Relationship between inorganic ion distribution, resting membrane potential, and the ΔG' of ATP hydrolysis: a new paradigm.

Cell membrane potential and inorganic ion distributions are currently viewed from a kinetic electric paradigm, which ignores thermodynamics. The resting membrane potential is viewed as a diffusion potential. The 9 major inorganic ions found in blood plasma (Ca2+, Na+, Mg2+, K+, H+, Cl-, HCO3-, H2PO4-, and HPO42-) are distributed unequally across the plasma membrane. This unequal distribution requires the energy of ATP hydrolysis through the action of the Na+-K+ ATPase. The cell resting membrane potential in each of 3 different tissues with widely different resting membrane potentials has been shown to be equal to the Nernst equilibrium potential of the most permeant inorganic ion. The energy of the measured distribution of the 9 major inorganic ions between extra- and intracellular phases was essentially equal to the independently measured energy of ATP hydrolysis, showing that the distribution of these 9 major ions was in near-equilibrium with the ΔG' of ATP. Therefore, thermodynamics does appear to play an essential role in the determination of the cell resting membrane potential and the inorganic ion distribution across the plasma membrane.-Veech, R. L., King, M. T., Pawlosky, R., Bradshaw, P. C., Curtis, W. Relationship between inorganic ion distribution, resting membrane potential, and the ΔG' of ATP hydrolysis: a new paradigm.

The "great" controlling nucleotide coenzymes.

Nucleotide coenzymes dot the map of metabolic pathways providing energy to drive the reactions of the pathway and play an important role in regulating and controlling energy metabolism through their shared potential energy, which is widely unobserved due to the paradox that the energy in the coenzyme pools cannot be determined from the concentration of the coenzyme couples. The potential energy of the nucleotide couples in the mitochondria or the cytoplasm is expressed in the enzyme reactions in which they take part. The energy in these couples, [NAD+]/[NADH], [NADP+]/[NADPH], [acetyl CoA]/[CoA], and [ATP]/[ADP]x[Pi], regulates energy metabolism. The energy contained in the couples can be altered by suppling energy equivalents in the form of ketones, such as, D-ß-hydroxybutyrate to overcome insulin resistance, to restore antioxidants capacity, to form potential treatments for Alzheimer's and Parkinson's diseases, to enhance life span, and to increase physiological performance. © 2019 IUBMB Life, 71(5):565-579, 2019.

Effects of a high protein diet and liver disease in an in silico model of human ammonia metabolism.

BACKGROUND: After proteolysis, the majority of released amino acids from dietary protein are transported to the liver for gluconeogenesis or to peripheral tissues where they are used for protein synthesis and eventually catabolized, producing ammonia as a byproduct. High ammonia levels in the brain are a major contributor to the decreased neural function that occurs in several pathological conditions such as hepatic encephalopathy when liver urea cycle function is compromised. Therefore, it is important to gain a deeper understanding of human ammonia metabolism. The objective of this study was to predict changes in blood ammonia levels resulting from alterations in dietary protein intake, from liver disease, or from partial loss of urea cycle function. METHODS: A simple mathematical model was created using MATLAB SimBiology and data from published studies. Simulations were performed and results analyzed to determine steady state changes in ammonia levels resulting from varying dietary protein intake and varying liver enzyme activity levels to simulate liver disease. As a toxicity reference, viability was measured in SH-SY5Y neuroblastoma cells following differentiation and ammonium chloride treatment. RESULTS: Results from control simulations yielded steady state blood ammonia levels within normal physiological limits. Increasing dietary protein intake by 72% resulted in a 59% increase in blood ammonia levels. Simulations of liver cirrhosis increased blood ammonia levels by 41 to 130% depending upon the level of dietary protein intake. Simulations of heterozygous individuals carrying a loss of function allele of the urea cycle carbamoyl phosphate synthetase I (CPS1) gene resulted in more than a tripling of blood ammonia levels (from roughly 18 to 60 µM depending on dietary protein intake). The viability of differentiated SH-SY5Y cells was decreased by 14% by the addition of a slightly higher amount of ammonium chloride (90 µM). CONCLUSIONS: Data from the model suggest decreasing protein consumption may be one simple strategy to decrease blood ammonia levels and minimize the risk of developing hepatic encephalopathy for many liver disease patients. In addition, the model suggests subjects who are known carriers of disease-causing CPS1 alleles may benefit from monitoring blood ammonia levels and limiting the level of protein intake if ammonia levels are high.

