Muscle Dysfunction and Exercise Intolerance in Cardiometabolic HFpEF

Introduction: Cardiometabolic heart failure with preserved ejection fraction (HFpEF) results in severe exercise intolerance. Dysregulated skeletal muscle metabolism and mitochondrial function are key drivers of exercise intolerance in cardiometabolic HFpEF. Mitochondrial hydrogen sulfide (H 2 S) generated by 3-mercaptopyruvate sulfur transferase (3-MST) exerts potent bioenergetic properties. We have recently shown that H 2 S bioavailability is significantly attenuated in HFpEF. The role of H2S in skeletal muscle metabolism and exercise tolerance in HFpEF is currently unknown. Hypothesis: We tested the hypothesis that the loss of skeletal muscle H 2 S results in metabolic dysfunction in skeletal muscle and contributes to exercise intolerance in cardiometabolic HFpEF. Methods: Skeletal muscle (gastrocnemius) from L-NAME + High Fat diet (“two-hit”) treated, and chow-fed control male mice (n=10/group) were assessed for free H 2 S and 3-MST enzymatic activity using GC-chemiluminescence detection. Additionally, muscles were processed for RNA sequencing. Next, male global 3-MST knockout (KO) mice (n=12) and controls (n=10) were subjected to “two-hit” HFpEF for 10 weeks, and exercise tolerance was evaluated. Skeletal muscle (soleus and gastroc) was subjected to high-resolution respirometry and substrate oxidation of radiolabeled isotope ( 14 C) for leucine and palmitate. Results: Skeletal muscle H 2 S levels (0.73 ± 0.18 vs. 1.0 ± 0.24 nmol/g/tissue) and 3-MST activity (0.46 ± 0.01 vs 0.57 ± 0.01 µmol/g/tissue/min) were significantly (p < 0.05) reduced in “two-hit” HFpEF mice compared to control mice. At the transcriptome level, 3-MST was one of the top 50 differentially regulated genes (i.e., reduced expression) in “two-hit” mice (Adjusted p=0.01). Genetic deficiency of 3-MST KO mice exacerbated (p < 0.05) exercise intolerance as measured by treadmill running distance (53 ± 24 vs. 138 ± 104 meters) vs. “two-hit” HFpEF genotype controls. Mitochondrial Complex I (low ADP) respiration was reduced by 23% in 3-MST KO HFpEF mice (p < 0.05). Genetic deficiency of 3-MST also reduced skeletal muscle leucine oxidation (58 ± 19 vs 84 ± 8; p = 0.01) and dramatically increased incomplete palmitate oxidation by 2.5-fold (p < 0.001) without changing complete oxidation. Conclusion: We demonstrate that skeletal muscle mitochondrial H 2 S is a critical regulator of metabolic function and exercise capacity in cardiometabolic HFpEF.

Polyphosphate Plays a Significant Role in the Maturation of Spores in Myxococcus xanthus.

Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1 and Ppx2, respectively. M. xanthus polyP:AMP phosphotransferase (Pap) generates ADP from AMP and polyPs. Pap expression is induced by an elevation in intracellular polyP concentration. M. xanthus synthesized polyPs during the stationary phase; the ppk1 mutant died earlier than the wild-type strain after the stationary phase. In addition, M. xanthus cells cultured in phosphate-starved medium, H2O2-supplemented medium, or amino acid-deficient medium increased the intracellular polyP levels by six- to ninefold after 6h of incubation. However, the growth of ppk1 and ppx2 mutants in phosphate-starved medium and H2O2-supplemented medium was not significantly different from that of wild-type strain, nor was there a significant difference in fruiting body formation and sporulation in starvation condition. During development, no difference was observed in the adenylate energy charge (AEC) values in the wild-type, ppk1 mutant, and pap mutant strains until the second day of development. However, after day 3, the ppk1 and pap mutants had a lower ADP ratio and a higher AMP ratio compared to wild-type strain, and as a result, the AEC values of these mutants were lower than those of the wild-type strain. Spores of ppk1 and pap mutants in the nutrient medium germinated later than those of the wild-type strain. These results suggested that polyPs produced during development may play an important role in cellular energy homeostasis of the spores by being used to convert AMP to ADP via Pap.

