Elevated NADH Research Articles

Aged fragmented-polypropylene microplastics induced ageing statues-dependent bioenergetic imbalance and reductive stress: In vivo and liver organoids-based in vitro study

Ageing is a nature process of microplastics that occurrs daily, and human beings are inevitably exposed to aged microplastics. However, a systematic understanding of ageing status and its toxic effect is currently still lacking. In this study, plastic cup lids-originated polypropylene (PP) microplastics were UV-photoaged until the carbonyl index (CI), a canonical indicator for plastic ageing, achieved 0.08, 0.17, 0.22 and 0.28. The adverse hepatic effect of these aged PPs (aPPs) was evaluated in Balb/c mice (75 ng/mL water, about 200 particles/day) and human-originated liver organoids (LOs, 50 particles/mL, ranged from 5.94 to 13.15 ng/mL) at low-dose equivalent to human exposure level. Low-dose of aged PP could induce hepatic reductive stress both in vitro and in vivo, by elevating the NADH/NAD+ratio in a CI-dependent manner, together with hepatoxicity (indicated by increased AST secretion and cytotoxicity), and disrupted the genes encoding the nutrients transporters and NADH subunits accompanied by the restricted ATP supply, declined mitochondrial membrane potential and mitochondrial complexI/IV activities, without significant increase in MDA levels in the liver. These changes in the liver disrupted systematic metabolism, representing a circulatory panel of increases in the lactate, triglyceride, Fgf21 levels, and decreases in the pyruvate level, linked the reductive stress to the declined body weight gain but elevated hepatic NADH contents following aPPs exposure. Additionally, assessing by the LOs, it was found that digestion drastically accelerated the ageing of aPPs and worsen the energy supply upon mitochondria, representing a “scattergun effect” induced by the formation of micro- and nano-plastics mixture toward NADH/NAD+imbalance.

Uncovering the impact of alcohol on internal organs and reproductive health: Exploring TLR4/NF-kB and CYP2E1/ROS/Nrf2 pathways.

This review delves into the detrimental impact of alcohol consumption on internal organs and reproductive health, elucidating the underlying mechanisms involving the Toll-like receptor 4 (TLR4)/Nuclear factor kappa light chain enhancer of activated B cells (NF-kB) pathway and the Cytochrome P450 2E1 (CYP2E1)/reactive oxygen species (ROS)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathways. The TLR4/NF-kB pathway, crucial for inflammatory and immune responses, triggers the production of pro-inflammatory agents and type-1 interferon, disrupting the balance between inflammatory and antioxidant responses when tissues are chronically exposed to alcohol. Alcohol-induced dysbiosis in gut microbes heightens gut wall permeability to pathogen-associated molecular patterns (PAMPs), leading to liver cell infection and subsequent inflammation. Concurrently, CYP2E1-mediated alcohol metabolism generates ROS, causing oxidative stress and damaging cells, lipids, proteins, and deoxyribonucleic acid (DNA). To counteract this inflammatory imbalance, Nrf2 regulates gene expression, inhibiting inflammatory progression and promoting antioxidant responses. Excessive alcohol intake results in elevated liver enzymes (ADH, CYP2E1, and catalase), ROS, NADH, acetaldehyde, and acetate, leading to damage in vital organs such as the heart, brain, and lungs. Moreover, alcohol negatively affects reproductive health by inhibiting the hypothalamic-pituitary-gonadal axis, causing infertility in both men and women. These findings underscore the profound health concerns associated with alcohol-induced damage, emphasizing the need for public awareness regarding the intricate interplay between immune responses and the multi-organ impacts of alcohol consumption.