In silico prediction of novel residues involved in amyloid primary nucleation of human I56T and D67H lysozyme.

BACKGROUND: Amyloidogenic proteins are most often associated with neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, but there are more than two dozen human proteins known to form amyloid fibrils associated with disease. Lysozyme is an antimicrobial protein that is used as a general model to study amyloid fibril formation. Studies aimed at elucidating the process of amyloid formation of lysozyme tend to focus on partial unfolding of the native state due to the relative instability of mutant amyloidogenic variants. While this is well supported, the data presented here suggest the native structure of the variants may also play a role in primary nucleation. RESULTS: Three-dimensional structural analysis identified lysozyme residues 21, 62, 104, and 122 as displaced in both amyloidogenic variants compared to wild type lysozyme. Residue interaction network (RIN) analysis found greater clustering of residues 112-117 in amyloidogenic variants of lysozyme compared to wild type. An analysis of the most energetically favored predicted dimers and trimers provided further evidence for a role for residues 21, 62, 104, 122, and 112-117 in amyloid formation. CONCLUSIONS: This study used lysozyme as a model to demonstrate the utility of combining 3D structural analysis with RIN analysis for studying the general process of amyloidogenesis. Results indicated that binding of two or more amyloidogenic lysozyme mutants may be involved in amyloid nucleation by placing key residues (21, 62, 104, 122, and 112-117) in proximity before partial unfolding occurs. Identifying residues in the native state that may be involved in amyloid formation could provide novel drug targets to prevent a range of amyloidoses.

Ketone bodies mimic the life span extending properties of caloric restriction.

The extension of life span by caloric restriction has been studied across species from yeast and Caenorhabditis elegans to primates. No generally accepted theory has been proposed to explain these observations. Here, we propose that the life span extension produced by caloric restriction can be duplicated by the metabolic changes induced by ketosis. From nematodes to mice, extension of life span results from decreased signaling through the insulin/insulin-like growth factor receptor signaling (IIS) pathway. Decreased IIS diminishes phosphatidylinositol (3,4,5) triphosphate (PIP3 ) production, leading to reduced PI3K and AKT kinase activity and decreased forkhead box O transcription factor (FOXO) phosphorylation, allowing FOXO proteins to remain in the nucleus. In the nucleus, FOXO proteins increase the transcription of genes encoding antioxidant enzymes, including superoxide dismutase 2, catalase, glutathione peroxidase, and hundreds of other genes. An effective method for combating free radical damage occurs through the metabolism of ketone bodies, ketosis being the characteristic physiological change brought about by caloric restriction from fruit flies to primates. A dietary ketone ester also decreases circulating glucose and insulin leading to decreased IIS. The ketone body, d-ß-hydroxybutyrate (d-ßHB), is a natural inhibitor of class I and IIa histone deacetylases that repress transcription of the FOXO3a gene. Therefore, ketosis results in transcription of the enzymes of the antioxidant pathways. In addition, the metabolism of ketone bodies results in a more negative redox potential of the NADP antioxidant system, which is a terminal destructor of oxygen free radicals. Addition of d-ßHB to cultures of C. elegans extends life span. We hypothesize that increasing the levels of ketone bodies will also extend the life span of humans and that calorie restriction extends life span at least in part through increasing the levels of ketone bodies. An exogenous ketone ester provides a new tool for mimicking the effects of caloric restriction that can be used in future research. The ability to power mitochondria in aged individuals that have limited ability to oxidize glucose metabolites due to pyruvate dehydrogenase inhibition suggests new lines of research for preventative measures and treatments for aging and aging-related disorders. © 2017 The Authors IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 69(5):305-314, 2017.

Calorie restriction does not restore brain mitochondrial function in P301L tau mice, but it does decrease mitochondrial F0F1-ATPase activity.