Low on-clopidogrel ADP- and TRAP-6-induced platelet aggregation in patients with atrial fibrillation undergoing percutaneous coronary intervention: an observational pilot study

High on-clopidogrel platelet reactivity (HPR) associates with ischemic risk in patients after percutaneous intervention (PCI). This study aimed to evaluate the association of HPR as assessed by multiple electrode aggregometry (MEA) with ischemic, thromboembolic, and bleeding risk in patients with atrial fibrillation (AF) undergoing PCI. Patients with AF and an indication for oral anticoagulation (OAC) were included in this prospective cohort study on day 1–3 after PCI. Platelet aggregation [U] was analyzed by MEA. HPR and low platelet reactivity (LPR) were defined as ADP-induced aggregation ≥ 46 U and ≤ 18 U, respectively. TRAP-6-induced aggregation reference was 94–156 U. The primary outcome was time to all-cause death, myocardial infarction, or stroke at 6 months. The secondary outcome was time to non-major clinically relevant bleedings or major bleedings. 159 patients were enrolled between May 2020 and May 2021. The median age was 78 years (interquartile range 72–82) and 111 (70%) were male. Median ADP- and TRAP-induced aggregation were 12 (6–17) and 49 (35–68) U, respectively. 147 (93%) patients had a low overall aggregability. HPR was detected in 2 patients (1%) and 125 (79%) had LPR. ADP-induced aggregation did not significantly associate with the primary outcome (r = 0.081, p = 0.309) but correlated inversely with bleeding risk (r = − 0.201, p = 0.011). HPR status as assessed by MEA among patients with AF after PCI was rare and overall aggregability was low. Conventional cut-off values for HPR might be inappropriate for these patients. ADP-induced aggregation might be helpful to identify patients at risk for bleeding.

Factors Determining Ticagrelor-Induced Dyspnea in Patients with Acute Coronary Syndrome

(1) Background: The aim of this study was to determine clinical and genetic factors predicting the development of dyspnea in patients receiving ticagrelor. (2) Methods: A total of 277 patients with acute myocardial infarction (with and without ST-segment elevation), who underwent coronary angiography and PTCA with stent implantation and treated with antiplatelet drugs (ticagrelor and aspirin), were enrolled in this study. Platelet aggregation (induction with high-sensitivity ADP, ADP HS) testing was performed using a MULTIPLATE analyzer and reagents for the determination of P2Y12 receptor activity. Venous blood samples were collected for genotyping. (3) Results: Patients experiencing ticagrelor-related dyspnea had lower ADP HS. ROC curve analysis showed that an ADP HS cut-off of ≤19.5 U was associated with the development of dyspnea. The ADP HS value of ≤19.5 U and any dose of atorvastatin lower than 80 mg (or no atorvastatin) increased the risk of dyspnea by more than 4 and 2 times, respectively (OR = 4.07, p ≤ 0.001 and OR = 2.25; p = 0.008). (4) Conclusion: A lower ADP HS value possibly indicates greater ticagrelor activity and a higher plasma concentration of this drug. Atorvastatin might have an impact on the occurrence of ticagrelor-related dyspnea by affecting ticagrelor metabolism. No impact of any genetic variant on the development of dyspnea was determined.

Syncopal patients without prodromes exhibit a diverse pattern of adenosine release during head-up tilt test

Abstract Background/Introduction In a significant proportion of patients with neurally-mediated syncope (NMS), the mechanism of syncope remains largely undetermined. Adenosine has been proposed as a central humoral factor in various forms of NMS: high Adenosine Plasma Levels (ADP) are encountered in patients with vasovagal syncope (VVS), low ADP in non-prodromes syncope (NPS), while ADP in patients with situational syncope SS are less well determined. Purpose We sought to assess ADP in patients with different clinical forms of syncope, with an emphasis on the presence or absence of prodromal symptoms, as well as the relation between ADP and the outcomes of Head-Up Tilt Table Test (HUTT) and Adenosine test (ADT). Methods Patients with different clinical types of NMS (n=124), i.e., VVS, NPS, or SS, were investigated using a standard protocol including HUTT and ADT. During HUTT, ADP was measured in the supine position, at table tilting, and in syncope. Results Baseline ADP did not differ among groups. ADP at syncope were higher in NPS compared to VVS (1.55±1.29 vs 0.16±0.05 μM, p=0.03) and SS (0.15±0.05 μM, p=0.02). In NPS, ADP increased from the supine position to the time of syncope (0.47±0.25 to 1.55±1.29 μM, p=0.04). In VVS, ADP increased only from the supine to the tilt position (0.23±0.04 to 0.35±0.10 μMu, p=0.02), while in SS ADP did not change in any stage of HUTT. SS was associated with cardioinhibitory HUTT (OR 3.40, 95% CI 1.05 to 9.56, p=0.04) and positive ADT (OR 4.22, 95% CI 1.47 to 11.46, p=0.012). Conclusion(s) A distinct pattern of ADP increase is noted during HUTT in NPS, suggesting that an excessive increase of ADP may play the key role in triggering this type of clinical presentation of syncope independently of the baseline ADP. Cardioinhibition prevails in patients with SS without clear adenosine involvement. Such observations contribute to an enhanced understanding of the pathophysiology of different clinical forms of syncope which may offer the possibility to tailor the management approach appropriately. Funding Acknowledgement Type of funding sources: None.

