Impairment of an Endothelial NAD+-H2S Signaling Network Is a Reversible Cause of Vascular Aging.

A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.

Dynamic Acetylation of Phosphoenolpyruvate Carboxykinase Toggles Enzyme Activity between Gluconeogenic and Anaplerotic Reactions.

Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is considered a gluconeogenic enzyme; however, its metabolic functions and regulatory mechanisms beyond gluconeogenesis are poorly understood. Here, we describe that dynamic acetylation of PCK1 interconverts the enzyme between gluconeogenic and anaplerotic activities. Under high glucose, p300-dependent hyperacetylation of PCK1 did not lead to protein degradation but instead increased the ability of PCK1 to perform the anaplerotic reaction, converting phosphoenolpyruvate to oxaloacetate. Lys91 acetylation destabilizes the active site of PCK1 and favors the reverse reaction. At low energy input, we demonstrate that SIRT1 deacetylates PCK1 and fully restores the gluconeogenic ability of PCK1. Additionally, we found that GSK3ß-mediated phosphorylation of PCK1 decreases acetylation and increases ubiquitination. Biochemical evidence suggests that serine phosphorylation adjacent to Lys91 stimulates SIRT1-dependent deacetylation of PCK1. This work reveals an unexpected capacity of hyperacetylated PCK1 to promote anaplerotic activity, and the intersection of post-translational control of PCK1 involving acetylation, phosphorylation, and ubiquitination.

Reproductive Ageing: Metabolic contribution to age-related chromosome missegregation in mammalian oocytes.

In brief: Chromosome missegregation and declining energy metabolism are considered to be unrelated features of oocyte ageing that contribute to poor reproductive outcomes. Given the bioenergetic cost of chromosome segregation, we propose here that altered energy metabolism during ageing may be an underlying cause of age-related chromosome missegregation and aneuploidy. Abstract: Advanced reproductive age in women is a major cause of infertility, miscarriage and congenital abnormalities. This is principally caused by a decrease in oocyte quality and developmental competence with age. Oocyte ageing is characterised by an increase in chromosome missegregation and aneuploidy. However, the underlying mechanisms of age-related aneuploidy have not been fully elucidated and are still under active investigation. In addition to chromosome missegregation, oocyte ageing is also accompanied by metabolic dysfunction. In this review, we integrate old and new perspectives on oocyte ageing, chromosome segregation and metabolism in mammalian oocytes and make direct links between these processes. We consider age-related alterations to chromosome segregation machinery, including the loss of cohesion, microtubule stability and the integrity of the spindle assembly checkpoint. We focus on how metabolic dysfunction in the ageing oocyte disrupts chromosome segregation machinery to contribute to and exacerbate age-related aneuploidy. More specifically, we discuss how mitochondrial function, ATP production and the generation of free radicals are altered during ageing. We also explore recent developments in oocyte metabolic ageing, including altered redox reactions (NAD+ metabolism) and the interactions between oocytes and their somatic nurse cells. Throughout the review, we integrate the mechanisms by which changes in oocyte metabolism influence age-related chromosome missegregation.

Multispectral autofluorescence characteristics of reproductive aging in old and young mouse oocytes.

Increasing age has a major detrimental impact on female fertility, which, with an ageing population, has major sociological implications. This impact is primarily mediated through deteriorating quality of the oocyte. Deteriorating oocyte quality with biological age is the greatest rate-limiting factor to female fertility. Here we have used label-free, non-invasive multi-spectral imaging to identify unique autofluorescence profiles of oocytes from young and aged animals. Discriminant analysis demonstrated that young oocytes have a distinct autofluorescent profile which accurately distinguishes them from aged oocytes. We recently showed that treatment with the nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide mononucleotide (NMN) restored oocyte quality and fertility in aged animals, and when our analysis was applied to oocytes from aged animals treated with NMN, 85% of these oocytes were classified as having the autofluorescent signature of young animals. Spectral unmixing using the Robust Dependent Component Analysis (RoDECA) algorithm demonstrated that NMN treatment altered the metabolic profile of oocytes, increasing free NAD(P)H, protein bound NAD(P)H, redox ratio and the ratio of bound to free NAD(P)H. The frequency of oocytes with simultaneously high NAD(P)H and flavin content was also significantly increased in mice treated with NMN. Young and Aged + NMN oocytes had a smoother spectral distribution, with the distribution of NAD(P)H in young oocytes specifically differing from that of aged oocytes. Identifying the multispectral profile of oocyte autofluorescence during aging could have utility as a non-invasive and sensitive measure of oocyte quality.

