Exercise and ageing impact the kynurenine/tryptophan pathway and acylcarnitine metabolite pools in skeletal muscle of older adults.

Exercise-induced perturbation of skeletal muscle metabolites is a probable mediator of long-term health benefits in older adults. Although specific metabolites have been identified to be impacted by age, physical activity and exercise, the depth of coverage of the muscle metabolome is still limited. Here, we investigated resting and exercise-induced metabolite distribution in muscle from well-phenotyped older adults who were active or sedentary, and a group of active young adults. Percutaneous biopsies of the vastus lateralis were obtained before, immediately after and 3 h following a bout of endurance cycling. Metabolite profile in muscle biopsies was determined by tandem mass spectrometry. Mitochondrial energetics in permeabilized fibre bundles was assessed by high resolution respirometry and fibre type proportion was assessed by immunohistology. We found that metabolites of the kynurenine/tryptophan pathway were impacted by age and activity. Specifically, kynurenine was elevated in muscle from older adults, whereas downstream metabolites of kynurenine (kynurenic acid and NAD+ ) were elevated in muscle from active adults and associated with cardiorespiratory fitness and muscle oxidative capacity. Acylcarnitines, a potential marker of impaired metabolic health, were elevated in muscle from physically active participants. Surprisingly, despite baseline group difference, acute exercise-induced alterations in whole-body substrate utilization, as well as muscle acylcarnitines and ketone bodies, were remarkably similar between groups. Our data identified novel muscle metabolite signatures that associate with the healthy ageing phenotype provoked by physical activity and reveal that the metabolic responsiveness of muscle to acute endurance exercise is retained [NB]:AUTHOR: Please ensure that the appropriate material has been provide for Table S2, as well as for Figures S1 to S7, as also cited in the text with age regardless of activity levels. KEY POINTS: Kynurenine/tryptophan pathway metabolites were impacted by age and physical activity in human muscle, with kynurenine elevated in older muscle, whereas downstream products kynurenic acid and NAD+ were elevated in exercise-trained muscle regardless of age. Acylcarnitines, a marker of impaired metabolic health when heightened in circulation, were elevated in exercise-trained muscle of young and older adults, suggesting that muscle act as a metabolic sink to reduce the circulating acylcarnitines observed with unhealthy ageing. Despite the phenotypic differences, the exercise-induced response of various muscle metabolite pools, including acylcarnitine and ketone bodies, was similar amongst the groups, suggesting that older adults can achieve the metabolic benefits of exercise seen in young counterparts.

Comprehensive interrogation of human skeletal muscle reveals a dissociation between insulin resistance and mitochondrial capacity.

Insulin resistance and blunted mitochondrial capacity in skeletal muscle are often synonymous, however, this association remains controversial. The aim of this study was to perform an in-depth multifactorial comparison of skeletal muscle mitochondrial capacity between individuals who were lean and active (Active, n = 9), individuals with obesity (Obese, n = 9), and individuals with obesity, insulin resistance, and type 2 diabetes (T2D, n = 22). Mitochondrial capacity was assessed by ex vivo mitochondrial respiration with fatty-acid and glycolytic-supported protocols adjusted for mitochondrial content (mtDNA and citrate synthase activity). Supercomplex assembly was measured by Blue Native (BN)-PAGE and immunoblot. Tricarboxylic (TCA) cycle intermediates were assessed with targeted metabolomics. Exploratory transcriptomics and DNA methylation analyses were performed to uncover molecular differences affecting mitochondrial function among the three groups. We reveal no discernable differences in skeletal muscle mitochondrial content, mitochondrial capacity, supercomplex assembly, TCA cycle intermediates, and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (body mass index, age, and aerobic capacity). We highlight that lean, active individuals have greater mitochondrial content, mitochondrial capacity, supercomplex assembly, and TCA cycle intermediates. These phenotypical changes are reflected at the level of DNA methylation and gene transcription. The collective observation of comparable muscle mitochondrial capacity in individuals with obesity and T2D (vs. individuals without T2D) underscores a dissociation from skeletal muscle insulin resistance. Clinical trial number: NCT01911104.NEW & NOTEWORTHY Whether impaired mitochondrial capacity contributes to skeletal muscle insulin resistance is debated. Our multifactorial analysis shows no differences in skeletal muscle mitochondrial content, mitochondrial capacity, and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (BMI, age, aerobic capacity). We highlight that lean, active individuals have enhanced skeletal muscle mitochondrial capacity that is also reflected at the level of DNA methylation and gene transcription.