Calorie restriction (CR) has been shown to increase lifespan and delay aging phenotypes in many diverse eukaryotic species. In mouse models of Alzheimer's disease (AD), CR has been shown to decrease amyloid-beta and hyperphosphorylated tau levels and preserve cognitive function. Overexpression of human mutant tau protein has been shown to induce deficits in mitochondrial electron transport chain complex I activity. Therefore, experiments were performed to determine the effects of 4-month CR on brain mitochondrial function in Tg4510 mice, which express human P301L tau. Expression of mutant tau led to decreased ADP-stimulated respiratory rates, but not uncoupler-stimulated respiratory rates. The membrane potential was also slightly higher in mitochondria from the P301L tau mice. As shown previously, tau expression decreased mitochondrial complex I activity. The decreased complex I activity, decreased ADP-stimulated respiratory rate, and increased mitochondrial membrane potential occurring in mitochondria from Tg4510 mice were not restored by CR. However, the CR diet did result in a genotype independent decrease in mitochondrial F0F1-ATPase activity. This decrease in F0F1-ATPase activity was not due to lowered levels of the alpha or beta subunits of F0F1-ATPase. The possible mechanisms through which CR reduces the F0F1-ATPase activity in brain mitochondria are discussed.

Mechanisms of amino acid-mediated lifespan extension in Caenorhabditis elegans.

BACKGROUND: Little is known about the role of amino acids in cellular signaling pathways, especially as it pertains to pathways that regulate the rate of aging. However, it has been shown that methionine or tryptophan restriction extends lifespan in higher eukaryotes and increased proline or tryptophan levels increase longevity in C. elegans. In addition, leucine strongly activates the TOR signaling pathway, which when inhibited increases lifespan. RESULTS: Therefore each of the 20 proteogenic amino acids was individually supplemented to C. elegans and the effects on lifespan were determined. All amino acids except phenylalanine and aspartate extended lifespan at least to a small extent at one or more of the 3 concentrations tested with serine and proline showing the largest effects. 11 of the amino acids were less potent at higher doses, while 5 even decreased lifespan. Serine, proline, or histidine-mediated lifespan extension was greatly inhibited in eat-2 worms, a model of dietary restriction, in daf-16/FOXO, sir-2.1, rsks-1 (ribosomal S6 kinase), gcn-2, and aak-2 (AMPK) longevity pathway mutants, and in bec-1 autophagy-defective knockdown worms. 8 of 10 longevity-promoting amino acids tested activated a SKN-1/Nrf2 reporter strain, while serine and histidine were the only amino acids from those to activate a hypoxia-inducible factor (HIF-1) reporter strain. Thermotolerance was increased by proline or tryptophan supplementation, while tryptophan-mediated lifespan extension was independent of DAF-16/FOXO and SKN-1/Nrf2 signaling, but tryptophan and several related pyridine-containing compounds induced the mitochondrial unfolded protein response and an ER stress response. High glucose levels or mutations affecting electron transport chain (ETC) function inhibited amino acid-mediated lifespan extension suggesting that metabolism plays an important role. Providing many other cellular metabolites to C. elegans also increased longevity suggesting that anaplerosis of tricarboxylic acid (TCA) cycle substrates likely plays a role in lifespan extension. CONCLUSIONS: Supplementation of C. elegans with 18 of the 20 individual amino acids extended lifespan, but lifespan often decreased with increasing concentration suggesting hormesis. Lifespan extension appears to be caused by altered mitochondrial TCA cycle metabolism and respiratory substrate utilization resulting in the activation of the DAF-16/FOXO and SKN-1/Nrf2 stress response pathways.

The Roles of White Adipose Tissue and Liver NADPH in Dietary Restriction-Induced Longevity.

