Nicotinamide riboside (NR) is in wide use as an NAD
precursor vitamin. Here we determine the time and dose-dependent effects of NR on blood NAD
metabolism in humans. We report that human blood NAD
...can rise as much as 2.7-fold with a single oral dose of NR in a pilot study of one individual, and that oral NR elevates mouse hepatic NAD
with distinct and superior pharmacokinetics to those of nicotinic acid and nicotinamide. We further show that single doses of 100, 300 and 1,000 mg of NR produce dose-dependent increases in the blood NAD
metabolome in the first clinical trial of NR pharmacokinetics in humans. We also report that nicotinic acid adenine dinucleotide (NAAD), which was not thought to be en route for the conversion of NR to NAD
, is formed from NR and discover that the rise in NAAD is a highly sensitive biomarker of effective NAD
repletion.
NAD
is a vital redox cofactor and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Supplementation with NAD
precursors, such as ...nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we show that nicotinamide riboside kinase 1 (NRK1) is necessary and rate-limiting for the use of exogenous NR and NMN for NAD
synthesis. Using genetic gain- and loss-of-function models, we further demonstrate that the role of NRK1 in driving NAD
synthesis from other NAD
precursors, such as nicotinamide or nicotinic acid, is dispensable. Using stable isotope-labelled compounds, we confirm NMN is metabolized extracellularly to NR that is then taken up by the cell and converted into NAD
. Our results indicate that mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD
synthesis, explaining the overlapping metabolic effects observed with the two compounds.
Aim/hypothesis
Urocortin-3 (UCN3) is a glucoregulatory peptide produced in the gut and pancreatic islets. The aim of this study was to clarify the acute effects of UCN3 on glucose regulation ...following an oral glucose challenge and to investigate the mechanisms involved.
Methods
We studied the effect of UCN3 on blood glucose, gastric emptying, glucose absorption and secretion of gut and pancreatic hormones in male rats. To supplement these physiological studies, we mapped the expression of UCN3 and the UCN3-sensitive receptor, type 2 corticotropin-releasing factor receptor (CRHR2), by means of fluorescence in situ hybridisation and by gene expression analysis.
Results
In rats, s.c. administration of UCN3 strongly inhibited gastric emptying and glucose absorption after oral administration of glucose. Direct inhibition of gastrointestinal motility may be responsible because UCN3’s cognate receptor, CRHR2, was detected in gastric submucosal plexus and in interstitial cells of Cajal. Despite inhibited glucose absorption, post-challenge blood glucose levels matched those of rats given vehicle in the low-dose UCN3 group, because UCN3 concomitantly inhibited insulin secretion. Higher UCN3 doses did not further inhibit gastric emptying, but the insulin inhibition progressed resulting in elevated post-challenge glucose and lipolysis. Incretin hormones and somatostatin (SST) secretion from isolated perfused rat small intestine was unaffected by UCN3 infusion; however, UCN3 infusion stimulated secretion of somatostatin from delta cells in the isolated perfused rat pancreas which, unlike alpha cells and beta cells, expressed
Crhr2
. Conversely, acute antagonism of CRHR2 signalling increased insulin secretion by reducing SST signalling. Consistent with these observations, acute drug-induced inhibition of CRHR2 signalling improved glucose tolerance in rats to a similar degree as administration of glucagon-like peptide-1. UCN3 also powerfully inhibited glucagon secretion from isolated perfused rat pancreas (perfused with 3.5 mmol/l glucose) in a SST-dependent manner, suggesting that UCN3 may be involved in glucose-induced inhibition of glucagon secretion.
Conclusions/interpretation
Our combined data indicate that UCN3 is an important glucoregulatory hormone that acts through regulation of gastrointestinal and pancreatic functions.
Graphical abstract
Myocardial metabolic impairment is a major feature in chronic heart failure. As the major coenzyme in fuel oxidation and oxidative phosphorylation and a substrate for enzymes signaling energy stress ...and oxidative stress response, nicotinamide adenine dinucleotide (NAD
) is emerging as a metabolic target in a number of diseases including heart failure. Little is known on the mechanisms regulating homeostasis of NAD
in the failing heart.
To explore possible alterations of NAD
homeostasis in the failing heart, we quantified the expression of NAD
biosynthetic enzymes in the human failing heart and in the heart of a mouse model of dilated cardiomyopathy (DCM) triggered by Serum Response Factor transcription factor depletion in the heart (SRF
) or of cardiac hypertrophy triggered by transverse aorta constriction. We studied the impact of NAD
precursor supplementation on cardiac function in both mouse models.