The Enzymatic Function of Kvβ2 Controls Vascular Potassium Channel Activity

Voltage-gated potassium (Kv) channels expressed in vascular smooth muscle control membrane potential and thereby regulate vascular tone and organ perfusion. Native Kv1 channels in the resistance vasculature consist of an alpha pore domain in complex with intracellular beta (Kvβ) proteins, which are active aldo-keto reductases (AKR6A). Recent work suggests that the AKR function of Kvβ2 is required for vascular Kv1 channel NAD(P)(H) redox sensing and O2 sensitivity of resistance arterial tone. Nonetheless, whether acute changes in Kvβ2 AKR function impact vascular Kv1 activity is unknown. We propose that Kv1 gating is sensitive to changes in the levels of endogenous substrates and post-translational modification of Kvβ2. Here, we tested the hypothesis that the availability of the glycolytic byproduct and purported Kvβ substrate, methylglyoxal, and protein kinase C-dependent phosphorylation differentially regulate vascular Kvβ2 function. Using the inside-out configuration of the patch clamp technique, we measured the open probability (nPo) of Kv channels present in patches excised from human coronary smooth muscle cells. Perfusion of methylglyoxal (5 μM) resulted in significantly increased Kv channel nPo (3.3x10−5 ± 9.5x10−6 to 3.2x10−4 ± 2.1x10−4; n=5) and this effect was enhanced (~8-fold) in the presence of 100 μM NADPH, suggesting that Kv sensitivity to methylglyoxal is cofactor-dependent. Based on this, we next tested whether Kvβ phosphorylation regulates AKR catalysis and influences vascular Kv1 redox sensitivity. Using in situ proximity ligation, we found that serine phosphorylation of Kvβ2 in smooth muscle was increased upon treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA; 100 nM), indicating that smooth muscle Kvβ2 is a phosphorylation target under these conditions. In vitro phosphorylation of purified Kvβ2 with PKCα significantly slowed catalysis (11.3 ± 0.3 vs. 6.5 ± 0.2 μmoles/min/mg protein, control and PKCα-treated rat Kvβ2.1, respectively). Pretreatment of cells with 10 nM PMA before excising inside-out membrane patches eliminated the increase in Kv channel nP(o) evoked by methylglyoxal (±NADPH). Moreover, ex vivo treatment of isolated coronary arteries with PMA (10 nM) abolished vasodilation in response to elevated NADH:NAD+ via application of 2–5 mM external L-lactate. Together, these results suggest that vascular smooth muscle Kv1 channels are functionally regulated by endogenous substrates and phosphorylation of intracellular Kvβ proteins. We propose that targeting vascular Kvβ AKR function may represent a novel strategy to modify K+ channel redox sensitivity and thus strengthen the coupling between metabolic demand and organ perfusion. T32-ES011564 and R01HL163818. 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.

Sex-specific metabolic adaptations in transgenic mice overexpressing cytochrome b5 reductase-3

Cytochrome b5 reductase 3 (CYB5R3) activates respiratory metabolism in cellular systems and exerts a prolongevity action in transgenic mice overexpressing this enzyme, mimicking some of the beneficial effects of calorie restriction. The aim of our study was to investigate the role of sex on metabolic adaptations elicited by CYB5R3 overexpression, and how key markers related with mitochondrial function are modulated in skeletal muscle, one of the major contributors to resting energy expenditure. Young CYB5R3 transgenic mice did not exhibit the striking adaptations in carbon metabolism previously detected in older animals. CYB5R3 was efficiently overexpressed and targeted to mitochondria in skeletal muscle from transgenic mice regardless sex. Overexpression significantly elevated NADH in both sexes, although differences were not statistically significant for NAD+, and increased the abundance of cytochrome c and the fission protein DRP-1 in females but not in males. Moreover, while mitochondrial biogenesis and function markers (as TFAM, NRF-1 and cleaved SIRT3) were markedly upregulated by CYB5R3 overexpression in females, a downregulation was observed in males. Ultrastructural changes were also highlighted, with an increase in the number of mitochondria per surface unit, and in the size of intermyofibrillar mitochondria in transgenic females compared with their wild-type controls. Our results support that CYB5R3 overexpression upregulates markers consistent with enhanced mitochondrial biogenesis and function, and increases mitochondrial abundance in skeletal muscle, producing most of these potentially beneficial actions in females.

Abstract 6512: Label-free autofluorescence imaging reveals different metabolic responses to adverse growth conditions between normal and breast cancer cells lines

