ADP Ratio Research Articles

Loss of mitochondrial Ca2+ response and CaMKII/ERK activation by LRRK2R1441G mutation correlate with impaired depolarization-induced mitophagy

BackgroundStress-induced activation of ERK/Drp1 serves as a checkpoint in the segregation of damaged mitochondria for autophagic clearance (mitophagy). Elevated cytosolic calcium (Ca2+) activates ERK, which is pivotal to mitophagy initiation. This process is altered in Parkinson’s disease (PD) with mutations in leucine-rich repeat kinase 2 (LRRK2), potentially contributing to mitochondrial dysfunction. Pathogenic LRRK2 mutation is linked to dysregulated cellular Ca2+ signaling but the mechanism involved remains unclear.MethodsMitochondrial damages lead to membrane depolarization. To investigate how LRRK2 mutation impairs cellular response to mitochondrial damages, mitochondrial depolarization was induced by artificial uncoupler (FCCP) in wild-type (WT) and LRRK2R1441G mutant knockin (KI) mouse embryonic fibroblasts (MEFs). The resultant cytosolic Ca2+ flux was assessed using live-cell Ca2+ imaging. The role of mitochondria in FCCP-induced cytosolic Ca2+ surge was confirmed by co-treatment with the mitochondrial sodium-calcium exchanger (NCLX) inhibitor. Cellular mitochondrial quality and function were evaluated by Seahorse™ real-time cell metabolic analysis, flow cytometry, and confocal imaging. Mitochondrial morphology was visualized using transmission electron microscopy (TEM). Activation (phosphorylation) of stress response pathways were assessed by immunoblotting.ResultsAcute mitochondrial depolarization induced by FCCP resulted in an immediate cytosolic Ca2+ surge in WT MEFs, mediated predominantly via mitochondrial NCLX. However, such cytosolic Ca2+ response was abolished in LRRK2 KI MEFs. This loss of response in KI was associated with impaired activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and MEK, the two upstream kinases of ERK. Treatment of LRRK2 inhibitor did not rescue this phenotype indicating that it was not caused by mutant LRRK2 kinase hyperactivity. KI MEFs exhibited swollen mitochondria with distorted cristae, depolarized mitochondrial membrane potential, and reduced mitochondrial Ca2+ store and mitochondrial calcium uniporter (MCU) expression. These mutant cells also exhibited lower cellular ATP: ADP ratio albeit higher basal respiration than WT, indicating compensation for mitochondrial dysfunction. These defects may hinder cellular stress response and signals to Drp1-mediated mitophagy, as evident by impaired mitochondrial clearance in the mutant.ConclusionsPathogenic LRRK2R1441G mutation abolished mitochondrial depolarization-induced Ca2+ response and impaired the basal mitochondrial clearance. Inherent defects from LRRK2 mutation have weakened the cellular ability to scavenge damaged mitochondria, which may further aggravate mitochondrial dysfunction and neurodegeneration in PD.

Compartmentalization in cardiomyocytes modulates creatine kinase and adenylate kinase activities.

Intracellular molecules are transported by motor proteins or move by diffusion resulting from random molecular motion. Cardiomyocytes are packed with structures that are crucial for function, but also confine the diffusional spaces, providing cells with a means to control diffusion. They form compartments in which local concentrations are different from the overall, average concentrations. For example, calcium and cyclic AMP are highly compartmentalized, allowing these versatile second messengers to send different signals depending on their location. In energetic compartmentalization, the ratios of AMP and ADP to ATP are different from the average ratios. This is important for the performance of ATPases fuelling cardiac excitation-contraction coupling and mechanical work. A recent study suggested that compartmentalization modulates the activity of creatine kinase and adenylate kinase in situ. This could have implications for energetic signaling through, for example, AMP-activated kinase. It highlights the importance of taking compartmentalization into account in our interpretation of cellular physiology and developing methods to assess local concentrations of AMP and ADP to enhance our understanding of compartmentalization in different cell types.

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.

