NADH Fluorescence Research Articles

Kisspeptin Amplifies GLP‐1 Potentiation of Insulin Secretion by a cAMP Independent Pathway

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic b‐cells, and activation of both receptors potentiates glucose stimulated insulin secretion (GSIS). Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 GLP‐1 ~20 nM vs KP >1 uM). The effects of GLP‐1 and a 10 amino acid analog of KP (KP10) on insulin secretion and cell signaling were studied in the INS 832/3 cell line. By itself KP10 has no effect on insulin secretion in the absence of glucose, but doubled GSIS (GSIS = 37.7 +/− 6.7; +KP10 = 69.2 +/− 1.9 ng/ml/80min) with an EC50 of ~5–10 mM. At saturating doses, KP10 (100 mm) and GLP‐1 (100 nM) had similar effects on GSIS, and in combination had an additive effect (GLP‐1 = 49.0 +/− 2.0, GLP‐1 + KP10 = 63.5 +/− 5.6 ng/ml/80min) consistent with activation of independent downstream signaling pathways. Neither GLP‐1 or KP‐10 altered intracellular Ca2+ levels at basal or activating glucose concentrations. GLP‐1 increased cAMP production (165.0 +/− 17.4 % over control), whereas KP‐10 was without effect. Conversely, pretreatment with KP‐10 enhanced glucose stimulated oxygen consumption and mitochondrial NADH fluorescence, although both responses were inconsistent. Therefore, the effects of KP on b‐cell metabolic activity needs to be investigated in more detail.We also developed a bivalent ligand composed of GLP‐17‐36 and KP10 binding domains, linked with a 3‐mer of proline/glycine repeats, to evaluate if linking KP10 to a high affinity ligand could promote KP10 activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers with an EC50 ~200nM; that is, an additive response was not obvious. Currently, we are determining the ability of GLP‐1/KP10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP10 is not.Support or Funding InformationFunded by the American Diabetes Association and Juvenile Diabetes Research Foundation

Changes in mitochondrial NAD redox state drive acidosis induced acute kidney injury

The kidney has an important role in maintaining normal blood pH. Mitochondria in the proximal tubule (PT) produce ammonia and bicarbonate from glutamine, and this pathway (ammoniagenesis) is acutely upregulated in PTs during metabolic acidosis (MA). MA is associated with an accelerated renal function decline in patients with chronic kidney disease (CKD). MA is also frequently found in patients with acute kidney injury (AKI) and correction of acid‐base status improves outcome, suggesting a direct relationship between MA and AKI. However, there is a surprising lack of experimental knowledge on the molecular mechanism responsible for cellular dysfunction associated with MA. Using live cell imaging and other complementary techniques we aimed to increase understanding of how PTs respond acutely to MA.Acute exposure of mouse kidney cortical slices to acidic extracellular pH caused a decrease in mitochondrial NADH fluorescence signal specifically in PTs, without changing total NADH content, consistent with a switch to a more oxidized state. Mitochondria remained energized and baseline oxygen consumption rate (OCR) was unchanged. Electron entry into the respiratory chain (RC) is thought to be predominantly via complex II (CII) in the kidney. However, under acidosis maximal OCR in isolated PTs was decreased and response to rotenone was exaggerated, suggesting a switch to complex I, which oxidizes NADH. Large vacuole like structures subsequently appeared in S2 PT segments, which were identified as lipid filled multi‐lamellar bodies (MLBs) on electron microscopy. Supplementation with nicotinamide or reducing NAD with lactate both inhibited the appearance of vacuoles. Moreover, increasing pH back to 7.4 reversed changes in NADH signal and led to disappearance of vacuoles.MA was induced in vivo using an established protocol (0.8 g/kg BW NH4Cl). Blood pH decreased to 7.0–7.1, which is comparable to patients with MA and AKI. Histological analysis showed thinning of PTs and shedding of debris, consistent with an acute metabolic insult. Large vacuoles were observed in S2 PT segments. Intravital imaging revealed that mitochondria in PTs remained energized, but uptake of fluorescently labeled dextrans by endocytosis was severely inhibited. Glomerular filtration rate (GFR) and tubular flow were unaltered during MA, excluding major hemodynamic effects.In summary, we have found evidence that MA induces an acute alteration in mitochondrial NAD redox state and lipid metabolism in PTs, which is explained by a switch to increased complex I activity. While these changes are potentially beneficial for ammoniagenesis, they are associated with major defects in PT cell structure and transport function. Thus, we show that MA directly causes AKI, and experiments are ongoing to assess whether bicarbonate supplementation has beneficial effects.Support or Funding InformationSwiss National Science Foundation and The Swiss National Centre for Competence in Research (NCCR) Kidney Control of Homeostasis. JRM is a IKPP2‐fellow funded by the EU 7th Framework Programme for research, technological development and demonstration under grant agreement 608847.