Abstract Cancer cells are metabolically distinct from normal cells, and can adapt differently to adverse growth conditions. We sought to compare normal MCF10A and Ras-transformed MCF10AT cells under various conditions (regular growth media, doxorubicin-treatment, and glucose-deprived media) by imaging endogenous cellular FAD and NADH fluorescence and comparing it to proliferation rates (measured by MTS assay) and chromatin structure (measured by Hoescht staining). Under normal growth conditions, MCF10AT cells displayed similar levels of FAD and higher levels of mitochondrial NADH than MCF10A cells. Elevated NADH in MCF10AT cells seems to reflect the preference for anaerobic glycolysis rather than oxidative phosphorylation (the so-called Warburg effect). Our MTS data showed that doxorubicin treatment did not significantly affect MCF10AT cell proliferation but led to a modest reduction in cell proliferation in the normal cell line. Likewise, in MCF10AT cells, doxorubicin treatment led to an increase in FAD and a decrease in NADH. Conversely, in MCF10A cells, doxorubicin led to a decrease in FAD levels and an increase in mitochondrial NADH. These results suggest that the cancer line may be switching to oxidative phosphorylation to resist doxorubicin-induced toxicity, while the normal cells reduce oxidative phosphorylation with growth inhibition. In contrast to the doxorubicin response, glucose deprivation produced significant growth inhibition and cell death in MCF10AT cells while having a modest growth-inhibitory effect on the normal line. Interestingly, low glucose decreased FAD and mitochondrial NADH in MCF10AT cells, suggesting that both aerobic and anaerobic respiration was reduced and consistent with the marked reduction in observed cell viability. In contrast, MCF10A cells showed a slight decrease in FAD and an increase in NADH, suggesting a decrease in oxidative phosphorylation consistent with the modest growth inhibition observed. Finally, MCF10ATs displayed higher levels of Hoechst staining than MCF10A cells, consistent with a higher proportion of condensed chromatin observed in cancer cells. In addition, we found a larger increase in Hoechst staining in MCF10AT cells in response to both doxorubicin and low glucose, as compared to MCF10A cells. This data suggests that the chromatin state of the two lines is also differentially affected by these treatments. In conclusion, label-free measurement of autofluorescence of cellular FAD and mitochondrial NADH reveals the different metabolic and chromatin responses to adverse growth conditions between cancer and non-cancer cells. These differences are associated with the different proliferation rates and may have important clinical implications for cancer risk stratification. Citation Format: Katherine Junkins, Angel Perez Martinez, Margaret Rodgers, Shelley A. Phelan, Min Xu. Label-free autofluorescence imaging reveals different metabolic responses to adverse growth conditions between normal and breast cancer cells lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6512.

TMET-20. TERT ACTS VIA FOXO1 TO ALTER REDOX METABOLISM IN BRAIN TUMORS

Abstract Telomere maintenance is a fundamental hallmark of cancer. Most tumors maintain telomere length via reactivation of telomerase reverse transcriptase (TERT) expression. Identifying imaging biomarkers of TERT can enable non-invasive assessment of tumor proliferation and response to therapy. Deuterium magnetic resonance spectroscopy (DMRS) following administration of 2H-labeled substrates recently emerged as a novel, clinically translatable method of monitoring metabolic activity in vivo. The goal of this study was to delineate metabolic reprogramming associated with TERT expression and to leverage this information for non-invasive imaging of tumor burden and treatment response in gliomas. Our results indicate that TERT expression is associated with elevated levels of the redox metabolite NADH in glioblastomas and oligodendrogliomas. Mechanistically, TERT expression is associated with inhibitory phosphorylation and cytosolic sequestration of FOXO1. FOXO1, in turn, negatively regulates nicotinamide phosphoribosyl transferase (NAMPT), which is the rate-limiting enzyme in NAD+ biosynthesis. As a result, TERT upregulates NAMPT, resulting in elevated steady-state pools of NAD+ and NADH. Concomitantly, FOXO1 negatively regulates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which converts NAD+ to NADH. As a result, TERT upregulates the NADH/NAD+ ratio. Since elevated NADH and NADH/NAD+ ratio drive pyruvate conversion to lactate, we then examined whether DMRS-based imaging of [U-2H]-pyruvate metabolism reports on TERT expression in gliomas. Our results indicate that doxycycline-inducible TERT silencing significantly reduces lactate production from [U-2H]-pyruvate in tumor-bearing mice. Importantly, [U-2H]-pyruvate metabolism to lactate differentiates tumor from normal brain in vivo, including at clinical field strength (3T). Furthermore, [U-2H]-pyruvate reports on early response to treatment with TERT inhibitors or with radiochemotherapy in mice bearing orthotopic patient-derived gliomas at early timepoints before radiographic alterations can be visualized by magnetic resonance imaging. Collectively, our studies integrate a mechanistic understanding of TERT biology with innovative imaging that has the potential to improve assessment of tumor burden and treatment response for glioma patients.