We observed a 30% loss in levels of NAD
in the murine failing heart of both DCM and transverse aorta constriction mice that was accompanied by a decrease in expression of the nicotinamide phosphoribosyltransferase enzyme that recycles the nicotinamide precursor, whereas the nicotinamide riboside kinase 2 (NMRK2) that phosphorylates the nicotinamide riboside precursor is increased, to a higher level in the DCM (40-fold) than in transverse aorta constriction (4-fold). This shift was also observed in human failing heart biopsies in comparison with nonfailing controls. We show that the
gene is an AMP-activated protein kinase and peroxisome proliferator-activated receptor α responsive gene that is activated by energy stress and NAD
depletion in isolated rat cardiomyocytes. Nicotinamide riboside efficiently rescues NAD
synthesis in response to FK866-mediated inhibition of nicotinamide phosphoribosyltransferase and stimulates glycolysis in cardiomyocytes. Accordingly, we show that nicotinamide riboside supplementation in food attenuates the development of heart failure in mice, more robustly in DCM, and partially after transverse aorta constriction, by stabilizing myocardial NAD
levels in the failing heart. Nicotinamide riboside treatment also robustly increases the myocardial levels of 3 metabolites, nicotinic acid adenine dinucleotide, methylnicotinamide, and N1-methyl-4-pyridone-5-carboxamide, that can be used as validation biomarkers for the treatment.
The data show that nicotinamide riboside, the most energy-efficient among NAD precursors, could be useful for treatment of heart failure, notably in the context of DCM, a disease with few therapeutic options.
As catabolites of nicotinamide possess physiological relevance, pyridones are often included in metabolomics measurements and associated with pathological outcomes in acute kidney injury (AKI). ...Pyridones are oxidation products of nicotinamide, its methylated form, and its ribosylated form. While they are viewed as markers of over-oxidation, they are often wrongly reported or mislabeled. To address this, we provide a comprehensive characterization of these catabolites of vitamin B3, justify their nomenclature, and differentiate between the biochemical pathways that lead to their generation. Furthermore, we identify an enzymatic and a chemical process that accounts for the formation of the ribosylated form of these pyridones, known to be cytotoxic. Finally, we demonstrate that the ribosylated form of one of the pyridones, the 4-pyridone-3-carboxamide riboside (4PYR), causes HepG3 cells to die by autophagy; a process that occurs at concentrations that are comparable to physiological concentrations of this species in the plasma in AKI patients.
Farnesoid X receptor (FXR) and Takeda G-protein coupled receptor 5 (TGR5) are the two known bile acid (BA) sensitive receptors and are expressed in the intestine and liver as well as in ...extra-enterohepatic tissues. The physiological effects of extra-enterohepatic FXR/TRG5 remain unclear. Further, the extent BAs escape liver reabsorption and how they interact with extra-enterohepatic FXR/TGR5 is understudied. We investigated if hepatic BA reuptake differed between BAs agonistic for FXR and TGR5 compared to non-agonists in the rat. Blood was collected from the portal vein and inferior caval vein from anesthetized rats before and 5, 20, 30, and 40 min post stimulation with sulfated cholecystokinin-8. Plasma concentrations of 20 different BAs were assessed by liquid chromatography coupled to mass spectrometry. Total portal vein BA AUC was 3-4 times greater than in the vena cava inferior (2.7 ± 0.6 vs. 0.7 ± 0.2 mM x min,
< 0.01, n = 8) with total unconjugated BAs being 2-3-fold higher than total conjugated BAs (AUC 8-10 higher
< 0.05 for both). However, in both cases, absolute ratios varied greatly among different BAs. The average hepatic reuptake of BAs agonistic for FXR/TGR5 was similar to non-agonists. However, as the sum of non-agonist BAs in vena portae was 2-3-fold higher than the sum agonist (
< 0.05), the peripheral BA pool was composed mostly of non-agonist BAs. We conclude that hepatic BA reuptake varies substantially by type and does not favor FXR/TGR5 BAs agonists.
The interdependent crises of climate change and biodiversity losses require strategic policies to protect, manage, and restore essential ecosystems. Here, we evaluate the relative importance of US ...national forests (NFs) for protection and conservation as natural climate and biodiversity solutions. We compared landscape integrity (degree of modification by humans), habitat for three keystone species, forest carbon density, accumulation, and total biomass carbon stocks across 154 NFs in the United States. Southern Alaska's Tongass and Chugach NFs hold disproportionally large amounts of high landscape integrity area among all NFs with 25.3% and 5.6% (total 30.9%) of all high (≥9.6) landscape integrity found on NF lands. The Tongass and Chugach store approximately 33% and 3% of all biomass carbon stocks that occur in NFs with high landscape integrity. These two NFs together account for about 49%, 37%, and 18% of all bald eagle, brown bear, and gray wolf habitat found on NF lands. Gray wolf habitat extent was 4% of the total or less on remaining NFs. The Tongass and Chugach were historically wetter and cooler among NFs, and are projected to experience much larger increases in precipitation and much lower increases in maximum temperatures over the coming century. Combined with relatively low recent occurrence of wildfire, this makes permanence more likely. The Tongass and Chugach forests, along with the Pacific Northwest's high carbon density forests should be a high priority for protection and conservation to meet climate and biodiversity goals given their landscape‐scale scarcity and high value.