Zastosowanie pomiaru fluorescencji NADH w ocenie mikrokrążenia

There is compelling evidence for a link between microvascular and endothelial dysfunction and the pathogenesis of cardiovascular disease. Endothelial dysfunction is a globalized systemic disease process consisting of attenuated vasodilation and augmented vasoconstriction. Till now the assessment of microvascular endothelial function was mainly based on perfusion methods perform in response to various stimuli. Recently, there is a new method to assess NADH fluorescence that allows to follow metabolic changes depending on the blood flow.

Metabolic mapping of glioblastoma stem cells reveals NADH fluxes associated with glioblastoma phenotype and survival.

.Significance: Glioblastoma multiforme (GBM) is the most frequently diagnosed adult primary brain malignancy with poor patient prognosis. GBM can recur despite aggressive treatment due to therapeutically resistant glioblastoma stem cells (GSCs) that may exhibit metabolic plasticity.Aim: Intrinsic nicotinamide adenine dinucleotide (NADH) fluorescence can be acquired with fluorescence lifetime imaging microscopy (FLIM) to examine its bound and free metabolic states in GSC and GBM tissues.Approach: We compared the mean NADH fluorescence lifetime in live human GSCs and normal neural stem cells and validated those results by measuring oxygen consumption rates (OCRs). We also examined the role that invasive versus less-invasive GSCs had on tumor metabolism by measuring the mean NADH lifetimes and the relative amount of the longer-lived component of NADH and correlated these results with survival in an orthotopic mouse xenograft model.Results: Mean NADH lifetime, amount of bound NADH, and OCR were increased in GSCs. Compared with normal mouse brain, mean NADH lifetimes were longer for all GBM tissues. Invasive xenografts had higher relative amounts of the longer-lived NADH component, and this correlated with decreased survival.Conclusions: FLIM offers cellular resolution quantification of metabolic flux in GBM phenotypes, potentially informing biomedical researchers on improved therapeutic approaches.

Quantifying Age-Related Changes in Skin Wound Metabolism Using In Vivo Multiphoton Microscopy.

Objective: The elderly are at high risk for developing chronic skin wounds, but the effects of intrinsic aging on skin healing are difficult to isolate due to common comorbidities like diabetes. Our objective is to use multiphoton microscopy (MPM) to find endogenous, noninvasive biomarkers to differentiate changes in skin wound healing metabolism between young and aged mice in vivo. Approach: We utilized MPM to monitor skin metabolism at the edge of full-thickness, excisional wounds in 24- and 4-month-old mice of both sexes for 10 days. MPM can assess quantitative biomarkers of cellular metabolism in vivo by utilizing autofluorescence from the cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Results: An optical redox ratio of FAD/(NADH+FAD) autofluorescence and NADH fluorescence lifetime imaging revealed dynamic changes in keratinocyte function during healing. Aged female mice demonstrated an attenuation of keratinocyte proliferation during wound healing detectable optically through a higher redox ratio and longer NADH fluorescence lifetime. By measuring the correlation between NADH lifetime and the optical redox ratio at each day, we also demonstrate sensitivity to the proliferative phase of wound healing. Innovation: Label-free MPM was used to longitudinally monitor individual wounds in vivo, which revealed age-dependent differences in wound metabolism. Conclusion: These results indicate in vivo MPM can provide quantitative biomarkers of age-related delays in healing, which can be used in the future to provide patient-specific wound care.

Methotrexate use and NAD+/NADH metabolism in psoriatic keratinocytes.