Deuterium Metabolic Imaging Reports on TERT Expression and Early Response to Therapy in Cancer.

Telomere maintenance is a hallmark of cancer. Most tumors maintain telomere length via reactivation of telomerase reverse transcriptase (TERT) expression. Identifying clinically translatable imaging biomarkers of TERT can enable noninvasive assessment of tumor proliferation and response to therapy. We used RNAi, doxycycline-inducible expression systems, and pharmacologic inhibitors to mechanistically delineate the association between TERT and metabolism in preclinical patient-derived tumor models. Deuterium magnetic resonance spectroscopy (2H-MRS), which is a novel, translational metabolic imaging modality, was used for imaging TERT in cells and tumor-bearing mice in vivo. Our results indicate that TERT expression is associated with elevated NADH in multiple cancers, including glioblastoma, oligodendroglioma, melanoma, neuroblastoma, and hepatocellular carcinoma. Mechanistically, TERT acts via the metabolic regulator FOXO1 to upregulate nicotinamide phosphoribosyl transferase, which is the key enzyme for NAD+ biosynthesis, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which converts NAD+ to NADH. Because NADH is essential for pyruvate flux to lactate, we show that 2H-MRS-based assessment of lactate production from [U-2H]-pyruvate reports on TERT expression in preclinical tumor models in vivo, including at clinical field strength (3T). Importantly, [U-2H]-pyruvate reports on early response to therapy in mice bearing orthotopic patient-derived gliomas at early timepoints before radiographic alterations can be visualized by MRI. Elevated NADH is a metabolic consequence of TERT expression in cancer. Importantly, [U-2H]-pyruvate reports on early response to therapy, prior to anatomic alterations, thereby providing clinicians with a novel tool for assessment of tumor burden and treatment response in cancer.

NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

In mammals, circadian rhythms are entrained to the light cycle and drive daily oscillations in levels of NAD+, a cosubstrate of the class III histone deacetylase sirtuin 1 (SIRT1) that associates with clock transcription factors. Although NAD+ also participates in redox reactions, the extent to which NAD(H) couples nutrient state with circadian transcriptional cycles remains unknown. Here we show that nocturnal animals subjected to time-restricted feeding of a calorie-restricted diet (TRF-CR) only during night-time display reduced body temperature and elevated hepatic NADH during daytime. Genetic uncoupling of nutrient state from NADH redox state through transduction of the water-forming NADH oxidase from Lactobacillus brevis (LbNOX) increases daytime body temperature and blood and liver acyl-carnitines. LbNOX expression in TRF-CR mice induces oxidative gene networks controlled by brain and muscle Arnt-like protein 1 (BMAL1) and peroxisome proliferator-activated receptor alpha (PPARα) and suppresses amino acid catabolic pathways. Enzymatic analyses reveal that NADH inhibits SIRT1 in vitro, corresponding with reduced deacetylation of SIRT1 substrates during TRF-CR in vivo. Remarkably, Sirt1 liver nullizygous animals subjected to TRF-CR display persistent hypothermia even when NADH is oxidized by LbNOX. Our findings reveal that the hepatic NADH cycle links nutrient state to whole-body energetics through the rhythmic regulation of SIRT1.

Reducing agents assisted fed-batch fermentation to enhance ABE yields

Acetone-butanol-ethanol (ABE) fermentation process is a promising bioenergy option amid rising concerns over the environmental impact of fossil fuel usage. However, the commercialization of the ABE process has been marred by challenges of low product yield and titer, thereby non-competitive process economics. Here, we coupled cost competitive reducing agents with a controlled feeding strategy to improve both product titer and yield. Reducing agents promote cofactor dependent butanol production, while fed-batch operation enhances glucose consumption, final ABE titer, and partly mitigates product toxicity. The effects of ascorbic acid, L-cysteine, and dithiothreitol (DTT) on ABE fed-batch production using Clostridium acetobutylicum was investigated in current study. NADH, ATP, extracellular amino acid secretion, and NADH-dependent butanol dehydrogenase (BDH) assays were performed to study the metabolic modifications triggered by reducing agents. Incidentally, L-cysteine and DTT improved ABE solvent titer by 2-fold, producing 24.33 and 22.98 g/L ABE with solvent yields of 0.38 and 0.37 g/g, respectively. Elevated NADH, BDH, and ATP levels in fermentation broth reflected in enhanced ABE titer and yield. Furthermore, histidine secretion emerged as an important factor in Clostridial acid stress tolerance in this study. The results demonstrate that addition of reducing agents in fed-batch ABE fermentation operation enables efficient utilization of glucose with significant improvement in solvent production.