Dietary restriction (DR) protocols frequently employ intermittent fasting. Following a period of fasting, meal consumption increases lipogenic gene expression, including that of NADPH-generating enzymes that fuel lipogenesis in white adipose tissue (WAT) through the induction of transcriptional regulators SREBP-1c and CHREBP. SREBP-1c knockout mice, unlike controls, did not show an extended lifespan on the DR diet. WAT cytoplasmic NADPH is generated by both malic enzyme 1 (ME1) and the pentose phosphate pathway (PPP), while liver cytoplasmic NADPH is primarily synthesized by folate cycle enzymes provided one-carbon units through serine catabolism. During the daily fasting period of the DR diet, fatty acids are released from WAT and are transported to peripheral tissues, where they are used for beta-oxidation and for phospholipid and lipid droplet synthesis, where monounsaturated fatty acids (MUFAs) may activate Nrf1 and inhibit ferroptosis to promote longevity. Decreased WAT NADPH from PPP gene knockout stimulated the browning of WAT and protected from a high-fat diet, while high levels of NADPH-generating enzymes in WAT and macrophages are linked to obesity. But oscillations in WAT [NADPH]/[NADP+] from feeding and fasting cycles may play an important role in maintaining metabolic plasticity to drive longevity. Studies measuring the WAT malate/pyruvate as a proxy for the cytoplasmic [NADPH]/[NADP+], as well as studies using fluorescent biosensors expressed in the WAT of animal models to monitor the changes in cytoplasmic [NADPH]/[NADP+], are needed during ad libitum and DR diets to determine the changes that are associated with longevity.

Characterization of the respiration-induced yeast mitochondrial permeability transition pore.

When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration-induced mannitol permeability in mitochondria from this strain. Potassium (K+) had varied effects on the respiration-induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K+ inhibited respiration-induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K+ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration-induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration-induced yPTP was also inhibited by NAD, Mg2+, NH4 + or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration-induced yPTP as those previously described for the ATP-induced yPTP and reconcile previous strain-dependent differences in yPTP solute selectivity.

Nicotinamide riboside functions during development while beta-hydroxybutyrate functions during adulthood to extend C. elegans lifespan.

Nicotinamide riboside (NR), a form of vitamin B3 and a nicotinamide adenine dinucleotide (NAD + ) precursor, has been shown to activate the mitochondrial unfolded protein response (UPR mt ) and extend the lifespan when supplemented to C. elegans. The ketone body and histone deacetylase (HDAC) inhibitor beta-hydroxybutyrate (BHB) has also been shown to extend C. elegans lifespan. Experiments were performed that showed that NR extended lifespan by acting almost exclusively during larval development, while BHB extended lifespan by acting during adulthood, and the combination of NR during development and BHB during adulthood unexpectedly decreased lifespan. This suggests that hormesis is involved in the lifespan-altering effects of BHB and NR and that they are inducing parallel longevity pathways that converge on a common downstream target.

NADPH and Mitochondrial Quality Control as Targets for a Circadian-Based Fasting and Exercise Therapy for the Treatment of Parkinson's Disease.

Dysfunctional mitochondrial quality control (MQC) is implicated in the pathogenesis of Parkinson's disease (PD). The improper selection of mitochondria for mitophagy increases reactive oxygen species (ROS) levels and lowers ATP levels. The downstream effects include oxidative damage, failure to maintain proteostasis and ion gradients, and decreased NAD+ and NADPH levels, resulting in insufficient energy metabolism and neurotransmitter synthesis. A ketosis-based metabolic therapy that increases the levels of (R)-3-hydroxybutyrate (BHB) may reverse the dysfunctional MQC by partially replacing glucose as an energy source, by stimulating mitophagy, and by decreasing inflammation. Fasting can potentially raise cytoplasmic NADPH levels by increasing the mitochondrial export and cytoplasmic metabolism of ketone body-derived citrate that increases flux through isocitrate dehydrogenase 1 (IDH1). NADPH is an essential cofactor for nitric oxide synthase, and the nitric oxide synthesized can diffuse into the mitochondrial matrix and react with electron transport chain-synthesized superoxide to form peroxynitrite. Excessive superoxide and peroxynitrite production can cause the opening of the mitochondrial permeability transition pore (mPTP) to depolarize the mitochondria and activate PINK1-dependent mitophagy. Both fasting and exercise increase ketogenesis and increase the cellular NAD+/NADH ratio, both of which are beneficial for neuronal metabolism. In addition, both fasting and exercise engage the adaptive cellular stress response signaling pathways that protect neurons against the oxidative and proteotoxic stress implicated in PD. Here, we discuss how intermittent fasting from the evening meal through to the next-day lunch together with morning exercise, when circadian NAD+/NADH is most oxidized, circadian NADP+/NADPH is most reduced, and circadian mitophagy gene expression is high, may slow the progression of PD.