Platelet Hemostasis Reactions at Different Temperatures Correlate with Intracellular Calcium Concentration.

Hypo- and hyperthermia affect both primary and secondary hemostasis; however, there are controversial data concerning platelet activation and the underlying mechanisms under hypo- and hyperthermia. The discrepancies in the data could be partly explained by different approaches to hemostatic reactions analysis. We applied a new LaSca-TMF laser particle analyzer for a simultaneous fluorescence and laser scattering analysis of platelet responses at different temperatures. Human platelets were activated by ADP in a wide range of temperatures, and platelet transformations (e.g., a shape change reaction, aggregation and clot formation) and the intracellular calcium concentration ([Ca2+]i) were analyzed by LaSca-TMF and confocal microscopy. The platelet shape change reaction gradually increased with a rising temperature. The platelet aggregation strongly decreased at low ADP concentrations with the augmentation of the temperature and was independent of the temperature at high ADP concentrations. In contrast, the clotting time decreased with a temperature increase. Similar to the aggregation response, a rise in [Ca2+]i triggered by low ADP concentrations was higher under hypothermic conditions and the differences were independent of the temperature at high ADP concentrations. We showed that the key reactions of cellular hemostasis are differentially regulated by temperature and demonstrated for the first time that an accelerated aggregation under hypothermic conditions directly correlated with an increased level in [Ca2+]i in platelets.

New Mechanistic Insights into Purine Biosynthesis with Second Messenger c-di-AMP in Relation to Biofilm-Related Persistent Methicillin-Resistant Staphylococcus aureus Infections.

ABSTRACTPersistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinically challenging subset of invasive, life-threatening S. aureus infections. We have recently demonstrated that purine biosynthesis plays an important role in such persistent infections. Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger that regulates many cellular pathways in bacteria. However, whether there is a regulatory connection between the purine biosynthesis pathway and c-di-AMP impacting persistent outcomes was not known. Here, we demonstrated that the purine biosynthesis mutant MRSA strain, the ΔpurF strain (compared to its isogenic parental strain), exhibited the following significant differences in vitro: (i) lower ADP, ATP, and c-di-AMP levels; (ii) less biofilm formation with decreased extracellular DNA (eDNA) levels and Triton X-100-induced autolysis paralleling enhanced expressions of the biofilm formation-related two-component regulatory system lytSR and its downstream gene lrgB; (iii) increased vancomycin (VAN)-binding and VAN-induced lysis; and (iv) decreased wall teichoic acid (WTA) levels and expression of the WTA biosynthesis-related gene, tarH. Substantiating these data, the dacA (encoding diadenylate cyclase enzyme required for c-di-AMP synthesis) mutant strain (dacAG206S strain versus its isogenic wild-type MRSA and dacA-complemented strains) showed significantly decreased c-di-AMP levels, similar in vitro effects as seen above for the purF mutant and hypersusceptible to VAN treatment in an experimental biofilm-related MRSA endovascular infection model. These results reveal an important intersection between purine biosynthesis and c-di-AMP that contributes to biofilm-associated persistence in MRSA endovascular infections. This signaling pathway represents a logical therapeutic target against persistent MRSA infections.

Is monitoring of antiplatelet therapy by light transmission aggregometry dependent on instrument and reagent used?