Methotrexate inhibits tetrahydrofolic acid production and influences mitochondrial oxygen uptake and activity of several enzymes in the respiratory chain reactions, which utilize nicotinamide adenine dinucleotide-linked (NAD-linked) substrates. Hyperproliferation of keratinocytes in psoriasis requires oxidative phosphorylation, in which the reduced form of nicotinamide adenine dinucleotide (NADH) is an electron donor. One hypothesis links increased cellular metabolism to the increased NADH/NAD+ ratio; as expected, the topical application of NAD+ (oxidized form of nicotinamide-adenine dinucleotide) resulted in a clinical improvement of psoriatic lesions in one study. Nevertheless, another report revealed reduced fluorescence of NADH in psoriatic plaques. The biological activity of NADH is not limited only to serving as the electron donor. It was also found to regulate gene transcription.

Changes of NADH Fluorescence from the Skin of Patients with Systemic Lupus Erythematosus.

Introduction The blood circulation of the skin is an accessible and representative vascular bed for examining the mechanisms of microcirculatory function. Endothelial function is impaired in systemic lupus erythematosus (SLE), which implies disorders in cell metabolism dependent on blood circulation; however, noninvasive monitoring of metabolism at the tissue and cell level is absent in daily clinical practice. Objective The aim of the study was to examine changes of NADH fluorescence from the epidermis of a forearm measured with the flow mediated skin fluorescence (FMSF) technique in patients with SLE and to investigate whether they are associated with clinical manifestation of the disease. Materials and Methods The study enrolled 36 patients with SLE and 34 healthy individuals. Changes of NADH fluorescence were measured using FMSF on the forearm in response to blocking and releasing of blood flow. The results were represented as ischemic (IR max and IR auc) and hyperemic response maximum and area under the curve (HR max and HR auc). Results IR max, IR auc, HR max, and HR auc were all lower in patients with SLE (p < 0.05) compared with controls. All four parameters were negatively correlated (p < 0.05) with patient age. No difference was found in NADH fluorescence between SLE patients with malar rash, discoid rash, photosensitivity, oral ulcers, nonerosive arthritis, renal disorder, hematologic disorder, or immunologic disorder and those without. No correlation was revealed between the SLEDAI score and NADH fluorescence. Conclusion Changes of NADH fluorescence indicate the reduction in NADH restoration, observed especially during reperfusion, and suggest the occurrence of disorders in the microcirculation of the skin and/or at the mitochondrial level. Such changes of NADH during reperfusion in patients with SLE could be associated with their possible lower sensitivity to hypoxia and possibly with endothelial dysfunction.

Catalytic effect of riboflavin on electron transfer from NADH to aquacobalamin.

Reduction of cobalamin by non-dedicated cellular reductases has been reported in earlier work, however, the sources of reducing power and the mechanisms are unknown. This study reports results of kinetic and mechanistic investigation of the reaction between aquacobalamin, H2OCbl, and reduced β-nicotinamide adenine dinucleotide, NADH. This interaction leads to the formation of one-electron reduced cobalamin, cob(II)alamin, and proceeds via water substitution on aquacobalamin by NADH and further decomposition of NADH-Co(III) complex to cob(II)alamin and NADH·+. Riboflavin catalyzes the reduction of aquacobalamin by NADH both in free form and with aquacobalamin bound to the cobalamin processing enzyme CblC. The rate-determining step of this catalytic reaction is the interaction between riboflavin and NADH to produce a charge transfer complex that reacts with aquacobalamin. Aquacobalamin quenches the fluorescence of NADH and riboflavin predominantly via a static mechanism.

MitoRACE: evaluating mitochondrial function in vivo and in single cells with subcellular resolution using multiphoton NADH autofluorescence