Melatonin treatment restores mitochondrial function in Alzheimer's mice: a mitochondrial protective role of melatonin membrane receptor signaling.

Mitochondrial dysfunction is a hallmark of Alzheimer's disease (AD) and is observed in mutant amyloid precursor protein (APP) transgenic mouse models of familial AD. Melatonin is a potent antioxidant, can prevent toxic aggregation of Alzheimer's beta-amyloid (Aß) peptide and, when taken long term, can protect against cognitive deficits in APP transgenic mice. To study the effects of melatonin on brain mitochondrial function in an AD model, APP/PS1 transgenic mice were treated for 1 month with melatonin. Analysis of isolated brain mitochondria from mice indicated that melatonin treatment decreased mitochondrial Aß levels by two- to fourfold in different brain regions. This was accompanied by a near complete restoration of mitochondrial respiratory rates, membrane potential, and ATP levels in isolated mitochondria from the hippocampus, cortex, or striatum. When isolated mitochondria from untreated young mice were given melatonin, a slight increase in respiratory rate was observed. No such effect was observed in mitochondria from aged mice. In APP-expressing neuroblastoma cells in culture, mitochondrial function was restored by melatonin or by the structurally related compounds indole-3-propionic acid or N(1)-acetyl-N(2)-formyl-5-methoxykynuramine. This restoration was partially blocked by melatonin receptor antagonists indicating melatonin receptor signaling is required for the full effect. Therefore, treatments that stimulate melatonin receptor signaling may be beneficial for restoring mitochondrial function in AD, and preservation of mitochondrial function may an important mechanism by which long term melatonin treatment delays cognitive dysfunction in AD mice.

Acetyl-CoA Metabolism and Histone Acetylation in the Regulation of Aging and Lifespan.

Acetyl-CoA is a metabolite at the crossroads of central metabolism and the substrate of histone acetyltransferases regulating gene expression. In many tissues fasting or lifespan extending calorie restriction (CR) decreases glucose-derived metabolic flux through ATP-citrate lyase (ACLY) to reduce cytoplasmic acetyl-CoA levels to decrease activity of the p300 histone acetyltransferase (HAT) stimulating pro-longevity autophagy. Because of this, compounds that decrease cytoplasmic acetyl-CoA have been described as CR mimetics. But few authors have highlighted the potential longevity promoting roles of nuclear acetyl-CoA. For example, increasing nuclear acetyl-CoA levels increases histone acetylation and administration of class I histone deacetylase (HDAC) inhibitors increases longevity through increased histone acetylation. Therefore, increased nuclear acetyl-CoA likely plays an important role in promoting longevity. Although cytoplasmic acetyl-CoA synthetase 2 (ACSS2) promotes aging by decreasing autophagy in some peripheral tissues, increased glial AMPK activity or neuronal differentiation can stimulate ACSS2 nuclear translocation and chromatin association. ACSS2 nuclear translocation can result in increased activity of CREB binding protein (CBP), p300/CBP-associated factor (PCAF), and other HATs to increase histone acetylation on the promoter of neuroprotective genes including transcription factor EB (TFEB) target genes resulting in increased lysosomal biogenesis and autophagy. Much of what is known regarding acetyl-CoA metabolism and aging has come from pioneering studies with yeast, fruit flies, and nematodes. These studies have identified evolutionary conserved roles for histone acetylation in promoting longevity. Future studies should focus on the role of nuclear acetyl-CoA and histone acetylation in the control of hypothalamic inflammation, an important driver of organismal aging.

Residue Interaction Network Analysis Predicts a Val24-Ile31 Interaction May be Involved in Preventing Amyloid-Beta (1-42) Primary Nucleation.