Transposable Elements Cross Kingdom Boundaries and Contribute to Inflammation and Ageing: Somatic Acquisition of Foreign Transposable Elements as a Catalyst of Genome Instability, Epigenetic Dysregulation, Inflammation, Senescence, and Ageing.

The de-repression of transposable elements (TEs) in mammalian genomes is thought to contribute to genome instability, inflammation, and ageing, yet is viewed as a cell-autonomous event. In contrast to mammalian cells, prokaryotes constantly exchange genetic material through TEs, crossing both cell and species barriers, contributing to rapid microbial evolution and diversity in complex communities such as the mammalian gut. Here, it is proposed that TEs released from prokaryotes in the microbiome or from pathogenic infections regularly cross the kingdom barrier to the somatic cells of their eukaryotic hosts. It is proposed this horizontal transfer of TEs from microbe to host is a stochastic, ongoing catalyst of genome destabilization, resulting in structural and epigenetic variations, and activation of well-evolved host defense mechanisms contributing to inflammation, senescence, and biological ageing. It is proposed that innate immunity pathways defend against the horizontal acquisition of microbial TEs, and that activation of this pathway during horizontal transposon transfer promotes chronic inflammation during ageing. Finally, it is suggested that horizontal acquisition of prokaryotic TEs into mammalian genomes has been masked and subsequently under-reported due to flaws in current sequencing pipelines, and new strategies to uncover these events are proposed.

Exercise-induced benefits on glucose handling in a model of diet-induced obesity are reduced by concurrent nicotinamide mononucleotide.

Almost 40% of adults worldwide are classified as overweight or obese. Exercise is a beneficial intervention in obesity, partly due to increases in mitochondrial activity and subsequent increases in nicotinamide adenine dinucleotide (NAD+), an important metabolic cofactor. Recent studies have shown that increasing NAD+ levels through pharmacological supplementation with precursors such as nicotinamide mononucleotide (NMN) improved metabolic health in high-fat-diet (HFD)-fed mice. However, the effects of combined exercise and NMN supplementation are unknown. Thus, here we examined the combined effects of NMN and treadmill exercise in female mice with established obesity after 10 wk of diet. Five-week-old female C57BL/6J mice were exposed to a control diet (n = 16) or HFD. Mice fed a HFD were either untreated (HFD; n = 16), received NMN in drinking water (400 mg/kg; HNMN; n = 16), were exposed to treadmill exercise 6 days/wk (HEx; n = 16), or were exposed to exercise combined with NMN (HNEx; n = 16). Although some metabolic benefits of NMN have been described, at this dose, NMN administration impaired several aspects of exercise-induced benefits in obese mice, including glucose tolerance, glucose-stimulated insulin secretion from islets, and hepatic triglyceride accumulation. HNEx mice also exhibited increased antioxidant and reduced prooxidant gene expression in both islets and muscle, suggesting that altered redox status is associated with the loss of exercise-induced health benefits with NMN cotreatment. Our data show that NMN treatment impedes the beneficial metabolic effects of exercise in a mouse model of diet-induced obesity in association with disturbances in redox metabolism.NEW & NOTEWORTHY NMN dampened exercise-induced benefits on glucose handling in diet-induced obesity. NMN administration alongside treadmill exercise enhanced the ratio of antioxidants to prooxidants. We suggest that NMN administration may not be beneficial when NAD+ levels are replete.