Light transmission aggregometry (LTA), used to detect clopidogrel resistance in patients under antiplatelet therapy, is prone to multiple variables potentially influencing results and interpretation. Currently, no attention is given to type of aggregometer or reagent used. The aim of this study was to evaluate the interassay variability between two aggregometers (Chronolog700 and TA-8V), using two different ADP reagents (Chrono-Par® and Agro-Bio ADP) in patients under clopidogrel therapy. Additionally, LTA results were correlated to CYP2C19-polymorphism. Light transmission aggregometry was performed in 20 healthy individuals and 30 patients using both aggregometers, and applying two different reagents (2.5 and 5µmol/L). The maximum platelet aggregation (ADPmax ), the platelet aggregation at 6minutes (ADP6min ), and the percentage of disaggregation at 6min (ADP%disaggr ) were compared between four applied combinations. Additionally, 23 clopidogrel-resistant patients according to Chronolog700-Chrono-par® ADP reagent analysis were tested for CYP2C19*2 polymorphism. Comparison of the LTA of healthy individuals revealed a significant lower ADPmax , lower ADP6min , and higher ADP%disaggr with the TA-8V aggregometer compared to Chronolog700, regardless of the reagent. In contrast, LTA results in patients are depending on the reagent, with significant higher ADPmax and ADP6min and lower ADP%disaggr using Chrono-Par® compared to Agro-Bio ADP reagent. All intermediate clopidogrel metabolizers (CYP2C19*2 carriers) were correctly classified as clopidogrel resistant using Chrono-Par® , in contrast to the Agro-Bio ADP reagent. Light transmission aggregometry in clopidogrel-treated patients is mainly depending on the type of ADP reagent. Comparison of LTA with genotype reveals that the choice of instrument seems less influencing. In contrast, in the healthy individuals, differences could be attributed to the instrument.

Point-of-care thrombocyte function testing using multiple-electrode aggregometry in dengue patients: an explorative study

BackgroundDengue virus (DENV) causes the hospitalisation of an estimated 500,000 people every year. Outbreaks can severely stress healthcare systems, especially in rural settings. It is difficult to discriminate patients who need to be hospitalized from those that do not. Earlier work identified thrombocyte count and subsequent function as a promising prognostic marker of DENV severity. Herein, we investigated the potential of quantitative thrombocyte function tests in those admitted in the very early phase of acute DENV infections, using Multiplate™ multiple-electrode aggregometry to explore its potential in triage.MethodsIn this prospective cohort study all patients aged ≥13 admitted to Universitas Airlangga Hospital in Surabaya, Indonesia with a fever (≥38 °C) between 25 January and 1 August 2018 and with a clinical suspicion of DENV, were eligible for inclusion. Exclusion criteria were a thrombocyte count below 100 × 109/L and the use of any medication with a known anticoagulant effect, nonsteroidal anti-inflammatory drugs and acetyl salicylic acid. Clinical data was collected and blood was taken on admission, day 1 and day 7. Samples were tested for acute DENV, using Panbio NS1 ELISA. Platelet aggregation using ADP-, TRAP- and COL-test were presented as Area Under the aggregation Curve (AUC). Significance was tested between DENV+, probably DENV, fever of another origin, and healthy controls (HC).ResultsA total of 59 patients (DENV+ n = 10, DENV probable n = 25, fever other origin n = 24) and 20 HC were included. We found a significantly lower thrombocyte aggregation in the DENV+ group, compared with both HCs and the fever of another origin group (p < .001). Low ADP AUC values on baseline correlated to a longer hospital stay in DENV+ and probable DENV cases.ConclusionThrombocyte aggregation induced by Adenosine diphosphate, Collagen and Thrombin receptor activating peptide-6 is impaired in human DENV cases, compared with healthy controls and other causes of fever. This explorative study provides insights to thrombocyte function in DENV patients and could potentially serve as a future marker in DENV disease.

Time-Resolved Oxygen Exchange Measurements Offer Novel Mechanistic Insights into Enzyme-Catalyzed ATP Synthesis during Photophosphorylation.