Differences in Brain Region Mitochondrial Bioenergetics Detected Using CK Clamp

The brain is a non-uniform organ comprised of discrete structures of varying function and cell composition. The differing function of brain regions ostensibly would necessitate disparate energetic demands, thereby suggesting brain bioenergetics would be different among brain regions. This study aimed to address whether brain regions indeed have different bioenergetics by evaluating substrate preference, fuel-specific mitochondrial function at physiologically relevant energy demands as well as associated membrane potential and electron conductance using high-resolution respirometry (Oroboros O2k) in the frontal cortex, hypothalamus, and hippocampus. We hypothesize there will be different bioenergetics among brain regions. Substrate preference was established using a substrate-uncoupler-inhibitor-titration (SUIT) with additions, in order: ADP, Pyruvate/Malate, UK5099, Octanoylcarnitine, Rotenone, Succinate, and Antimycin A. There was no statistically significant interaction between region and substrate (p=0.15) nor a main effect of region (p=0.91), however, there was a main effect of substrate in which pyruvate/malate (+275%) and succinate/rotenone (+170%) had significantly greater respiration than octanoylcarnitine, independent of region (p<0.001). Given the results of the SUIT test, pyruvate/malate was used for subsequent tests. The SUIT method relies upon saturating ADP levels and non-physiological ATP:ADP concentration ratios, the extent to which this test obscures the interpretation of brain region respiration is unclear. Instead, an alternative approach that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiological ATP:ADP ratios, “CK Clamp,” was used. Function of mitochondria with a pyruvate fuel was then evaluated in all three brain regions in which energetic demand was clamped at ΔGATP = -12.87 (high demand), -14.08, -14.5, and -14.85 (low demand). There was a statistically significant interaction between brain region and clamped ΔGATP values (p = 0.02). Frontal cortex displayed higher respiration than hippocampus at -12.87 ΔGATP (33%). There was no statistically significant interaction between region and clamp for mitochondrial membrane potential assessed during the CK clamp (p=0.16), however there was a significant main effect of clamped ΔGATP (p<0.001) in which membrane potential increased (i.e., hyperpolarized) as energetic demand decreased. This agrees with the bioenergetic model that a decrease in energetic demand increases the proton motive force (aka, hyperpolarization), thereby applying a backpressure on the electron transport chain, slowing respiration. Electron conductance was then assessed by calculating the slope of respiration versus clamped ΔGATP. Electron conductance was greater in the frontal cortex than hypothalamus (+70%, p = 0.015). The conclusion of this study is there are differences in mitochondrial bioenergetics among brain regions and the CK clamp was more sensitive at detecting these differences than the SUIT method. None. 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.

Comparison of hole expansion ratios in a dual phase steel for holes prepared by conventional punching and fine piercing

Dual phase steels generally exhibit poorer stretch flangeability compared to lower strength steels. Stretch flangeability is normally evaluated using hole expansion testing, wherein a specimen with a 10 mm central hole is expanded using a conical punch till onset of cracking at the edge of the hole. The percentage increase in hole diameter is referred to as the hole expansion ratio (HER). In this study, hole expansion ratios of a DP 600 steel were evaluated. Holes were prepared on a standard 90 mm x 90 mm specimen using two different hole preparation conditions: i) conventional punching where the hole edge exhibits both fracture and shear regions and ii) fine piercing where the hole edge exhibits almost completely smooth shear. Hole expansion testing was carried out on these specimens as per standard ISO 16630. The specimens with holes produced by fine piercing exhibited lower values of HER in spite of having a hole edge with a smooth surface. The specimens with holes prepared by conventional punching exhibited higher HER values. The influence of strain hardening of the cut edge during hole preparation and the surface roughness of the cut edge on the HER are discussed.

Metformin Effects on SHIP2, AMPKs and Gut Microbiota: Recent Updates on Pharmacology.