Impact of nicotinamide mononucleotide on transplanted mouse ovarian tissue.

Ovarian tissue cryopreservation and future transplantation is the only strategy to preserve the fertility of young female adolescent and prepubertal patients. The primary challenge to ovarian graft longevity is the substantial loss of primordial follicles during the period of ischaemia post-transplantation. Nicotinamide mononucleotide (NMN), a precursor of the essential metabolite NAD+, is known to reduce ischaemic damage. Therefore, the objective of the current study was to assess the impact of short- and long-term NMN administration on follicle number and health following ovarian tissue transplantation. Hemi-ovaries from C57Bl6 mice (n = 8-12/group) were transplanted under the kidney capsule of bilaterally ovariectomised severe combined immunodeficient (SCID) mice. Recipient mice were administered either normal drinking water or water supplemented with NMN (2 g/L) for either 14 or 56 days. At the end of each treatment period, ovarian transplants were collected. There was no effect of NMN on the resumption of oestrous or length of oestrous cycles. Transplantation significantly reduced the total number of follicles with the greatest impact observed at the primordial follicle stage. We report that NMN did not prevent this loss. While NMN did not significantly impact the proportion of apoptotic follicles, NMN normalised PCNA expression at the primordial and intermediate stages but not at later stages. In conclusion, NMN administration did not prevent ovarian follicle loss under the conditions of this study.

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan.

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1(H/H)) live shorter and show signs of accelerated aging. As wild-type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age-related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1-7) are a family of NAD(+)-dependent deacetylases that can delay age-related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD(+) and the ability of SIRT2 to maintain lysine-668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD(+) precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1(H/H) animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD(+) to delay diseases of aging in mammals is warranted.

Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach.

INTRODUCTION: Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as a key hydride transfer coenzyme for several oxidoreductases. It is also the substrate for intracellular secondary messenger signalling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase, and epigenetic regulation of gene expression by a class of histone deacetylase enzymes known as sirtuins. The measurement of NAD+ and its related metabolites (hereafter, the NAD+ metabolome) represents an important indicator of cellular function. OBJECTIVES: A study was performed to develop a sensitive, selective, robust, reproducible, and rapid method for the concurrent quantitative determination of intracellular levels of the NAD+ metabolome in glial and oocyte cell extracts using liquid chromatography coupled to mass spectrometry (LC/MS/MS). METHODS: The metabolites were separated on a versatile amino column using a dual HILIC-RP gradient with heated electrospray (HESI) tandem mass spectrometry detection in mixed polarity multiple reaction monitoring mode. RESULTS: Quantification of 17 metabolites in the NAD+ metabolome in U251 human astroglioma cells could be achieved. Changes in NAD+ metabolism in U251 cell line, and murine oocytes under different culture conditions were also investigated. CONCLUSION: This method can be used as a sensitive profiling tool, tailoring chromatography for metabolites that express significant pathophysiological changes in several disease conditions and is indispensable for targeted analysis.

Niclosamide blocks glucagon phosphorylation of Ser552 on ß-catenin in primary rat hepatocytes via PKA signalling.