Serine Catabolism Feeds NADH when Respiration Is Impaired

NADH provides electrons for aerobic ATP production. In cells deprived of oxygen or with impaired electron transport chain activity, NADH accumulation can be toxic. To minimize such toxicity, elevated NADH inhibits the classical NADH-producing pathways: glucose, glutamine, and fat oxidation. Here, through deuterium-tracing studies in cultured cells and mice, we show that folate-dependent serine catabolism also produces substantial NADH. Strikingly, when respiration is impaired, serine catabolism through methylene tetrahydrofolate dehydrogenase (MTHFD2) becomes a major NADH source. In cells whose respiration is slowed by hypoxia, metformin, or genetic lesions, mitochondrial serine catabolism inhibition partially normalizes NADH levels and facilitates cell growth. In mice with engineered mitochondrial complex I deficiency (NDUSF4-/-), serine's contribution to NADH is elevated, and progression of spasticity is modestly slowed by pharmacological blockade of serine degradation. Thus, when respiration is impaired, serine catabolism contributes to toxic NADH accumulation.

Abstract 16845: Functional Regulation of Native Coronary Kv1 Channels by Pyridine Nucleotides

Introduction: Recent studies indicate that vasodilation and enhanced blood flow to the heart following increases in myocardial O 2 demand are mediated by voltage-gated Kv1 channels in coronary smooth muscle cells (CVSM). Periods of high O 2 demand alter the intracellular pyridine nucleotide (PN) redox (e.g., elevated NADH:NAD + ), which, in heterologous systems, differentially modulates the gating properties of Kv1 channels by binding to intracellular Kvβ subunits. Hypothesis: Elevated levels of reduced PNs increase Kv1 channel activity in CVSM via the Kvβ complex. Methods: We performed patch clamp electrophysiology on CVSM freshly isolated from the left anterior descending coronary arteries (1 st and 2 nd order) of wild type and Kvβ2-null (Kvβ2 -/- ) mice. In the conventional whole cell configuration, we measured outward K + current density and the voltage-dependence of activation and inactivation (V 0.5, act, V 0.5,inact , respectively) in the absence and presence of extracellular lactate or pyruvate (10 mM), which alter intracellular PN levels. In inside-out membrane patches, we measured the open probability (nPo) of single K + -permeable channels that are sensitive to the Kv1 channel inhibitors 4-aminopyridine (4-AP; 1 mM) and psora-4 (500 nM), in the absence and presence of NADH (1 mM). Results: Treatment of wild type CVSM with lactate to elevate [NADH] I significantly increased whole cell Kv current density and shifted V 0.5, act to more negative membrane potentials (n=4, P<0.05). This response was effectively reversed upon increasing [NAD + ] i by treatment with pyruvate. Recordings performed in the inside-out configuration revealed K + currents that were sensitive to inhibition by psora-4 and 4-AP and had a unitary conductance of 70.2 ± 2.4 pS. Perfusion with a bath solution that contained 1 mM NADH lead to a significant increase (~2-fold) in the nPo of 4-AP-sensitive channels (n=25-30 cells, P<0.05). However, this effect was largely attenuated in membrane patches from Kvβ2 -/- CVSM. Conclusions: Native Kv1 channels in CVSM are functionally regulated by the intracellular NADH:NAD + ratio via their auxiliary Kvβ subunit complex.

Elevated mitochondria-coupled NAD(P)H in endoplasmic reticulum of dopamine neurons.