Alzheimer's disease (AD) patients could benefit from a more effective treatment than the current FDA-approved options. Because amyloid-beta (Aß) is thought to play a central role in AD pathogenesis, many experimental drugs attempt to reduce Aß-induced pathology. Preventing amyloid accumulation may be a more effective strategy than clearing Aß plaques after they form. If preventing Aß accumulation can treat or prevent AD, then understanding Aß primary nucleation may aid rational drug design. This study examines Aß residue interaction networks and reports network and structural observations that may provide insight into primary nucleation. While many studies identify structural features of Aß that promote aggregation, this study reports features that may resist primary nucleation by examining Aß42 studies in more and less polar solvents. In Aß42 in a less polar solvent (PDB ID: 1IYT), Val24 and Ile31 have higher betweenness and residue centrality values. This may be due to a predicted interaction between Val24 and Ile31. Residues in the central hydrophobic cluster (CHC) of Aß40 and Aß42 had significantly higher betweenness values compared to the average betweenness of the structures, highlighting the CHC's reported role in oligomerization. The predicted interaction between Val24 and Ile31 may reduce the likelihood of primary nucleation of Aß.

The role of mitochondrial DNA mutations in mammalian aging.

Mitochondrial DNA (mtDNA) accumulates both base-substitution mutations and deletions with aging in several tissues in mammals. Here, we examine the evidence supporting a causative role for mtDNA mutations in mammalian aging. We describe and compare human diseases and mouse models associated with mitochondrial genome instability. We also discuss potential mechanisms for the generation of these mutations and the means by which they may mediate their pathological consequences. Strategies for slowing the accumulation and attenuating the effects of mtDNA mutations are discussed.

COVID-19: Proposing a Ketone-Based Metabolic Therapy as a Treatment to Blunt the Cytokine Storm.

Human SARS-CoV-2 infection is characterized by a high mortality rate due to some patients developing a large innate immune response associated with a cytokine storm and acute respiratory distress syndrome (ARDS). This is characterized at the molecular level by decreased energy metabolism, altered redox state, oxidative damage, and cell death. Therapies that increase levels of (R)-beta-hydroxybutyrate (R-BHB), such as the ketogenic diet or consuming exogenous ketones, should restore altered energy metabolism and redox state. R-BHB activates anti-inflammatory GPR109A signaling and inhibits the NLRP3 inflammasome and histone deacetylases, while a ketogenic diet has been shown to protect mice from influenza virus infection through a protective γδ T cell response and by increasing electron transport chain gene expression to restore energy metabolism. During a virus-induced cytokine storm, metabolic flexibility is compromised due to increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that damage, downregulate, or inactivate many enzymes of central metabolism including the pyruvate dehydrogenase complex (PDC). This leads to an energy and redox crisis that decreases B and T cell proliferation and results in increased cytokine production and cell death. It is hypothesized that a moderately high-fat diet together with exogenous ketone supplementation at the first signs of respiratory distress will increase mitochondrial metabolism by bypassing the block at PDC. R-BHB-mediated restoration of nucleotide coenzyme ratios and redox state should decrease ROS and RNS to blunt the innate immune response and the associated cytokine storm, allowing the proliferation of cells responsible for adaptive immunity. Limitations of the proposed therapy include the following: it is unknown if human immune and lung cell functions are enhanced by ketosis, the risk of ketoacidosis must be assessed prior to initiating treatment, and permissive dietary fat and carbohydrate levels for exogenous ketones to boost immune function are not yet established. The third limitation could be addressed by studies with influenza-infected mice. A clinical study is warranted where COVID-19 patients consume a permissive diet combined with ketone ester to raise blood ketone levels to 1 to 2 mM with measured outcomes of symptom severity, length of infection, and case fatality rate.

Cytoplasmic and Mitochondrial NADPH-Coupled Redox Systems in the Regulation of Aging.