Metformin, a biguanide on the WHO's list of essential medicines has a long history of 50 years or more in treating hyperglycemia, and its therapeutic saga continues beyond diabetes treatment. Glucoregulatory actions are central to the physiological effects of metformin; surprisingly, the precise mechanism with which metformin regulates glucose metabolism is not thoroughly understood yet. The main aim of this review is to explore the recent implications of metformin in hepatic gluconeogenesis, AMPKs, and SHIP2 and subsequently to elucidate the metformin action across intestine and gut microbiota. We have searched PubMed, google scholar, Medline, eMedicine, National Library of Medicine (NLM), clinicaltrials.gov (registry), and ReleMed for the implications of metformin with its updated role in AMPKs, SHIP2, and hepatic gluoconeogenesis, and gut microbiota. In this review, we have described the efficacy of metformin as a drug repurposing strategy in modulating the role of AMPKs and lysosomal-AMPKs, and controversies associated with metformin. Research suggests that biguanide exhibits hormetic effects depending on the concentrations used (micromolar to millimolar). The primary mechanism attributed to metformin action is the inhibition of mitochondrial complex I, and subsequent reduction of cellular energy state, as observed with increased AMP or ADP ratio, thereby metformin can also activate the cellular energy sensor AMPK to inhibit hepatic gluconeogenesis. However, new mechanistic models have been proposed lately to explain the pleiotropic actions of metformin; at low doses, metformin can activate lysosomal-AMPK via the AXIN-LKB1 pathway. Conversely, in an AMPK-independent mechanism, metformin-induced elevation of AMP suppresses adenylate cyclase and glucagon-activated cAMP production to inhibit hepatic glucose output by glucagon. Metformin inhibits mitochondrial glycerophosphate dehydrogenase; mGPDH, and increases the cytosolic NADH/NAD+, affecting the availability of lactate and glycerol for gluconeogenesis. Metformin can inhibit Src homology 2 domain-containing inositol 5-phosphatase 2; SHIP2 to increase the insulin sensitivity and glucose uptake by peripheral tissues. In addition, new exciting mechanisms suggest the role of metformin in promoting beneficial gut microbiome and gut health; metformin regulates duodenal AMPK activation, incretin hormone secretion, and bile acid homeostasis to improve intestinal glucose absorption and utilization.

Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals.

Motor neurons are the longest neurons in the body, with axon terminals separated from the soma by as much as a meter. These terminals are largely autonomous with regard to their bioenergetic metabolism and must burn energy at a high rate to sustain muscle contraction. Here, through computer simulation and drawing on previously published empirical data, we determined that motor neuron terminals in Drosophilalarvae experience highly volatile power demands. It might not be surprising then, that we discovered the mitochondria in the motor neuron terminals of both Drosophilaand mice to be heavily decorated with phosphagen kinases - a key element in an energy storage and buffering system well-characterized in fast-twitch muscle fibres. Knockdown of arginine kinase 1 (ArgK1) in Drosophila larval motor neurons led to several bioenergetic deficits, including mitochondrial matrix acidification and a faster decline in the cytosol ATP to ADP ratio during axon burst firing. KEY POINTS: Neurons commonly fire in bursts imposing highly volatile demands on the bioenergetic machinery that generates ATP. Using a computational approach, we built profiles of presynaptic power demand at the level of single action potentials, as well as the transition from rest to sustained activity. Phosphagen systems are known to buffer ATP levels in muscles and we demonstrate that phosphagen kinases, which support such phosphagen systems, also localize to mitochondria in motor nerve terminals of fruit flies and mice. By knocking down phosphagen kinases in fruit fly motor nerve terminals, and using fluorescent reporters of the ATP:ADP ratio, lactate, pH and Ca2+ , we demonstrate a role for phosphagen kinases in stabilizing presynaptic ATP levels. These data indicate that the maintenance of phosphagen systems in motor neurons, and not just muscle, could be a beneficial initiative in sustaining musculoskeletal health and performance.

Kinetic modelling of β-cell metabolism reveals control points in the insulin-regulating pyruvate cycling pathways.

Insulin, a key hormone in the regulation of glucose homoeostasis, is secreted by pancreatic β-cells in response to elevated glucose levels. Insulin is released in a biphasic manner in response to glucose metabolism in β-cells. The first phase of insulin secretion is triggered by an increase in the ATP:ADP ratio; the second phase occurs in response to both a rise in ATP:ADP and other key metabolic signals, including a rise in the NADPH:NADP+ ratio. Experimental evidence indicates that pyruvate-cycling pathways play an important role in the elevation of the NADPH:NADP+ ratio in response to glucose. The authors developed a kinetic model for the tricarboxylic acid cycle and pyruvate cycling pathways. The authors successfully validated the model against experimental observations and performed a sensitivity analysis to identify key regulatory interactions in the system. The model predicts that the dicarboxylate carrier and the pyruvate transporter are the most important regulators of pyruvate cycling and NADPH production. In contrast, the analysis showed that variation in the pyruvate carboxylase flux was compensated by a response in the activity of mitochondrial isocitrate dehydrogenase (ICDm ) resulting in minimal effect on overall pyruvate cycling flux. The model predictions suggest starting points for further experimental investigation, as well as potential drug targets for the treatment of type 2 diabetes.