We developed a novel metabolic imaging approach that provides direct measures of the rate of mitochondrial energy conversion with single-cell and subcellular resolution by evaluating NADH autofluorescence kinetics during the mitochondrial redox after cyanide experiment (mitoRACE). Measures of mitochondrial NADH flux by mitoRACE are sensitive to physiological and pharmacological perturbations in vivo. Metabolic imaging with mitoRACE provides a highly adaptable platform for evaluating mitochondrial function in vivo and in single cells with potential for broad applications in the study of energy metabolism. Mitochondria play a critical role in numerous cell types and diseases, and structure and function of mitochondria can vary greatly among cells or within different regions of the same cell. However, there are currently limited methodologies that provide direct assessments of mitochondrial function in vivo, and contemporary measures of mitochondrial energy conversion lack the spatial resolution necessary to address cellular and subcellular heterogeneity. Here, we describe a novel metabolic imaging approach that provides direct measures of mitochondrial energy conversion with single-cell and subcellular resolution by evaluating NADH autofluorescence kinetics during the mitochondrial redox after cyanide experiment (mitoRACE). MitoRACE measures the rate of NADH flux through the steady-state mitochondrial NADH pool by rapidly inhibiting mitochondrial energetic flux, resulting in an immediate, linear increase in NADH fluorescence proportional to the steady-state NADH flux rate, thereby providing a direct measure of mitochondrial NADH flux. The experiments presented here demonstrate the sensitivity of this technique to detect physiological and pharmacological changes in mitochondrial flux within tissues of living animals and reveal the unique capability of this technique to evaluate mitochondrial function with single-cell and subcellular resolution in different cell types in vivo and in cell culture. Furthermore, we highlight the potential applications of mitoRACE by showing that within single neurons, mitochondria in neurites have higher energetic flux rates than mitochondria in the cell body. Metabolic imaging with mitoRACE provides a highly adaptable platform for evaluating mitochondrial function in vivo and in single cells, with potential for broad applications in the study of energy metabolism.

Non-invasive evaluation of microcirculation and metabolic regulation using flow mediated skin fluorescence (FMSF): Technical aspects and methodology

This paper describes a new technique for noninvasive diagnostic analysis of metabolic regulation and the microcirculation. Flow Mediated Skin Fluorescence (FMSF) is based on monitoring the intensity of NADH fluorescence emitted from skin tissue on the forearm. The principles of the technique are discussed, in particular, concerning experimental procedures and the definition and interpretation of the measured parameters. The unique features and potential avenues for development of the FMSF technique are also outlined.

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

Effect of the oxygen transfer rate on oxygen-limited production of plasmid DNA by Escherichia coli

The influence of oxygen limitation on pDNA yields in cultures of Escherichia coli was studied. Cultures at maximum oxygen transfer rate (OTRmax) values of 10, 14, 30 and 45 mmol L−1 h−1 and an aerobic culture at an OTRmax of 110 mmol L−1 h−1 were performed in microtiter plates (MTPs). Dissolved oxygen tension (DOT), pH, biomass and NADH fluorescence were monitored online. An E. coli strain that constitutively expresses Vitreoscilla hemoglobin (VHb) was used and compared with its parent strain. pDNA yields were inversely proportional to the OTRmax for both strains and increased more than two-fold in cultures at the lowest OTRmax compared with that in aerobic cultures. Expression of VHb increased the specific growth rate at OTRmax values of 10, 14 and 30 mmol L−1 h−1 compared with that of the parent strain. NADH-specific fluorescence decreased in cultures of the engineered strain. The pDNA supercoiled fraction (SCF) was greatest in cultures at an OTRmax of 30 mmol L−1 h−1, reaching 92.9% for the wild type strain and 98.7% for the VHb-expressing strain, while no linearized pDNA was detected. This condition was replicated in a 1 L stirred tank bioreactor, and the results closely resembled those of cultures in MTPs.

Regulation of nuclear epigenome by mitochondrial DNA heteroplasmy

Diseases associated with mitochondrial DNA (mtDNA) mutations are highly variable in phenotype, in large part because of differences in the percentage of normal and mutant mtDNAs (heteroplasmy) present within the cell. For example, increasing heteroplasmy levels of the mtDNA tRNALeu(UUR) nucleotide (nt) 3243A > G mutation result successively in diabetes, neuromuscular degenerative disease, and perinatal lethality. These phenotypes are associated with differences in mitochondrial function and nuclear DNA (nDNA) gene expression, which are recapitulated in cybrid cell lines with different percentages of m.3243G mutant mtDNAs. Using metabolic tracing, histone mass spectrometry, and NADH fluorescence lifetime imaging microscopy in these cells, we now show that increasing levels of this single mtDNA mutation cause profound changes in the nuclear epigenome. At high heteroplasmy, mitochondrially derived acetyl-CoA levels decrease causing decreased histone H4 acetylation, with glutamine-derived acetyl-CoA compensating when glucose-derived acetyl-CoA is limiting. In contrast, α-ketoglutarate levels increase at midlevel heteroplasmy and are inversely correlated with histone H3 methylation. Inhibition of mitochondrial protein synthesis induces acetylation and methylation changes, and restoration of mitochondrial function reverses these effects. mtDNA heteroplasmy also affects mitochondrial NAD+/NADH ratio, which correlates with nuclear histone acetylation, whereas nuclear NAD+/NADH ratio correlates with changes in nDNA and mtDNA transcription. Thus, mutations in the mtDNA cause distinct metabolic and epigenomic changes at different heteroplasmy levels, potentially explaining transcriptional and phenotypic variability of mitochondrial disease.