Recently, it has been found that glucagon is able to activate the ß-catenin signalling pathway leading to increased cyclin D1 and c-Myc expression in liver. Therefore the main aim of the present study is to determine whether the effect of glucagon activating ß-catenin signalling leading to increased target gene expression is mediated through cAMP activation of PKA (protein kinase A). Primary rat hepatocytes were incubated with insulin, glucagon or adrenaline (epinephrine) and a range of inhibitors of PI3K (phosphoinositide 3-kinase), Wnt, mitochondrial uncoupler (niclosamide) or PKA inhibitors to dissect out the pathway leading to increased Ser(552) phosphorylation on ß-catenin following glucagon exposure. In primary rat hepatocytes, we found that short exposure to glucagon or adrenaline caused a rapid increase in Ser(552) phosphorylation on ß-catenin that leads to increased cyclin D1 and c-Myc expression. A range of PI3K and Wnt inhibitors were unable to block the effect of glucagon phosphorylating ß-catenin. Interestingly, both niclosamide and the PKA inhibitor H89 blocked the glucagon effect on ß-catenin signalling, leading to a reduction in target gene expression. Likewise, niclosamide inhibited cAMP levels and the direct addition of db-cAMP (dibutyryl-cAMP sodium salt) also resulted in Ser(552) phosphorylation of ß-catenin. We have identified a new pathway via glucagon signalling that leads to increased ß-catenin activity that can be reversed with the antihelminthic drug niclosamide, which has recently shown promise as a potential treatment of T2D (Type 2 diabetes). This novel finding could be useful in liver cancer treatment, particularly in the context of T2D with increased ß-catenin activity.

Calsyntenin-1 mediates hepatitis C virus replication.

The hepatitis C virus (HCV) RNA genome of 9.6 kb encodes only 10 proteins, and so is highly dependent on host hepatocyte factors to facilitate replication. We aimed to identify host factors involved in the egress of viral particles. By screening the supernatant of HCV-infected Huh7 cells using SILAC-based proteomics, we identified the transmembrane protein calsyntenin-1 as a factor specifically secreted by infected cells. Calsyntenin-1 has previously been shown to mediate transport of endosomes along microtubules in neurons, through interactions with kinesin light chain-1. Here we demonstrate for the first time, we believe, a similar role for calsyntenin-1 in Huh7 cells, mediating intracellular transport of endosomes. In HCV-infected cells we show that calsyntenin-1 contributes to the early stages of the viral replication cycle and the formation of the replication complex. Importantly, we demonstrate in our model that silencing calsyntenin-1 disrupts the viral replication cycle, confirming the reliance of HCV on this protein as a host factor. Characterizing the function of calsyntenin-1 will increase our understanding of the HCV replication cycle and pathogenesis, with potential application to other viruses sharing common pathways.

Carcinogenic Chromium(VI) Compounds Formed by Intracellular Oxidation of Chromium(III) Dietary Supplements by Adipocytes.

Chromium(III) nutritional supplements are widely consumed for their purported antidiabetic activities. X-ray fluorescence microscopy (XFM) and X-ray absorption near-edge structure (XANES) studies have now shown that non-toxic doses of [Cr3 O(OCOEt)6 (OH2 )3 ](+) (A), a prospective antidiabetic drug that undergoes similar H2 O2 induced oxidation reactions in the blood as other Cr supplements, was also oxidized to carcinogenic Cr(VI) and Cr(V) in living cells. Single adipocytes treated with A had approximately 1 µm large Cr hotspots containing Cr(III) , Cr(V) , and Cr(VI) (primarily Cr(VI) thiolates) species. These results strongly support the hypothesis that the antidiabetic activity of Cr(III) and the carcinogenicity of Cr(VI) compounds arise from similar mechanisms involving highly reactive Cr(VI) and Cr(V) intermediates, and highlight concerns over the safety of Cr(III) nutritional supplements.

Hepatic fat loss in advanced nonalcoholic steatohepatitis: are alterations in serum adiponectin the cause?