Plain Language Summary
Permanent protection of forests with relatively high carbon stocks, landscape integrity, and habitat extent would contribute substantially to climate mitigation and species adaptation. The Tongass and Chugach National Forests in southern Alaska rank highest among U.S. National Forests in all three areas. These forests also have relatively low near‐term vulnerability to wildfire and climate, higher connectivity for animal movement, and lower human impacts, making permanence more likely.
Key Points
The Tongass and Chugach are the largest and most intact of all US National Forests, with key bald eagle, brown bear, and gray wolf habitat
The Tongass and Chugach are cool and wet with forest carbon stocks minimally impacted by wildfire and likely to increase with climate change
The Tongass, Chugach and Pacific Northwest's National Forests are high priority for protection to meet climate and biodiversity goals
Bile acid‐CoA: amino acid N‐acyltransferase (BAAT) catalyzes bile acid conjugation, the last step in bile acid synthesis. BAAT gene mutation in humans results in hypercholanemia, growth retardation, ...and fat‐soluble vitamin insufficiency. The current study investigated the physiological function of BAAT in bile acid and lipid metabolism using Baat−/− mice. The bile acid composition and hepatic gene expression were analyzed in 10‐week‐old Baat−/− mice. They were also challenged with a westernized diet (WD) for additional 15 weeks to assess the role of BAAT in bile acid, lipid, and glucose metabolism. Comprehensive lab animal monitoring system and cecal 16S ribosomal RNA gene sequencing were used to evaluate the energy metabolism and microbiome structure of the mice, respectively. In Baat−/− mice, hepatic bile acids were mostly unconjugated and their levels were significantly increased compared with wild‐type mice. Bile acid polyhydroxylation was markedly up‐regulated to detoxify unconjugated bile acid accumulated in Baat−/− mice. Although the level of serum marker of bile acid synthesis, 7α‐hydroxy‐4‐cholesten‐3‐one, was higher in Baat−/− mice, their bile acid pool size was smaller. When fed a WD, the Baat−/− mice showed a compromised body weight gain and impaired insulin secretion. The gut microbiome of Baat−/− mice showed a low level of sulfidogenic bacteria Bilophila. Conclusion: Mouse BAAT is the major taurine‐conjugating enzyme. Its deletion protected the animals from diet‐induced obesity, but caused glucose intolerance. The gut microbiome of the Baat−/− mice was altered to accommodate the unconjugated bile acid pool.
BaatKO mice exhibited almost complete absence of taurine conjugated bile acids in the liver. The mutant mice thrived on this hydrophobic bile acid condition via induction of BA hydroxylation mechanism with compromised body weight gaining.
Nicotinamide adenine dinucleotide (NAD(+)) is a coenzyme for hydride transfer reactions and a substrate for sirtuins and other NAD(+)-consuming enzymes. The abundance of NAD (+), NAD(+) biosynthetic ...intermediates, and related nucleotides reflects the metabolic state of cells and tissues. High performance liquid chromatography (HPLC) followed by ultraviolet-visible (UV-Vis) spectroscopic analysis of NAD(+) metabolites does not offer the specificity and sensitivity necessary for robust quantification of complex samples. Thus, we developed a targeted, quantitative assay of the NAD(+) metabolome with the use of HPLC coupled to mass spectrometry. Here we discuss NAD(+) metabolism as well as the technical challenges required for reliable quantification of the NAD(+) metabolites. The new method incorporates new separations and improves upon a previously published method that suffered from the problem of ionization suppression for particular compounds.
Aim
Neurons in the arcuate nucleus of the hypothalamus are involved in regulation of food intake and energy expenditure, and dysregulation of signalling in these neurons promotes development of ...obesity. The role of the rate‐limiting enzyme in the NAD+ salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT), for regulation energy homeostasis by the hypothalamus has not been extensively studied.
Methods
We determined whether Nampt mRNA or protein levels in the hypothalamus of mice were affected by diet‐induced obesity, by fasting and re‐feeding, and by leptin and ghrelin treatment. Primary hypothalamic neurons were treated with FK866, a selective inhibitor of NAMPT, or rAAV carrying shRNA directed against Nampt, and levels of reactive oxygen species (ROS) and mitochondrial respiration were assessed. Fasting and ghrelin‐induced food intake was measured in mice in metabolic cages after intracerebroventricular (ICV)‐mediated FK866 administration.
Results
NAMPT levels in the hypothalamus were elevated by administration of ghrelin and leptin. In diet‐induced obese mice, both protein and mRNA levels of NAMPT decreased in the hypothalamus. NAMPT inhibition in primary hypothalamic neurons significantly reduced levels of NAD+, increased levels of ROS, and affected the expression of Agrp, Pomc and genes related to mitochondrial function. Finally, ICV‐induced NAMPT inhibition by FK866 did not cause malaise or anhedonia, but completely ablated fasting‐ and ghrelin‐induced increases in food intake.
Conclusion
Our findings indicate that regulation of NAMPT levels in hypothalamic neurons is important for the control of fasting‐ and ghrelin‐induced food intake.
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