Techniques to probe molecular mechanistic events occurring at a single catalytic site of multi-subunit enzymes in real time are few and are still under development. Here time-resolved information is extracted from measurements of the extensive oxygen exchange that occurs at an intermediate stage of adenosine triphosphate (ATP) synthesis during photophosphorylation by chloroplast thylakoids. A stochastic process-based approach for modeling exchange reactions is formulated that provides a physical basis for the kinetic theory. Compatible with the assumptions made in such a model of randomness, the formulation is shown to lead to a Poisson-type theory that enables kinetic analysis of oxygen-exchange data and offers novel physical insights. Parameters such as the apparent rate constant of exchange and the average lifetime of the exchanging intermediates during the synthesis of ATP by the chloroplast F1FO-ATP synthase have been determined over a 5000-fold range of ADP concentration. Experimental isotopomer distributions of [18O]ATP at high (0.5 mM), intermediate (10 μM), and low (0.2 μM) ADP concentrations have been quantified and compared to expected distributions from the theory. The observed distributions are shown to closely match the predicted distributions. A wealth of novel mechanistic insights such as the number of sites/pathways of oxygen exchange, the order of substrate binding steps at the enzyme catalytic site, and regulation of the process of energy coupling have been deduced, and the results are interpreted with the help of available high-resolution X-ray structures. The various biological implications for models of energy coupling have been discussed. Permutation of oxygen ligands about the phosphorus center is proposed as a possible and general but not well-recognized mechanism for oxygen exchange that is consistent with the principal results of this work, and several suggestions for future research are offered.

Modulation of complex II-energized respiration in muscle, heart, and brown adipose mitochondria by oxaloacetate and complex I electron flow.

We recently reported that membrane potential (ΔΨ) primarily determines the relationship of complex II-supported respiration by isolated skeletal muscle mitochondria to ADP concentrations. We observed that O2 flux peaked at low ADP concentration ([ADP]) (high ΔΨ) before declining at higher [ADP] (low ΔΨ). The decline resulted from oxaloacetate (OAA) accumulation and inhibition of succinate dehydrogenase. This prompted us to question the effect of incremental [ADP] on respiration in interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low because of uncoupling protein 1 (UCP1). We found that succinate-energized IBAT mitochondria, even in the absence of ADP, accumulate OAA and manifest limited respiration, similar to muscle mitochondria at high [ADP]. This could be prevented by guanosine 5'-diphosphate inhibition of UCP1. NAD+ cycling with NADH requires complex I electron flow and is needed to form OAA. Therefore, to assess the role of electron transit, we perturbed flow using a small molecule, N1-(3-acetamidophenyl)-N2-(2-(4-methyl-2-(p-tolyl)thiazol-5-yl)ethyl)oxalamide. We observed decreased OAA, increased NADH/NAD+, and increased succinate-supported mitochondrial respiration under conditions of low ΔΨ (IBAT) but not high ΔΨ (heart). In summary, complex II-energized respiration in IBAT mitochondria is tempered by complex I-derived OAA in a manner dependent on UCP1. These dynamics depend on electron transit in complex I.-Fink, B. D., Yu, L., Sivitz, W. I. Modulation of complex II-energized respiration in muscle, heart, and brown adipose mitochondria by oxaloacetate and complex I electron flow.

The signaling effects of ATP on melanoma-like skin cancer

Melanoma is a type of skin cancer originated by the malignant transformation of melanocytes. Increasing incidence and mortality require efforts focused on studies and research about this cancer. Its microenvironment is rich in extracellular ATP, but there are no studies evaluating the ectonucleotidases and ATP effects on tumor-derived melanoma cells with known amounts of ATP. This way, the objective of this work was to evaluate the purinergic signaling in the pathophysiology of in vivo melanoma and the in vitro effects of ATP signaling. We found increased and effective extracellular ATP hydrolysis in platelets and a significant decrease of extracellular ATP levels and adenosine hydrolysis. In addition, we cultured PBMCs of melanoma patients and used ATP salt with specific concentrations to evaluate its signaling effects. The enzymatic activity analysis revealed that even with higher ATP doses cells metabolize adenine nucleotides less efficiently, and present low ATP, ADP and AMP hydrolytic activity in CM compared to CT cells. In summary, we showed for the first time important data about the purinergic signaling in the pathophysiology of melanoma and ATP signaling exercising immunosuppressive effects. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for melanoma management and treatment and could offer novel therapeutic prospects.

Differential Sensitivity of Various Markers of Platelet Activation with Adenosine Diphosphate

A number of techniques have been available to assess platelet activation, but their relative sensitivity is unknown and their usage is variable and not based on any rational criteria. Here, we compared the ability of several techniques based on morphological and biochemical markers to detect the first signs of ADP-induced platelet activation. Scanning electron microscopy of platelets was performed in parallel with flow cytometry to quantify the surface expression of P-selectin (marked by labeled anti-CD62P antibodies), active αIIbβ3-intergrin (assessed by the binding of labeled fibrinogen) and phosphatidylserine (assessed by the binding of labeled Annexin V). When expressed as a fraction of activated platelets, shape changes were the most sensitive to a low ADP concentration compared to the biochemical markers in the following order of sensitivity: morphological changes>fibrinogen binding capacity>P-selectin expression> phosphatidylserine exposure. These results suggest the greater sensitivity of platelet microscopy and the importance of its combination with flow cytometry used to detect surface expression of the molecular markers of platelet activation.