Fatty acid and energy (ATP-related compounds) metabolism of Eurasian perch (Perca fluviatilis) sperm: Endogenous metabolic strategy

Structural (fatty acids) and energetic (ATP-related compounds) processes of spermatozoa of Eurasian perch (Perca fluviatilis) has been studied. Present study identified changes in fatty acids and adenosine phosphate stoichiometries as sperm ascended from basal metabolism (pre-activation quiescent state) to active metabolism (post-activation exhaustion). Results suggest long chain unsaturated (C14:0, C16:0) and monounsaturated fatty acids (C16:1) were significantly utilized (p < 0.05) for energy yielding purposes. Polyunsaturated fatty acids, C20:5n-3 (EPA) content was reduced from basal metabolism (1.09–2.57 mg g−1) to post-activation (0.69–2.39 mg g−1), while C20:4n-6 (ARA) and C22:6n-3 (DHA) remained preferentially conserved. In a way that ARA: EPA and DHA: EPA ratios became significantly higher (p < 0.05) in exhausted than quiescent state, for potential maintenance of membrane fluidity by DHA or eicosanoid activity (anti-inflammatory EPA to pro-inflammatory ARA balancing) during motility. Although endogenous fatty acid bioconversion capabilities could not be proved by the present study, these findings encourage future research. In terms of nucleotides, K-value (relative degradation of ATP; metabolic loss) was 30% at basal metabolism. ATP consumption equalled production at quiescent state, as the ratio of forward and backward reaction products in adenylate kinase reaction was maintained at ≈1 (equilibrium). With activation, 0.21–0.65 nmol ATP second−1 per 109 spermatozoa was metabolized and K-value showed 85–90% of pre-stored ATP pool degraded to non-calorific inosine and hypoxanthine. There was missing AMP, lowered ATP: ADP ratio and adenylate kinase reaction equilibrium was lost. We conclude a potential mismatch between glycolysis and substrates led to exhaustion in P. fluviatilis sperm, despite a tendency towards selective FA utilization to ensure membrane fluidity (DHA conservation) or prostaglandin and anti-inflammatory responses (ARA and EPA) till its last moments.

Actin cytoskeletal dynamics do not impose an energy drain on growth cone bioenergetics.

The regulation of the intracellular level of ATP is a fundamental aspect of bioenergetics. Actin cytoskeletal dynamics have been reported to be an energetic drain in developing neurons and platelets. We addressed the role of actin dynamics in primary embryonic chicken neurons using luciferase assays, and by measurement of the ATP/ADP ratio using the ratiometric reporter PercevalHR and the ATP level using the ratiometric reporter mRuby-iATPSnFR. None of the methods revealed an effect of suppressing actin dynamics on the decline in the neuronal ATP level or the ATP/ADP ratio following shutdown of ATP production. Similarly, we find that treatments that elevate or suppress actin dynamics do not alter the ATP/ADP ratio in growth cones, the subcellular domain with the highest actin dynamics in developing neurons. Collectively, the data indicate that actin cytoskeletal dynamics are not a significant energy drain in developing neurons and that the ATP/ADP ratio is maintained when energy utilization varies. Discrepancies between prior work and the current data are discussed with emphasis on methodology and interpretation of the data.

Analogy or fallacy, unsafe chemical alternatives: Mechanistic insights into energy metabolism dysfunction induced by Bisphenol analogs in HepG2 cells

Bisphenol analogs (BPs) are widely used as industrial alternatives for Bisphenol A (BPA). Their toxicity assessment in humans has mainly focused on estrogenic activity, while other toxicity effects and mechanisms resulting from BPs exposure remain unclear. In this study, we investigated the effects of three BPs (Bisphenol AF (BPAF), Bisphenol G (BPG) and Bisphenol PH (BPPH)) on metabolic pathways of HepG2 cells. Results from comprehensive cellular bioenergetics analysis and nontarget metabolomics indicated that the most important process affected by BPs exposure was energy metabolism, as evidenced by reduced mitochondrial function and enhanced glycolysis. Compared to the control group, BPG and BPPH exhibited a consistent pattern of metabolic dysregulation, while BPAF differed from both, such as an increased ATP: ADP ratio (1.29-fold, p < 0.05) observed in BPAF and significantly decreased ATP: ADP ratio for BPG (0.28-fold, p < 0.001) and BPPH (0.45-fold, p < 0.001). Bioassay endpoint analysis revealed BPG/BPPH induced alterations in mitochondrial membrane potential and overproductions of reactive oxygen species. Taken together these data suggested that BPG/BPPH induced oxidative stress and mitochondrial damage in cells results in energy metabolism dysregulation. By contrast, BPAF had no effect on mitochondrial health, but induced a proliferation promoting effect on cells, which might contribute to the energy metabolism dysfunction. Interestingly, BPPH induced the greatest mitochondrial damage among the three BPs but did not exhibit Estrogen receptor alpha (ERα) activating effects. This study characterized the distinct metabolic mechanisms underlying energy metabolism dysregulation induced by different BPs in target human cells, providing new insight into the evaluation of the emerging BPA substitutes.