ROS-mediated relationships between metabolism and DAF-16 subcellular localization in Caenorhabditis elegans revealed by a novel fluorometric method

Signalling pathways provide a fine-tuned control network for catabolic and anabolic cellular processes under changing environmental conditions (e.g. changes in oxygen partial pressure, Po2). These pathways frequently activate or deactivate transcription factors (TFs) in the cytoplasm, with the subsequent nuclear translocation of activated TFs constituting a prerequisite for gene control and expression. This study introduces a newly developed fluorometric method for the quantification of relationships between environmental factors and the subcellular localization of reporter-coupled TFs in Caenorhabditis elegans (and possibly other transparent organisms). We applied this method to determine and analyse the relationship between Po2 and the subcellular localization of the GFP-coupled transcription factor DAF-16 (FoxO) of the DAF-2 (insulin/IGF-1) signalling pathway via the DAF-16::GFP fluorescence intensity of whole worms (Po2 characteristic). The Po2 characteristic resembled the Po2-specific metabolic rate of C. elegans, with a critical Po2 (Pco2) of 3.6 kPa separating two Po2 ranges, where either anaerobic metabolism and DAF-16::GFP nuclear occupancy strongly increased (i.e. decreasing DAF-16::GFP fluorescence intensity) (Po2 < Pco2) or aerobic metabolism and DAF-16::GFP cytoplasmic localization prevailed (Po2 > Pco2). These results and other data, which included the Po2-specific mitochondrial oxidation-reduction state of whole worms (as determined using the endogenous NADH fluorescence) and the effects of higher levels of reactive oxygen species (ROS) or RNAi-mediated knockdowns of catabolic or anabolic control genes (aak-2 or let-363) on the Po2 characteristic, suggest that ROS play a decisive role for DAF-16 nuclear translocation due to tissue hypoxia or higher anabolic activity induced by aak-2(RNAi). As DAF-16 and its target genes are of central importance for the cellular stress resistance, ROS-mediated relationships between metabolism and DAF-16 subcellular (i.e. nuclear) localization provide protection of the cell machinery against elevated ROS formation under challenging metabolic conditions.

Can we use rapid lifetime determination for fast, fluorescence lifetime based, metabolic imaging? Precision and accuracy of double-exponential decay measurements with low total counts.

Fluorescence lifetime imaging microscopy (FLIM) can assess cell’s metabolism through the fluorescence of the co-enzymes NADH and FAD, which exhibit a double-exponential decay, with components related to free and protein-bound conditions. In vivo real time clinical imaging applications demand fast acquisition. As photodamage limits excitation power, this is best achieved using wide-field techniques, like time-gated FLIM, and algorithms that require few images to calculate the decay parameters. The rapid lifetime determination (RLD) algorithm requires only four images to analyze a double-exponential decay. Using computational simulations, we evaluated the accuracy and precision of RLD when measuring endogenous fluorescence lifetimes and metabolic free to protein-bound ratios, for total counts per pixel (TC) lower than 104. The simulations were based on a time-gated FLIM instrument, accounting for its instrument response function, gain and noise. While the optimal acquisition setting depends on the values being measured, the accuracy of the free to protein-bound ratio α2/α1 is stable for low gains and gate separations larger than 1000 ps, while its precision is almost constant for gate separations between 1500 and 2500 ps. For the gate separations and free to protein-bound ratios considered, the accuracy error can be as high as 30% and the precision error can reach 60%. Precision errors lower than 10% cannot be obtained. The best performance occurs for low camera gains and gate separations near 1800 ps. When considering the narrow physiological ranges for the free to protein-bound ratio, the precision errors can be confined to an interval between 10% and 20%. RLD is a valid option when for real time FLIM. The simulations and methodology presented here can be applied to any time-gated FLIM instrument and are useful to obtain the accuracy and precision limits for RLD in the demanding conditions of TC lower than 104.