Loss of mitochondrial phosphate carrier in skeletal muscle: dissociation of muscle dysfunction from lower ADP phosphorylating potential

Many mutations in the mitochondrial genome and in nuclear genes that encode mitochondrial proteins hamper oxidative phosphorylation (oxphos) and lead to tissue dysfunction. Yet, whether an energy deficit is necessarily a direct cause of tissue dysfunction is not clear. We addressed this question in mice in which the mitochondrial phosphate carrier, PiC, was depleted from skeletal muscle (skm) by tamoxifen‐induced excision of nuclear gene SLC25A3 encoding PiC (Cre+). Three weeks after tamoxifen injections, PiC protein in skm was &lt;5% of levels found in control (Ctrl) wild‐type mice (tamoxifen‐treated). Oxphos, measured in mitochondria isolated from skm, was greatly suppressed to &lt;2%, 35% or 50% of Ctrl, depending on substrate, with essentially no oxphos when succinate (+rotenone) was supplied and the highest residual oxphos when fatty acid substrate and malate were supplied; the residual oxphos may be driven by alternate Pi uptake on the dicarboxylate carrier. Residual oxphos was similar at 3 and 9 wks of PiC depletion, suggesting that the oxphos defect was stable for this interval. Furthermore, subunits of the oxphos machinery were robustly upregulated (50–70% rise) at both time points. Nonetheless, treadmill running capacity worsened during this time. Time‐to‐exhaustion was ~50 mins in Cre+ mice with PiC absent for 3 wks, but ~25 mins when PiC was absent for 9 wks, whereas Ctrl mice ran for the scheduled 80 mins. Thus, deteriorating treadmill running in Cre+ mice seems unrelated to suppressed oxphos per se. Stress signaling via transcription factor ATF4 as well as elevated mTORC1 signaling have been associated with oxphos defects caused by mitochondrial DNA depletion in cell models and in mouse skm. These models have also suggested a causal relationship between persistent mTORC1 activation/stress signaling and cell dysfunction. Yet we found little evidence for stress signaling via ATF4 (or ATF2, ATF3, or ATF5) after 3 or 9 wks of PiC depletion, and evidence for only mild mTORC1 activation after 9 wks of PiC loss. Overall, these observations suggest that skm dysfunction is not related to an oxphos deficit in a simple way. Rather, the oxphos deficit may drive adaptive mechanisms that either become exhausted or turn maladaptive when ongoing over a longer term, or trigger additional processes that are deleterious for muscle function. Furthermore, the model presented here suggests that a mechanism other than stress signaling through ATFs is responsible for tissue dysfunction in the context of a severe oxphos defect.Support or Funding InformationFunding: R01 GM123771 to ELSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loss of Mitochondrial Phosphate Carrier in Skeletal Muscle: Dissociation of Muscle Dysfunction from Lower ADP Phosphorylating Potential

Loss of Mitochondrial Phosphate Carrier in Skeletal Muscle: Dissociation of Muscle Dysfunction from Lower ADP Phosphorylating Potential

Identification of the signals for glucose-induced insulin secretion in INS1 (832/13) β-cells using metformin-induced metabolic deceleration as a model