The bioenergetic "CK Clamp" technique detects substrate-specific changes in mitochondrial respiration and membrane potential during early VML injury pathology.

Volumetric muscle loss (VML) injuries are characterized by non-recoverable loss of tissue resulting in contractile and metabolic dysfunction. The characterization of metabolic dysfunction in volumetric muscle loss-injured muscle has been interpreted from permeabilized myofiber respiration experiments involving saturating ADP levels and non-physiologic ATP:ADP concentration ratios. The extent to which this testing condition obscures the analysis of mitochondrial (dys) function after volumetric muscle loss injury is unclear. An alternative approach is described that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiologic ATP:ADP ratios, "CK Clamp." The objective of this study was to validate the CK Clamp in volumetric muscle loss-injured muscle and to detect differences that may exist between volumetric muscle loss-injured and uninjured muscles at 1, 3, 5, 7, 10, and 14days post-injury. Volumetric muscle loss-injured muscle maintains bioenergetic features of the CK Clamp approach, i.e., mitochondrial respiration rate (JO2) titters down and mitochondrial membrane potential is more polarized with increasing ATP:ADP ratios. Pyruvate/malate/succinate-supported JO2 was significantly less in volumetric muscle loss-injured muscle at all timepoints compared to uninjured controls (-26% to -84%, p < 0.001) and electron conductance was less at day 1 (-60%), 5 (-52%), 7 (-35%), 10 (-59%), and 14 (-41%) (p < 0.001). Palmitoyl-carnitine/malate-supported JO2 and electron conductance were less affected following volumetric muscle loss injury. volumetric muscle loss-injury also corresponded with a more polarized mitochondrial membrane potential across the clamped ATP:ADP ratios at day 1 and 10 (pyruvate and palmitoyl-carnitine, respectively) (+5%, p < 0.001). This study supports previous characterizations of metabolic dysfunction and validates the CK Clamp as a tool to investigate bioenergetics in traumatically-injured muscle.

Analysis of Energy-Driven Leader-Follower Hierarchy During Collective Cancer Cell Invasion.

Many solid tumors can invade the surrounding three-dimensional (3D) tissue in a collective manner, and increasing evidence suggests that collective migration makes cancer cell clusters more invasive and metastatic than individual cells. A cohesive cohort of cancer cells can have many advantages over individual cells, including more efficient bioenergetics that have been recently identified. Minimization of bioenergetic costs during collective cell migration drives leader-follower dynamics and contributes to enhanced cancer invasion. Hence, it is critical to understand the migratory and bioenergetic dynamics of cancer collective invasion. While analysis of structures and dynamics in a 3D space has been a challenging task, here we describe a widely applicable method to analyze the energy-driven leader-follower hierarchy during cancer collective invasion. An in vitro tumor spheroid model is employed to reproduce the in vivo collective behaviors of cancer cells while allowing high spatiotemporal resolution imaging, where the leader-follower dynamics can be analyzed by tracking nuclear positions. As glucose is one of the main energy sources that fuel cancer cell migration, the quantification of glucose uptake along the invading strands provides an estimate of the energy demand associated with collective invasion. Finally, we describe a method to quantify the dynamics of intracellular energy level using the PercevalHR ATP:ADP ratio biosensor.