Discovery of 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea as a potent NAMPT (nicotinamide phosphoribosyltransferase) activator with attenuated CYP inhibition.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the NAD+ salvage pathway. Since NAD+ plays a pivotal role in many biological processes including metabolism and aging, activation of NAMPT is an attractive therapeutic target for treatment of diverse array of diseases. Herein, we report the continued optimization of novel urea-containing derivatives which were identified as potent NAMPT activators. Early optimization of HTS hits afforded compound 12, with a triazolopyridine core, as a lead compound. CYP direct inhibition (DI) was identified as an issue of concern, and was resolved through modulation of lipophilicity to culminate in 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea (21), which showed potent NAMPT activity accompanied with attenuated CYP DI towards multiple CYP isoforms.

Optimization of a urea-containing series of nicotinamide phosphoribosyltransferase (NAMPT) activators.

NAD+ is a crucial cellular factor that plays multifaceted roles in wide ranging biological processes. Low levels of NAD+ have been linked to numerous diseases including metabolic disorders, cardiovascular disease, neurodegeneration, and muscle wasting disorders. A novel strategy to boost NAD+ is to activate nicotinamide phosphoribosyltransferase (NAMPT), the putative rate-limiting step in the NAD+ salvage pathway. We previously showed that NAMPT activators increase NAD+ levels in vitro and in vivo. Herein we describe the optimization of our NAMPT activator prototype (SBI-0797812) leading to the identification of 1-(4-((4-chlorophenyl)sulfonyl)phenyl)-3-(oxazol-5-ylmethyl)urea (34) that showed far more potent NAMPT activation and improved oral bioavailability.

The Failing Heart Relies on Ketone Bodies as a Fuel.

BACKGROUND: Significant evidence indicates that the failing heart is energy starved. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure. METHODS AND RESULTS: Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, the amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of ß-hydroxybutyrate dehydrogenase 1, a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified. CONCLUSIONS: These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure.

Use of Ion Chromatography/Mass Spectrometry for Targeted Metabolite Profiling of Polar Organic Acids.

Organic acids (OAs) serve as metabolites that play pivotal roles in a host of different metabolic and regulatory pathways. The polar nature of many OAs poses a challenge to their measurement using widely practiced analytical methods. In this study, a targeted metabolomics method was developed using ion chromatography/triple quadrupole mass spectrometry (IC/MS) to quantitate 28 polar OAs with limits of quantitation ranging from 0.25 to 50 µM. The interday assay precisions ranged from 1% to 19%, with accuracies ranging from 82% to 115%. The IC/MS assay was used to quantitate OAs in quadriceps muscle from sedentary mice compared to fatigued mice subjected to either a low intensity, long duration (LILD) or high intensity, short duration (HISD) forced treadmill regimen. Among the OAs examined, significant differences were detected for hippuric acid, malic acid, fumaric acid, and 2-ketoglutaric acid between the sedentary and fatigued mice. In conclusion, the IC/MS method enabled the separation and quantitative survey of a broad range of polar OAs that are difficult to analyze by chromatographic techniques.

Genetic drivers of human plasma metabolites that determine mortality in heart failure patients with reduced ejection fraction.

Background: Heart failure with reduced ejection fraction (HFrEF) remains a significant public health issue, with the disease advancing despite neurohormonal antagonism. Energetic dysfunction is a likely contributor to residual disease progression, and we have previously reported a strong association of plasma metabolite profiles with survival among patients with HFrEF. However, the genetic and biologic mechanisms that underlie the metabolite-survival association in HFrEF were uncertain. Methods and results: We performed genetic mapping of the key metabolite parameters, followed by mediation analyses of metabolites and genotypes on survival, and genetic pathway analyses. Patients with HFrEF (n = 1,003) in the Henry Ford Pharmacogenomic Registry (HFPGR; 500 self-reported Black/African race patients [AA], 503 self-reported White/European race patients [EA], and 249 deaths over a median of 2.7 years) with genome-wide genotyping and targeted metabolomic profiling of plasma were included. We tested genome-wide association (GWA) of single nucleotide polymorphisms (SNPs) with the prognostic metabolite profile (PMP) and its components; first stratified by race, and then combined via meta-analysis for the entire cohort. Seven independent loci were identified as GWA significant hits in AA patients (3 for PMP and 4 for individual metabolites), one of which was also significant in the entire cohort (rs944469). No genome wide significant hits were found in White/EA patients. Among these SNPs, only rs35792152, (a hit for 3.HBA) tended to be associated with mortality in standard survival analysis (HR = 1.436, p = 0.052). The mediation analyses indicated several significant associations between SNPs, metabolites, and mortality in AA patients. Functional annotation mapping (FUMA) implicated inflammation, DNA metabolic, and mRNA splicing processes. Conclusions: GWAS of key metabolites and survival along with FUMA pathway analysis revealed new candidate genes which unveiled molecular pathways that contribute to HF disease progression via metabolic and energetic abnormalities.

The TAS1R2 G-protein-coupled receptor is an ambient glucose sensor in skeletal muscle that regulates NAD homeostasis and mitochondrial capacity.