Pyridine nucleotides are redox coenzymes that are critical in bioenergetics, metabolism, and neurodegeneration. Here we use brain slice multiphoton microscopy to show that substantia nigra dopamine neurons, which are sensitive to stress in mitochondria and the endoplasmic reticulum (ER), display elevated combined NADH and NADPH (i.e., NAD(P)H) autofluorescence. Despite limited mitochondrial mass, organellar NAD(P)H is extensive because much of the signal is derived from the ER. Remarkably, even though pyridine nucleotides cannot cross mitochondrial and ER membranes, inhibiting mitochondrial function with an uncoupler or interrupting the electron transport chain with cyanide (CN-) alters ER NAD(P)H. The ER CN- response can occur without a change in nuclear NAD(P)H, raising the possibility of redox shuttling via the cytoplasm locally between neuronal mitochondria and the ER. We propose that coregulation of NAD(P)H in dopamine neuron mitochondria and ER coordinates cell redox stress signaling by the two organelles.

Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver.

BackgroundAberrant mitochondrial function, including excessive reactive oxygen species (ROS) production, has been implicated in the pathogenesis of human diseases. The use of mitochondrial inhibitors to ascertain the sites in the electron transport chain (ETC) resulting in altered ROS production can be an important tool. However, the response of mouse mitochondria to ETC inhibitors has not been thoroughly assessed. Here we set out to characterize the differences in phenotypic response to ETC inhibitors between the more energetically demanding brain mitochondria and less energetically demanding liver mitochondria in commonly utilized C57BL/6J mice.ResultsWe show that in contrast to brain mitochondria, inhibiting distally within complex I or within complex III does not increase liver mitochondrial ROS production supported by complex I substrates, and liver mitochondrial ROS production supported by complex II substrates occurred primarily independent of membrane potential. Complex I, II, and III enzymatic activities and membrane potential were equivalent between liver and brain and responded to ETC. inhibitors similarly. Brain mitochondria exhibited an approximately two-fold increase in complex I and II supported respiration compared with liver mitochondria while exhibiting similar responses to inhibitors. Elevated NADH transport and heightened complex II–III coupled activity accounted for increased complex I and II supported respiration, respectively in brain mitochondria.ConclusionsWe conclude that important mechanistic differences exist between mouse liver and brain mitochondria and that mouse mitochondria exhibit phenotypic differences compared with mitochondria from other species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0051-8) contains supplementary material, which is available to authorized users.

Lactate up-regulates the expression of lactate oxidation complex-related genes in left ventricular cardiac tissue of rats.

BackgroundBesides its role as a fuel source in intermediary metabolism, lactate has been considered a signaling molecule modulating lactate-sensitive genes involved in the regulation of skeletal muscle metabolism. Even though the flux of lactate is significantly high in the heart, its role on regulation of cardiac genes regulating lactate oxidation has not been clarified yet. We tested the hypothesis that lactate would increase cardiac levels of reactive oxygen species and up-regulate the expression of genes related to lactate oxidation complex.Methods/Principal FindingsIsolated hearts from male adult Wistar rats were perfused with control, lactate or acetate (20mM) added Krebs-Henseleit solution during 120 min in modified Langendorff apparatus. Reactive oxygen species (O2 ●-/H2O2) levels, and NADH and NADPH oxidase activities (in enriched microsomal or plasmatic membranes, respectively) were evaluated by fluorimetry while SOD and catalase activities were evaluated by spectrophotometry. mRNA levels of lactate oxidation complex and energetic enzymes MCT1, MCT4, HK, LDH, PDH, CS, PGC1α and COXIV were quantified by real time RT-PCR. Mitochondrial DNA levels were also evaluated. Hemodynamic parameters were acquired during the experiment. The key findings of this work were that lactate elevated cardiac NADH oxidase activity but not NADPH activity. This response was associated with increased cardiac O2 ●-/H2O2 levels and up-regulation of MCT1, MCT4, LDH and PGC1α with no changes in HK, PDH, CS, COXIV mRNA levels and mitochondrial DNA levels. Lactate increased NRF-2 nuclear expression and SOD activity probably as counter-regulatory responses to increased O2 ●-/H2O2.ConclusionsOur results provide evidence for lactate-induced up-regulation of lactate oxidation complex associated with increased NADH oxidase activity and cardiac O2 ●-/H2O2 driving to an anti-oxidant response. These results unveil lactate as an important signaling molecule regulating components of the lactate oxidation complex in cardiac muscle.

The mitochondrial permeability transition pore regulates endothelial bioenergetics and angiogenesis.