IPSC-Derived Neurons from Patients with POLG Mutations Exhibit Decreased Mitochondrial Content and Dendrite Simplification

Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase gamma, result in clinical syndromes characterized by mitochondrial DNA (mtDNA) depletion in affected tissues with variable organ involvement. The brain is one of the most affected organs, and symptoms include intractable seizures, developmental delay, dementia, and ataxia. Patient-derived induced pluripotent stem cells (iPSCs) provide opportunities to explore mechanisms in affected cell types and potential therapeutic strategies. Fibroblasts from two patients were reprogrammed to create new iPSC models of POLG-related mitochondrial diseases. Compared with iPSC-derived control neurons, mtDNA depletion was observed upon differentiation of the POLG-mutated lines to cortical neurons. POLG-mutated neurons exhibited neurite simplification with decreased mitochondrial content, abnormal mitochondrial structure and function, and increased cell death. Expression of the mitochondrial kinase PTEN-induced kinase 1 (PINK1) mRNA was decreased in patient neurons. Overexpression of PINK1 increased mitochondrial content and ATP:ADP ratios in neurites, decreasing cell death and rescuing neuritic complexity. These data indicate an intersection of polymerase gamma and PINK1 pathways that may offer a novel therapeutic option for patients affected by this spectrum of disorders.

Electrolyzed reduced water could regulate muscular energy producing ability of heat-exposed broiler chickens

We have previously shown that alleviation in ROS-induced oxidative damage to skeletal muscle and improvement in production efficiencies are observed in broiler chickens supplied with electrolyzed-reduced water when exposed to 5 days-long chronic heat exposure. In this study, we investigated whether this reduced water may also improve those parameters in broiler chickens exposed to 14 days-long chronic heat exposure. Broiler chickens (Gallus gallus) were fed either a control diet with tape water or control diet supplied with electrolyzed-reduced water for 14 d after hatch, and then exposed either to heat stress (34oC for 14 days), or kept at a thermoneutral temperature (24oC). Skeletal muscle substrate metabolism (3HADH and CS activities), adenine nucleotides concentration, enzymatic scavenging systems were studied. We confirmed that heat-stressed broilers decreased weight gain and feed consumption compared to control broilers. In contrast, heat-exposed broilers supplied with electrolyzed-reduced water significantly and definitely improved performance when compared to heat-stressed control broilers. On exposure to long-term heat treatments, broiler supplied with reduced water showed higher CS activity, indicating that the reduced water could improve energy-producing ability via modulating TCA cycle. We also found that the reduced water could definitely improve the total adenine nucleotide (TAN), adenylated energy charge (AEC), and ATP to ADP ratios under such conditions. On the other hand, broiler chickens supplied with reduced water kept relatively constant the SOD, catalase, and GPx activities, implying that reduced water could support the enzymatic scavenging systems. Taken together, these results suggest that broiler chickens supplied with electrolyzed-reduced water could improve energy producing ability via modulating TCA-cycle enzyme, and lead to improving performance.

SPICE-Met: profiling and imaging energy metabolism at the single-cell level using a fluorescent reporter mouse.

The regulation of cellular energy metabolism is central to most physiological and pathophysiological processes. However, most current methods have limited ability to functionally probe metabolic pathways in individual cells. Here, we describe SPICE-Met (Single-cell Profiling and Imaging of Cell Energy Metabolism), a method for profiling energy metabolism in single cells using flow cytometry or imaging. We generated a transgenic mouse expressing PercevalHR, a fluorescent reporter for cellular ATP:ADP ratio. Modulation of PercevalHR fluorescence with metabolic inhibitors was used to infer the dependence of energy metabolism on oxidative phosphorylation and glycolysis in defined cell populations identified by flow cytometry. We applied SPICE-Met to analyze T-cell memory development during vaccination. Finally, we used SPICE-Met in combination with real-time imaging to dissect the heterogeneity and plasticity of energy metabolism in single macrophages ex vivo and identify three distinct metabolic patterns. Functional probing of energy metabolism with single-cell resolution should greatly facilitate the study of immunometabolism at a steady state, during disease pathogenesis or in response to therapy.

Lithium Enhances Hippocampal Glucose Metabolism in an In Vitro Mice Model of Alzheimer's Disease.