UNLABELLED: Advanced liver fibrosis in nonalcoholic steatohepatitis (NASH) is often accompanied by a reduction in hepatic fat to the point of complete fat loss (burnt-out NASH), but the mechanisms behind this phenomenon have not been elucidated. Adiponectin is raised in cirrhosis of any cause and has potent antisteatotic activity. In this study we examined 65 patients with advanced biopsy-proven NASH (fibrosis stage 3-4) and 54 with mild disease (fibrosis stage 0-1) to determine if disappearance of steatosis correlated with changes in serum adiponectin. All patents had fasting blood tests and anthropometric measures at the time of liver biopsy. Liver fat was accurately quantitated by morphometry. Serum adiponectin was measured by immunoassay. When compared to those with early disease, patients with advanced NASH were more insulin-resistant, viscerally obese, and older, but there was no difference in liver fat content or adiponectin levels. Adiponectin had a significant negative correlation with liver fat percentage in the whole cohort (r = -0.28, P < 0.01), driven by patients with advanced NASH (r = -0.40, P < 0.01). In advanced NASH, for each 4 µg/L increase in adiponectin there was an odds ratio OR of 2.0 (95% confidence interval [CI]: 1.3-3.0, P < 0.01) for a 5% reduction in hepatic fat. Adiponectin was highly and significantly associated with almost complete hepatic fat loss or burnt-out NASH (12.1 versus 7.4 µg/L, P = 0.001) on multivariate analysis. A relationship between adiponectin, bile acids, and adipocyte fexaramine activation was demonstrated in vivo and in vitro, suggestive of hepatocyte-adipocyte crosstalk. CONCLUSION: Serum adiponectin levels in advanced NASH are independently associated with hepatic fat loss. Adiponectin may in part be responsible for the paradox of burnt-out NASH. (HEPATOLOGY 2012).

Beneficial effects of MGL-3196 and BAM15 combination in a mouse model of fatty liver disease.

BACKGROUND AND AIM: Metabolic dysfunction-associated steatohepatitis (MASH) is a metabolic disorder with limited treatment options. The thyroid hormone receptor (THR)-ß agonist resmetirom/MGL-3196 (MGL) increases liver fat oxidation and has been approved for treating adult MASH. However, over 60% of patients receiving MGL treatment do not achieve MASH resolution. Therefore, we investigated the potential for combination therapy of MGL with the mitochondrial uncoupler BAM15 to improve fatty liver disease outcomes in the GAN mouse model of MASH. METHODS: C57BL/6J male mice were fed GAN diet for 38 weeks before stratification and randomization to treatments including MGL, BAM15, MGL + BAM15, or no drug control for 8 weeks. Treatments were admixed in diet and mice were pair-fed to control for drug intake. Treatment effectiveness was assessed by body weight, body composition, energy expenditure, glucose tolerance, tissue lipid content, and histological analyses. RESULTS: MGL + BAM15 treatment resulted in better efficacy versus GAN control mice than either monotherapy in the context of energy expenditure, liver fat loss, glucose control, and fatty liver disease activity score. Improvements in ALT, liver mass, and plasma cholesterol were primarily driven by MGL, while improvements in body fat were primarily driven by BAM15. No treatments altered liver fibrosis. CONCLUSIONS: MGL + BAM15 treatment had overall better efficacy to improve metabolic outcomes in mice fed GAN diet than either monotherapy alone. These data warrant further investigation into combination therapies of THR-ß agonists and mitochondrial uncouplers for the potential treatment of disorders related to fatty liver, obesity, and insulin resistance.

Multispectral Imaging of Collagen, NAD(P)H and Flavin Autofluorescence in Mesenchymal Stem Cells Undergoing Trilineage Differentiation.

Understanding the molecular mechanisms of differentiation is important for regenerative medicine and developmental biology. This study aims to characterise the role of the glycolysis/oxidative phosphorylation balance as a driver of mesenchymal stem cell (MSC) differentiation. Cells were maintained in normal conditions or stimulated towards the MSC trilineage cell types over 21 days. Multispectral imaging of cell autofluorescence was applied as a non-invasive methodology to continuously image cultures in situ. Spectral signals for collagen, NAD(P)H, and flavins were unmixed. MSCs cultured under chondrogenic conditions exhibited increased collagen levels relative to controls. Following osteogenic induction, MSCs showed increased collagen levels relative to controls during the earlier stages of culture; however, control cells increased their collagen levels as they became confluent. MSCs cultured under adipogenic conditions exhibited lower levels of collagen than controls. The redox ratio (RR; NAD(P)H/flavins) immediately decreased during chondrogenesis, with this early effect persisting throughout the culture compared to control cells, which appeared to increase their RR, similar to osteogenesis. Adipogenesis resulted in a small increase in RR on day 2 relative to control cells, followed by a persistent decrease. Chondrogenic and adipogenic differentiation favoured oxidative phosphorylation, whereas osteogenesis and MSC overgrowth resulted in a glycolytic metabolism. Following consideration of these findings, as well as the diverse reports in the literature, it is concluded that neither enhanced oxidative phosphorylation nor glycolysis are fundamental to the canonical modes of differentiation, and researchers should avoid interpreting shifts as indicating differentiation.