The mitochondrial permeability transition pore is a well-known initiator of cell death that is increasingly recognized as a physiological modulator of cellular metabolism. We sought to identify how the genetic deletion of a key regulatory subunit of the mitochondrial permeability transition pore, cyclophilin D (CypD), influenced endothelial metabolism and intracellular signaling. In cultured primary human endothelial cells, genetic targeting of CypD using siRNA or shRNA resulted in a constitutive increase in mitochondrial matrix Ca(2+) and reduced nicotinamide adenine dinucleotide (NADH). Elevated matrix NADH, in turn, diminished the cytosolic NAD(+)/NADH ratio and triggered a subsequent downregulation of the NAD(+)-dependent deacetylase sirtuin 1 (SIRT1). Downstream of SIRT1, CypD-deficient endothelial cells exhibited reduced phosphatase and tensin homolog expression and a constitutive rise in the phosphorylation of angiogenic Akt. Similar changes in SIRT1, phosphatase and tensin homolog, and Akt were also noted in the aorta and lungs of CypD knockout mice. Functionally, CypD-deficient endothelial cells and aortic tissue from CypD knockout mice exhibited a dramatic increase in angiogenesis at baseline and when exposed to vascular endothelial growth factor. The NAD(+) precursor nicotinamide mononucleotide restored the cellular NAD(+)/NADH ratio and normalized the CypD-deficient phenotype. CypD knockout mice also presented accelerated wound healing and increased neovascularization on tissue injury as monitored by optical microangiography. Our study reveals the importance of the mitochondrial permeability transition pore in the regulation of endothelial mitochondrial metabolism and vascular function. The mitochondrial regulation of SIRT1 has broad implications in the epigenetic regulation of endothelial phenotype.

Rex (encoded by DVU_0916) in Desulfovibrio vulgaris Hildenborough is a repressor of sulfate adenylyl transferase and is regulated by NADH.

Although the enzymes for dissimilatory sulfate reduction by microbes have been studied, the mechanisms for transcriptional regulation of the encoding genes remain unknown. In a number of bacteria the transcriptional regulator Rex has been shown to play a key role as a repressor of genes producing proteins involved in energy conversion. In the model sulfate-reducing microbe Desulfovibrio vulgaris Hildenborough, the gene DVU_0916 was observed to resemble other known Rex proteins. Therefore, the DVU_0916 protein has been predicted to be a transcriptional repressor of genes encoding proteins that function in the process of sulfate reduction in D. vulgaris Hildenborough. Examination of the deduced DVU_0916 protein identified two domains, one a winged helix DNA-binding domain common for transcription factors, and the other a Rossman fold that could potentially interact with pyridine nucleotides. A deletion of the putative rex gene was made in D. vulgaris Hildenborough, and transcript expression studies of sat, encoding sulfate adenylyl transferase, showed increased levels in the D. vulgaris Hildenborough Rex (RexDvH) mutant relative to the parental strain. The RexDvH-binding site upstream of sat was identified, confirming RexDvH to be a repressor of sat. We established in vitro that the presence of elevated NADH disrupted the interaction between RexDvH and DNA. Examination of the 5' transcriptional start site for the sat mRNA revealed two unique start sites, one for respiring cells that correlated with the RexDvH-binding site and a second for fermenting cells. Collectively, these data support the role of RexDvH as a transcription repressor for sat that senses the redox status of the cell.

Effect of metformin and lipid emulsion on the circadian gene expression in muscle cells

The circadian clock influences nearly all aspects of metabolism. However, little is known regarding the effect of the energy status on circadian rhythms. Our aim was to test the effect of two opposing energy situations, metformin and lipid emulsion (LE), on clock and metabolic circadian expression in differentiated C2C12 myotubes. Metformin treatment led to depleted ATP levels accompanied by elevated NADH levels, whereas LE treatment led to increased ATP and NAD+ levels. Nevertheless, both LE and metformin treatments activated the AMP-activated protein kinase (AMPK) pathway. In contrast, the effect on circadian rhythms was completely different. LE led to disrupted clock and metabolic gene expression, whereas metformin led to mainly high-amplitude shifted rhythms. Combination of metformin and LE led to an antagonistic effect on circadian gene expression. Although metformin and LE have an opposing effect on circadian gene expression and on the cellular energy status, they both lead to AMPK activation.