CuZnSOD expressed specifically in neurons rescues mitochondrial function and calcium handling in muscles of Sod1KO mice

BackgroundAging is accompanied by loss of muscle mass and force. The mechanism of the loss is not fully understood, but oxidative stress and mitochondrial dysfunction have been implicated as potential contributors. Mice deficient in CuZnSOD (Sod1−/− mice) show decreased ATP production and increased generation of reactive oxygen species by muscle mitochondria as well as muscle atrophy and weakness early in life that is reminiscent of that observed in old age in wild type (WT) mice. Thus, Sod1−/− mice represent a model of accelerated neuromuscular aging. Surprisingly, SynTgSod1−/− mice, which have Sod1 expression restored specifically in neurons, show none of the neuromuscular deficits observed in Sod1−/− mice. To investigate how neuron‐specific Sod1 expression rescues muscle mass and force in Sod1−/− mice, we examined contractile properties and mitochondrial function in muscles of SynTgSod1−/− mice.MethodLumbrical muscles were removed from 7–9 months old wild‐type, Sod1−/−, and SynTgSod1−/− mice, attached to a force transducer, and placed in an experimental chamber for the measurement of muscle contractile parameters and NADH fluorescence was recorded as a measure of mitochondrial redox status. Because mitochondrial function is regulated by calcium, we also examined intracellular calcium transients (ICT) using the fluorescent dye Mag‐fura2. Immunofluorescence techniques were used to examine neuromuscular junction (NMJ) structure and fiber type composition.ResultCompared with WT muscles, mitochondria of muscles of Sod1−/− mice were in a more reduced state at rest and became transiently more oxidized following a 5s maximum isometric contraction. Consistent with the idea that recovery of NADH may be driven by calcium activation of the TCA cycle, the peak of the ICT was reduced and the width prolonged in muscles of Sod1−/− mice. For all measures, muscles of SynTgSod1−/− and WT mice were not different. A shift in muscle fiber type composition observed in Sod1−/− mice was also restored in SynTgSod1−/− mice. Moreover, NMJ denervation and fragmentation was rescued in SynTg Sod1−/− mice.ConclusionOur finding of improved mitochondrial function in muscles of SynTgSod1−/− compared with Sod1−/− mice is consistent with the hypothesis that the mitochondrial dysfunction is an important contributor to the development of atrophy and weakness in Sod1−/− mice. Since SynTgSod1−/− mice are still lacking CuZnSOD in muscle, we conclude that appropriate neuronal function and intact innervation are key to maintaining muscle mitochondrial function and overall muscle size and strength.Support or Funding InformationSupported by NIH Grant AG051442 and the Second Xiangya Hospital of Central South University, ChinaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A readily usable two-photon fluorescence lifetime microendoscope.

A two-photon fluorescence lifetime (2P-FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence, at sub-cellular resolution, is demonstrated. It exhibits readily usable characteristics such as convenient endoscope probe diameter (≈2 mm), fiber length (>5 m) and data accumulation rate (16 frames per second (fps)), leading to a FLIM refreshing rate of ≈0.1 to 1 fps depending on the sample. The spiral scanning image formation does not influence the instrument response function (IRF) characteristics of the system. Near table-top microscope performances are achieved through a comprehensive system including a home-designed spectro-temporal pulse shaper and a custom air-silica double-clad photonic crystal fiber, which enables to reach up to 40 mW of ≈100 fs pulses @ 760 nm with a 80 MHz repetition rate. A GRadient INdex (GRIN) lens provides a lateral resolution of 0.67 μm at the focus of the fiber probe. Intracellular NADH fluorescence lifetime data are finally acquired on cultured cells at 16 fps.

Switchable sniff-cam (gas-imaging system) based on redox reactions of alcohol dehydrogenase for ethanol and acetaldehyde in exhaled breath