Metabolic deceleration in pancreatic β-cells is associated with inhibition of glucose-induced insulin secretion (GIIS), but only in the presence of intermediate/submaximal glucose concentrations. Here, we used acute metformin treatment as a tool to induce metabolic deceleration in INS1 (832/13) β-cells, with the goal of identifying key pathways and metabolites involved in GIIS. Metabolites and pathways previously implicated as signals for GIIS were measured in the cells at 2-25 mm glucose, with or without 5 mm metformin. We defined three criteria to identify candidate signals: 1) glucose-responsiveness, 2) sensitivity to metformin-induced inhibition of the glucose effect at intermediate glucose concentrations, and 3) alleviation of metformin inhibition by elevated glucose concentrations. Despite the lack of recovery from metformin-induced impairment of mitochondrial energy metabolism (glucose oxidation, O2 consumption, and ATP production), insulin secretion was almost completely restored at elevated glucose concentrations. Meeting the criteria for candidates involved in promoting GIIS were the following metabolic indicators and metabolites: cytosolic NAD+/NADH ratio (inferred from the dihydroxyacetone phosphate:glycerol-3-phosphate ratio), mitochondrial membrane potential, ADP, Ca2+, 1-monoacylglycerol, diacylglycerol, malonyl-CoA, and HMG-CoA. On the contrary, most of the purine and nicotinamide nucleotides, acetoacetyl-CoA, H2O2, reduced glutathione, and 2-monoacylglycerol were not glucose-responsive. Overall these results underscore the significance of mitochondrial energy metabolism-independent signals in GIIS regulation; in particular, the candidate lipid signaling molecules 1-monoacylglycerol, diacylglycerol, and malonyl-CoA; the predominance of KATP/Ca2+ signaling control by low ADP·Mg2+ rather than by high ATP levels; and a role for a more oxidized state (NAD+/NADH) in the cytosol during GIIS that favors high glycolysis rates.

Neuromedin U potentiates ADP- and epinephrine-induced human platelet activation

Neuromedin U (NmU) is a pleiotropic hypothalamic neuropeptide involved in the gut–brain axis. It acts via both a Gαq/11-coupled receptor (NMUR1) and a Gαi-coupled receptor (NMUR2) in different cell types. Expression of both receptors was reported in platelets, but their significance for NmU signaling remains elusive. We studied the potential effects of NmU on human platelet activation. In platelet-rich plasma (PRP), NmU alone (up to 10μM) did not induce any measurable aggregation, but at nanomolar concentrations, it potentiated platelet aggregation by low (mean 0.47μM) ADP concentrations (from 25.9±3.6% to 74.8±2.7% maximal aggregation for ADP vs. ADP+NmU, 100nM, mean±SEM, n=13), accompanied by platelet P-selectin expression and intracellular calcium mobilization. Accordingly, platelet preincubation with NmU for 2min sensitized platelets for subsequent activation by ADP. When P2Y1 was inactivated by 50μM MRS2179, NmU comparably potentiated ADP-induced PRP aggregation, suggestive of cooperative activation with Gαi-coupled P2Y12. Likewise, NmU potentiated platelet aggregation by Gαi-operated epinephrine at subthreshold concentrations (99ng/ml, mean), but not that by Gαq-dependent serotonin (20μM). Platelet aggregation by NmU/epinephrine combination was fully inhibited by the Gαq inhibitor YM-254890 (1μM). qPCR detection and western blot analysis substantiated platelet expression of NMUR1 in different donors, a finding collectively complying with functionally relevant Gαq/11-mediated activation of platelet NMUR1 by NmU. Our findings advocate further studies on platelet sensitization by NmU, released during vascular activation and injury, to define its role as a modifier of platelet responsiveness to the physiological activation signals, operational in cardiovascular health and disease.

Nucleotide Dependence of Subunit Rearrangements in Short-Form Rubisco Activase from Spinach

Higher-plant Rubisco activase (Rca) plays a critical role in regulating the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). In vitro, Rca is known to undergo dynamic assembly-disassembly processes, with several oligomer stoichiometries coexisting over a broad concentration range. Although the hexamer appears to be the active form, changes in quaternary structure could play a role in Rubisco regulation. Therefore, fluorescent labels were attached to the C-termini of spinach β-Rca, and the rate of subunit mixing was monitored by measuring energy transfer as a function of nucleotide and divalent cation. Only dimeric units appeared to exchange. Poorly hydrolyzable substrate analogues provided locked complexes with high thermal stabilities (apparent Tm = 60 °C) and an estimated t1/2 of at least 7 h, whereas ATP-Mg provided tight assemblies with t1/2 values of 30-40 min and ADP-Mg loose assemblies with t1/2 values of <15 min. Accumulation of ADP to 20% of the total level of adenine nucleotide substantially accelerated equilibration. An initial lag period was observed with ATP·Mg, indicating inhibition of subunit exchange at low ADP concentrations. The ADP Ki value was estimated to exceed the Km for ATP (0.772 ± 96 mM), suggesting that the equilibration rate is a function of the relative contributions of high- and low-affinity states. C-Terminal cross-linking generated covalent dimers, required the N-terminal extension to the AAA+ domain, and provided evidence of different classes of sites. We propose that oligomer reorganization may be stalled during periods of high Rubisco reactivation activity, whereas changes in quaternary structure are stimulated by the accumulation of ADP at low light levels.