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) protects against redox stress by providing reducing equivalents to antioxidants such as glutathione and thioredoxin. NADPH levels decline with aging in several tissues, but whether this is a major driving force for the aging process has not been well established. Global or neural overexpression of several cytoplasmic enzymes that synthesize NADPH have been shown to extend lifespan in model organisms such as Drosophila suggesting a positive relationship between cytoplasmic NADPH levels and longevity. Mitochondrial NADPH plays an important role in the protection against redox stress and cell death and mitochondrial NADPH-utilizing thioredoxin reductase 2 levels correlate with species longevity in cells from rodents and primates. Mitochondrial NADPH shuttles allow for some NADPH flux between the cytoplasm and mitochondria. Since a decline of nicotinamide adenine dinucleotide (NAD⁺) is linked with aging and because NADP⁺ is exclusively synthesized from NAD⁺ by cytoplasmic and mitochondrial NAD⁺ kinases, a decline in the cytoplasmic or mitochondrial NADPH pool may also contribute to the aging process. Therefore pro-longevity therapies should aim to maintain the levels of both NAD⁺ and NADPH in aging tissues.

In Silico Preliminary Association of Ammonia Metabolism Genes GLS, CPS1, and GLUL with Risk of Alzheimer's Disease, Major Depressive Disorder, and Type 2 Diabetes.

Ammonia is a toxic by-product of protein catabolism and is involved in changes in glutamate metabolism. Therefore, ammonia metabolism genes may link a range of diseases involving glutamate signaling such as Alzheimer's disease (AD), major depressive disorder (MDD), and type 2 diabetes (T2D). We analyzed data from a National Institute on Aging study with a family-based design to determine if 45 single nucleotide polymorphisms (SNPs) in glutaminase (GLS), carbamoyl phosphate synthetase 1 (CPS1), or glutamate-ammonia ligase (GLUL) genes were associated with AD, MDD, or T2D using PLINK software. HAPLOVIEW software was used to calculate linkage disequilibrium measures for the SNPs. Next, we analyzed the associated variations for potential effects on transcriptional control sites to identify possible functional effects of the SNPs. Of the SNPs that passed the quality control tests, four SNPs in the GLS gene were significantly associated with AD, two SNPs in the GLS gene were associated with T2D, and one SNP in the GLUL gene and three SNPs in the CPS1 gene were associated with MDD before Bonferroni correction. The in silico bioinformatic analysis suggested probable functional roles for six associated SNPs. Glutamate signaling pathways have been implicated in all these diseases, and other studies have detected similar brain pathologies such as cortical thinning in AD, MDD, and T2D. Taken together, these data potentially link GLS with AD, GLS with T2D, and CPS1 and GLUL with MDD and stimulate the generation of testable hypotheses that may help explain the molecular basis of pathologies shared by these disorders.

The effects of AICAR and rapamycin on mitochondrial function in immortalized mitochondrial DNA mutator murine embryonic fibroblasts.

Mitochondrial DNA mutations accumulate with age and may play a role in stem cell aging as suggested by the premature aging phenotype of mitochondrial DNA polymerase gamma (POLG) exonuclease-deficient mice. Therefore, E1A immortalized murine embryonic fibroblasts (MEFs) from POLG exonuclease-deficient and wild-type (WT) mice were constructed. Surprisingly, when some E1A immortalized MEF lines were cultured in pyruvate-containing media they slowly became addicted to the pyruvate. The POLG exonuclease-deficient MEFs were more sensitive to several mitochondrial inhibitors and showed increased reactive oxygen species (ROS) production under standard conditions. When cultured in pyruvate-containing media, POLG exonuclease-deficient MEFs showed decreased oxygen consumption compared to controls. Increased AMP-activated protein kinase (AMPK) signaling and decreased mammalian target of rapamycin (mTOR) signaling delayed aging and influenced mitochondrial function. Therefore, the effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, or rapamycin, an mTOR inhibitor, on measures of mitochondrial function were determined. Rapamycin treatment transiently increased respiration only in WT MEFs and, under most conditions, increased ATP levels. Short term AICAR treatment transiently increased ROS production and, under most conditions, decreased ATP levels. Chronic AICAR treatment decreased respiration and ROS production in WT MEFs. These results demonstrate the context-dependent effects of AICAR and rapamycin on mitochondrial function.