Measuring the volatile organic compounds (VOCs) released from a human is a promising method for noninvasive disease screening and metabolism assessment. Selectively imaging multiple VOCs derived from human breath and skin gas is expected to improve current gas analysis techniques. In this study, a gas-imaging system (sniff-cam) that can be used to simultaneously image the concentration distribution of multiple VOCs, namely, ethanol (EtOH) and acetaldehyde (AcH), was developed. The sniff-cam was based on the pH-dependent redox reactions of nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase (ADH). The sniff-cam was constructed with a camera, two ADH-immobilized meshes, and a UV-LED array sheet. The ADH-immobilized mesh containing a solution of the oxidized form of NAD (NAD+) or reduced form (NADH) was used as an EtOH-imaging mesh and an AcH-imaging mesh, respectively. The distributions of the EtOH and AcH concentrations were visualized through the fluorescence of NADH (the excitation wavelength was 340 nm; the emission wavelength was 490 nm) occurring by the ADH-mediated redox reaction. First, the influence of pH on the activity of the redox reaction of ADH was measured, and then the quantitativeness and selectivity of the sniff-cam were evaluated. The ADH-mediated reactions of EtOH and AcH showed maximum activities at pH 9.0 and pH 6.5, respectively. The sniff-cam demonstrated not only a dynamic range (0.1–1000 ppm for EtOH and 0.2–10 ppm for AcH) for measuring EtOH and AcH in breath after drinking alcohol, but also displayed a high selectivity against other breath VOCs. Finally, EtOH and AcH in breath after drinking alcohol were measured simultaneously. A group with high activity of aldehyde dehydrogenase type 2 (EtOH = 143.3 ± 13.5 ppm, AcH = 1.7 ± 0.2 ppm) and a group with low activity (EtOH = 163.3 ± 28.0 ppm, AcH = 8.4 ± 0.5 ppm) displayed differences in the concentrations of EtOH and AcH contained in their breath samples, and the effectiveness of the developed method was confirmed and compared with previous results. It is suggested that the multiplexed sniff-cam in the future may be capable of selectively and simultaneously imaging various VOCs in human breath and skin gas by using multiple NADH-dependent enzymes.

Increased nicotinamide adenine dinucleotide pool promotes colon cancer progression by suppressing reactive oxygen species level.

Nicotinamide adenine dinucleotide (NAD) exists in an oxidized form (NAD+) and a reduced form (NADH). NAD + plays crucial roles in cancer metabolism, including in cellular signaling, energy production and redox regulation. However, it remains unclear whether NAD(H) pool size (NAD + and NADH) could be used as biomarker for colon cancer progression. Here, we showed that the NAD(H) pool size and NAD +/NADH ratio both increased during colorectal cancer (CRC) progression due to activation of the NAD + salvage pathway mediated by nicotinamide phosphoribosyltransferase (NAMPT). The NAMPT expression was upregulated in adenoma and adenocarcinoma tissues from CRC patients. The NADH fluorescence intensity measured by two‐photon excitation fluorescence (TPEF) microscopy was consistently increased in CRC cell lines, azoxymethane/dextran sodium sulfate (AOM/DSS)‐induced CRC tissues and tumor tissues from CRC patients. The increases in the NAD(H) pool inhibited the accumulation of excessive reactive oxygen species (ROS) levels and FK866, a specific inhibitor of NAMPT, treatment decreased the CRC nodule size by increasing ROS levels in AOM/DSS mice. Collectively, our results suggest that NAMPT‐mediated upregulation of the NAD(H) pool protects cancer cells against detrimental oxidative stress and that detecting NADH fluorescence by TPEF microscopy could be a potential method for monitoring CRC progression.

Cerebral metabolism in a mouse model of Alzheimer's disease characterized by two-photon fluorescence lifetime microscopy of intrinsic NADH.

.Disruptions and alterations to cerebral energy metabolism play a vital role in the onset and progression of many neurodegenerative disorders and cerebral pathologies. In order to precisely understand the complex alterations underlying Alzheimer’s disease (AD) progression, in vivo imaging at the microscopic level is required in preclinical animal models. Utilizing two-photon fluorescence lifetime imaging microscopy and the phasor analysis method, we have observed AD-related variations of endogenous fluorescence of reduced nicotinamide adenine dinucleotide (NADH) in vivo. We collected NADH FLIM images from the cerebral cortices of both APPswe:PS1dE9 mice to model amyloid plaque accumulation and corresponding age-matched wildtype controls. Distinct variations in NADH fluorescence lifetime between wildtype and AD mice, as well as variations related to proximity from amyloid plaques, are obvervable via the phasor method. The combination of NADH FLIM and phasor analysis allows for a minimally invasive, high-resolution technique to characterize the adverse effects of amyloid accumulation on mitochondrial energy metabolism and could guide our understanding of preclinical AD pathology.