Evaluation of mitochondrial respiratory function in highly glycolytic glioma cells reveals low ADP phosphorylation in relation to oxidative capacity.

High-grade gliomas are aggressive and intensely glycolytic tumors. In the present study, we evaluated the mitochondrial respiratory function of glioma cells (T98G and U-87MG) and fresh human glioblastoma (GBM) tissue. To this end, measurements of oxygen consumption rate (OCR) were performed under various experimental conditions. The OCR of T98G and U-87MG cells was well coupled to ADP phosphorylation based on the ratio of ATP produced per oxygen consumed of ~2.5. In agreement, the basal OCR of GBM tissue was also partially associated with ADP phosphorylation. The basal respiration of intact T98G and U-87MG cells was not limited by the supply of endogenous substrates, as indicated by the increased OCR in response to a protonophore. These cells also displayed a high affinity for oxygen, as evidenced by the values of the partial pressure of oxygen when respiration is half maximal (p 50). In permeabilized glioma cells, ADP-stimulated OCR was only approximately 50% of that obtained in the presence of protonophore, revealing a significant limitation in oxidative phosphorylation (OXPHOS) relative to the activity of the electron transport system (ETS). This characteristic was maintained when the cells were grown under low glucose conditions. Flux control coefficient analyses demonstrated that the impaired OXPHOS was associated with the function of both mitochondrial ATP synthase and the adenine nucleotide translocator, but not the phosphate carrier. Altogether, these data indicate that the availability and metabolism of respiratory substrates and mitochondrial ETS are preserved in T98G and U-87MG glioma cells even though these cells possess a relatively restrained OXPHOS capability.

High Fat Diet Impairs Mitochondrial ADP Sensitivity and Attenuates Respiration in the Presence of Submaximal, but Not Maximal, ADP Concentrations

The consumption of a high fat (HF) diet is a major contributor of ectopic lipid accumulation in skeletal muscle and insulin resistance. While the underlying mechanisms are not well‐defined, mitochondrial dysfunction, including lower ADP sensitivity through the voltage dependent anion channel (VDAC)/adenine nucleotide transport (ANT) axis, has been implicated in this process. It is currently unknown how a HF diet impairs mitochondrial function, however, lipid moieties (palmitoyl‐CoA; P‐CoA) have been shown to inhibit ANT and reduce ADP sensitivity in situ. Therefore, lipid accumulation following a HF diet could promote inhibition on ANT, explaining these mitochondrial abnormalities and possibly insulin resistance. Since we have previously shown mitochondrial creatine kinase (Mi‐CK) knockout (KO) mice have greater ADP sensitivity through the VDAC/ANT axis when challenged by exercise, a situation that also increases intramuscular lipid supply, we determined whether this would be protective against HF‐induced mitochondrial dysfunction and insulin resistance. Mi‐CK wildtype (WT) and KO mice were fed a control (CON) or HF diet (60% fat) for 4 weeks. High‐resolution respirometry was used to assess ADP sensitivity in the presence or absence of P‐CoA in permeabilized muscle fibres, glucose‐tolerance tests were performed, and western blotting was used to determine VDAC and ANT protein content. Our results show on a CON diet that ADP sensitivity in the absence of P‐CoA was not affected by ablating Mi‐CK, while in contrast these mice displayed attenuated P‐CoA‐mediated inhibition of ADP sensitivity. Therefore, we determined if KO mice would be protected from HF diet impairments in ADP sensitivity, and insulin resistance, through improved ADP transport. However, regardless of genotype, consumption of a HF diet resulted in modest reductions in ADP sensitivity in the absence of P‐CoA, which corresponded with reduced insulin sensitivity. This occurred in the absence of altered VDAC or ANT protein content, highlighting external regulation of these proteins following a HF diet. While Mi‐CK KO mice were not protected from HF diet‐induced insulin resistance, this may occur because the improved ADP sensitivity in the presence of P‐CoA was lost during the HF diet. Altogether, these data demonstrate that reductions in ADP sensitivity may be an early event contributing to insulin resistance, while an increase in intramuscular P‐CoA concentrations and an improved sensitivity to P‐CoA inhibition may exacerbate this response.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada; Canadian Foundation for Innovation/Ontario Research Fund.