Long-term NMN treatment increases lifespan and healthspan in mice in a sex dependent manner.

Nicotinamide adenine dinucleotide (NAD) is essential for many enzymatic reactions, including those involved in energy metabolism, DNA repair and the activity of sirtuins, a family of defensive deacylases. During aging, levels of NAD + can decrease by up to 50% in some tissues, the repletion of which provides a range of health benefits in both mice and humans. Whether or not the NAD + precursor nicotinamide mononucleotide (NMN) extends lifespan in mammals is not known. Here we investigate the effect of long-term administration of NMN on the health, cancer burden, frailty and lifespan of male and female mice. Without increasing tumor counts or severity in any tissue, NMN treatment of males and females increased activity, maintained more youthful gene expression patterns, and reduced overall frailty. Reduced frailty with NMN treatment was associated with increases in levels of Anerotruncus colihominis, a gut bacterium associated with lower inflammation in mice and increased longevity in humans. NMN slowed the accumulation of adipose tissue later in life and improved metabolic health in male but not female mice, while in females but not males, NMN increased median lifespan by 8.5%, possible due to sex-specific effects of NMN on NAD + metabolism. Together, these data show that chronic NMN treatment delays frailty, alters the microbiome, improves male metabolic health, and increases female mouse lifespan, without increasing cancer burden. These results highlight the potential of NAD + boosters for treating age-related conditions and the importance of using both sexes for interventional lifespan studies.

Fertility protection during chemotherapy treatment by boosting the NAD(P)+ metabolome.

Chemotherapy induced ovarian failure and infertility is an important concern in female cancer patients of reproductive age or younger, and non-invasive, pharmacological approaches to maintain ovarian function are urgently needed. Given the role of reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an essential cofactor for drug detoxification, we sought to test whether boosting the NAD(P)+ metabolome could protect ovarian function. We show that pharmacological or transgenic strategies to replenish the NAD+ metabolome ameliorates chemotherapy induced female infertility in mice, as measured by oocyte yield, follicle health, and functional breeding trials. Importantly, treatment of a triple-negative breast cancer mouse model with the NAD+ precursor nicotinamide mononucleotide (NMN) reduced tumour growth and did not impair the efficacy of chemotherapy drugs in vivo or in diverse cancer cell lines. Overall, these findings raise the possibility that NAD+ precursors could be a non-invasive strategy for maintaining ovarian function in cancer patients, with potential benefits in cancer therapy.

Host-microbiome interactions in nicotinamide mononucleotide (NMN) deamidation.

The nicotinamide adenine dinucleotide (NAD+ ) precursor nicotinamide mononucleotide (NMN) is a proposed therapy for age-related disease, whereby it is assumed that NMN is incorporated into NAD+ through the canonical recycling pathway. During oral delivery, NMN is exposed to the gut microbiome, which could modify the NAD+ metabolome through enzyme activities not present in the mammalian host. We show that orally delivered NMN can undergo deamidation and incorporation in mammalian tissue via the de novo pathway, which is reduced in animals treated with antibiotics to ablate the gut microbiome. Antibiotics increased the availability of NAD+ metabolites, suggesting the microbiome could be in competition with the host for dietary NAD+ precursors. These findings highlight new interactions between NMN and the gut microbiome.