Impaired cerebral glucose metabolism is an early event that contributes to the pathogenesis of Alzheimer’s disease (AD). Importantly, restoring glucose availability by pharmacological agents or genetic manipulation has been shown to protect against Aβ toxicity, ameliorate AD pathology, and increase lifespan. Lithium, a therapeutic agent widely used as a treatment for mood disorders, has been shown to attenuate AD pathology and promote glucose metabolism in skeletal muscle. However, despite its widespread use in neuropsychiatric disorders, lithium’s effects on the brain have been poorly characterized. Here we evaluated the effect of lithium on glucose metabolism in hippocampal neurons from wild-type (WT) and APPSwe/PS1ΔE9 (APP/PS1) mice. Our results showed that lithium significantly stimulates glucose uptake and replenishes ATP levels by preferential oxidation of glucose through glycolysis in neurons from WT mice. This increase was also accompanied by a strong increase in glucose transporter 3 (Glut3), the major carrier responsible for glucose uptake in neurons. Similarly, using hippocampal slices from APP-PS1 mice, we demonstrate that lithium increases glucose uptake, glycolytic rate, and the ATP:ADP ratio in a process that also involves the activation of AMPK. Together, our findings indicate that lithium stimulates glucose metabolism and can act as a potential therapeutic agent in AD.

Development of Fire Retardant on Jute by Chemical Means

Jute is highly flammable in character. Due to its high degree of flammability, the versatile use of this fibre is handicapped to some extent, particularly in some specific purpose where jute products with flame resistance finishes and demanded. Considering this disadvantage, a research project was undertaken to make this fibre flameproof and therefore safer in specialized textile uses. The study was performed using yarn and fabrics which were desized with diastase and lissapol-N. Yarn and fabrics were scoured with sodium carbonate, sodium hydroxide under some standard conditions. These pretreated yarns and fabrics were used in the whole experimental work. The treated yarns were tested for flame retardant by subjecting them to the luminous flame of Bunsen burner and by observing the time of flaming (after flame) and time of glowing (flameless combustion, after glow), if any Percentage losses of strength of the treated yarn and fabrics were also measured by standard method. Different solutions of fire resistant chemicals were prepared to change the chemical, concentration and pH ratio of the solution. Jute fabrics and yarns treated with 65% solution of urea and ammonium dihydrogen phosphate (the ratio of urea and ADP being 3:2) together with 2% Turpex NP and 3-6% perapret PE-40% were found durably flame retardant causing minimum loss of the strength. This research was focused on fire resistant treatment of jute yarn and fabrics with different chemicals to make jute products for diversified textile uses.

Intermitochondrial signaling regulates the uniform distribution of stationary mitochondria in axons

In the central nervous system (CNS), many neurons develop axonal arbors that are crucial for information processing. Previous studies have demonstrated that premature axons contain motile and stationary mitochondria, and their balance is important for axonal arborization. However, the mechanisms by which neurons determine the positions of stationary mitochondria as well as their turnover remain to be elucidated. We observed that the distribution of stationary mitochondrial spots along the unmyelinated and nonsynaptic axons is not random but rather relatively uniform both in primary cultured neurons and in tissues. Intriguingly, whereas the positions of each mitochondrial spot changed over time, the overall distribution remained uniform. In addition, local inactivation of mitochondria by KillerRed mediated chromophore-assisted light inactivation (CALI) inhibited the translocation of mitochondrial spots in adjacent axonal regions, suggesting that functional mitochondria enhance the motility of other mitochondria in the vicinity. Signals of ATP:ADP sensor, PercevalHR indicated that the ATP:ADP ratio was relatively high around mitochondria, and treating axons with phosphocreatine (PCr), which supplies ATP, reduced the immobile mitochondria induced by the local mitochondrial inactivation. In a mathematical model, we found that the ATP gradient generated by mitochondria, and ATP dependent regulation of mitochondrial motility could establish uniform mitochondrial distribution. These observations suggest that axons in the CNS possess the system that distributes mitochondria uniformly, and intermitochondrial signaling contribute to the regulation. In addition, our results suggest the possibility that ATP might be one of the molecules mediating the signaling.

Novel cryo-EM structure of an ADP-bound GroEL\u2013GroES complex

The GroEL–GroES chaperonin complex is a bacterial protein folding system, functioning in an ATP-dependent manner. Upon ATP binding and hydrolysis, it undergoes multiple stages linked to substrate protein binding, folding and release. Structural methods helped to reveal several conformational states and provide more information about the chaperonin functional cycle. Here, using cryo-EM we resolved two nucleotide-bound structures of the bullet-shaped GroEL–GroES1 complex at 3.4 Å resolution. The main difference between them is the relative orientation of their apical domains. Both structures contain nucleotides in cis and trans GroEL rings; in contrast to previously reported bullet-shaped complexes where nucleotides were only present in the cis ring. Our results suggest that the bound nucleotides correspond to ADP, and that such a state appears at low ATP:ADP ratios.