Biochemical and Structural Studies of NADH-Dependent FabG Used To Increase the Bacterial Production of Fatty Acids under Anaerobic Conditions

Major efforts in bioenergy research have focused on producing fuels that can directly replace petroleum-derived gasoline and diesel fuel through metabolic engineering of microbial fatty acid biosynthetic pathways. Typically, growth and pathway induction are conducted under aerobic conditions, but for operational efficiency in an industrial context, anaerobic culture conditions would be preferred to obviate the need to maintain specific dissolved oxygen concentrations and to maximize the proportion of reducing equivalents directed to biofuel biosynthesis rather than ATP production. A major concern with fermentative growth conditions is elevated NADH levels, which can adversely affect cell physiology. The purpose of this study was to identify homologs of Escherichia coli FabG, an essential reductase involved in fatty acid biosynthesis, that display a higher preference for NADH than for NADPH as a cofactor. Four potential NADH-dependent FabG variants were identified through bioinformatic analyses supported by crystallographic structure determination (1.3- to 2.0-Å resolution). In vitro assays of cofactor (NADH/NADPH) preference in the four variants showed up to ≈ 35-fold preference for NADH, which was observed with the Cupriavidus taiwanensis FabG variant. In addition, FabG homologs were overexpressed in fatty acid- and methyl ketone-overproducing E. coli host strains under anaerobic conditions, and the C. taiwanensis variant led to a 60% higher free fatty acid titer and 75% higher methyl ketone titer relative to the titers of the control strains. With further engineering, this work could serve as a starting point for establishing a microbial host strain for production of fatty acid-derived biofuels (e.g., methyl ketones) under anaerobic conditions.

The Molecular Mechanism of Cardiac Sodium Channel Current Reduction with Metabolic Stress

Background Human cardiomyopathy is associated with decreased cardiac Na + channel current (I Na ), and Na + channel changes have been implicated in the increased risk of sudden death in heart failure. We have reported that in cardiomyopathy, elevated intracellular NADH downregulates I Na acutely to a magnitude seen in Brugada syndrome through protein kinase C (PKC) activation without changing the channel membrane expression. The decrease in I Na can be ameliorated by NAD + and protein kinase A (PKA) activators. Here, we investigate the molecular mechanism by which I Na is changed during metabolic stress. Methods HEK293 cells stably expressing human cardiac channel (Na v 1.5) were used to measure single channel currents at –40, –60, and –80 mV using the cell-attached voltage-clamp configuration. Results The single channel currents and conductance of Na v 1.5 were significantly decreased by NADH (100 µM) and phorbol 12-myristate 13-acetate (PMA, 30 nM) ( P + (500 µM) or forskolin (1 µM) restored the single channel currents and conductance to the untreated level ( P >.05 vs untreated group, P Conclusions Because we have already shown no reduction in cell surface channel number with metabolic stress, the decrease of macroscopic current of cardiac Na v 1.5 induced by NADH and PKC results from a decrease of single channel conductance, whereas NAD + and PKA elevate the single channel conductance. These results are consistent with the time course of the current changes seen with NADH/NAD + .

Protective effect of curcuminoids on age-related mitochondrial impairment in female Wistar rat brain

The present study demonstrated the neuroprotective effect of curcuminoids, the active polyphenols of Curcuma longa (L.) rhizomes on mitochondrial dysfunctioning in middle aged and aged female Wistar rat brain. Rats were orally treated with curcuminoids (100mg/kg) for 3months and their brain was collected for evaluation of mitochondrial enzymes and complexes activity, ultra structural changes in mitochondria, neuronal nitric oxide synthase (nNOS) protein expression, adenosine triphosphate (ATP) and lipofuscin content. Significant alterations were observed in all the tested parameters in highly aged rat brain when compared with young control. Long term curcuminoids administration prevented this age associated loss of mitochondrial enzymes and complexes activity in middle aged rat brain except for malate dehydrogenase, Complex II and IV activity when compared with young control. Among aged rats, curcuminoids treatment specifically elevated isocitrate and NADH dehydrogenase, cytochrome c oxidase, Complex I and total ATP content. A significant down-regulation of nNOS protein expression along with reduced lipofuscin content was also observed in curucminoids treated middle aged and aged rats. Thus, it was suggested that curcuminoids may act as a putative drug candidate for the prevention of deleterious effects of ageing and age associated neurodegenerative disorders through amelioration of aberrant mitochondrial functioning.