The bioavailability of nicotinamide adenine dinucleotide (NAD) is vital for skeletal muscle health, yet the mechanisms or signals regulating NAD homeostasis remain unclear. Here, we uncover a pathway connecting peripheral glucose sensing to the modulation of muscle NAD through TAS1R2, the sugar-sensing G protein-coupled receptor (GPCR) initially identified in taste perception. Muscle TAS1R2 receptor stimulation by glucose and other agonists induces ERK1/2-dependent phosphorylation and activation of poly(ADP-ribose) polymerase1 (PARP1), a major NAD consumer in skeletal muscle. Consequently, muscle-specific deletion of TAS1R2 (mKO) in male mice suppresses PARP1 activity, elevating NAD levels and enhancing mitochondrial capacity and running endurance. Plasma glucose levels negatively correlate with muscle NAD, and TAS1R2 receptor deficiency enhances NAD responses across the glycemic range, implicating TAS1R2 as a peripheral energy surveyor. These findings underscore the role of GPCR signaling in NAD regulation and propose TAS1R2 as a potential therapeutic target for maintaining muscle health.

Source of nicotinamide governs its metabolic fate in cultured cells, mice, and humans.

Metabolic routing of nicotinamide (NAM) to NAD+ or 1-methylnicotinamide (MeNAM) has impacts on human health and aging. NAM is imported by cells or liberated from NAD+. The fate of 2H4-NAM in cultured cells, mice, and humans was determined by stable isotope tracing. 2H4-NAM is an NAD+ precursor via the salvage pathway in cultured A549 cells and human PBMCs and in A549 cell xenografts and PBMCs from 2H4-NAM-dosed mice and humans, respectively. 2H4-NAM is a MeNAM precursor in A549 cell cultures and xenografts, but not isolated PBMCs. NAM released from NAD+ is a poor MeNAM precursor. Additional A549 cell tracer studies yielded further mechanistic insight. NAMPT activators promote NAD+ synthesis and consumption. Surprisingly, NAM liberated from NAD+ in NAMPT activator-treated A549 cells is also routed toward MeNAM production. Metabolic fate mapping of the dual NAM sources across the translational spectrum (cells, mice, humans) illuminates a key regulatory node governing NAD+ and MeNAM synthesis.

Metabolic dysregulation impairs lymphocyte function during severe SARS-CoV-2 infection.

Cellular metabolic dysregulation is a consequence of SARS-CoV-2 infection that is a key determinant of disease severity. However, how metabolic perturbations influence immunological function during COVID-19 remains unclear. Here, using a combination of high-dimensional flow cytometry, cutting-edge single-cell metabolomics, and re-analysis of single-cell transcriptomic data, we demonstrate a global hypoxia-linked metabolic switch from fatty acid oxidation and mitochondrial respiration towards anaerobic, glucose-dependent metabolism in CD8+Tc, NKT, and epithelial cells. Consequently, we found that a strong dysregulation in immunometabolism was tied to increased cellular exhaustion, attenuated effector function, and impaired memory differentiation. Pharmacological inhibition of mitophagy with mdivi-1 reduced excess glucose metabolism, resulting in enhanced generation of SARS-CoV-2- specific CD8+Tc, increased cytokine secretion, and augmented memory cell proliferation. Taken together, our study provides critical insight regarding the cellular mechanisms underlying the effect of SARS-CoV-2 infection on host immune cell metabolism, and highlights immunometabolism as a promising therapeutic target for COVID-19 treatment.

The TAS1R2 sweet taste receptor regulates skeletal muscle mass and fitness.

Muscle fitness and mass deteriorate under the conditions of obesity and aging for reasons yet to be fully elucidated. Herein, we describe a novel pathway linking peripheral nutrient sensing and skeletal muscle function through the sweet taste receptor TAS1R2 and the involvement of ERK2-PARP1-NAD signaling axis. Muscle-specific deletion of TAS1R2 (mKO) in mice produced elevated NAD levels due to suppressed PARP1 activity, improved mitochondrial function, increased muscle mass and strength, and prolonged running endurance. Deletion of TAS1R2 in obese or aged mice also ameliorated the decline in muscle mass and fitness arising from these conditions. Remarkably, partial loss-of-function of TAS1R2 (rs35874116) in older, obese humans recapitulated the healthier muscle phenotype displayed by mKO mice in response to exercise training. Our findings show that inhibition of the TAS1R2 signaling in skeletal muscle is a promising therapeutic approach to preserve muscle mass and function.

Enhanced clearance of Abeta in brain by sustaining the plasmin proteolysis cascade.

The amyloid hypothesis states that a variety of neurotoxic beta-amyloid (Abeta) species contribute to the pathogenesis of Alzheimer's disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Abeta production and clearance. Enzymes responsible for the degradation of Abeta are not well understood, and, thus far, it has not been possible to enhance Abeta catabolism by pharmacological manipulation. We provide evidence that Abeta catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Abeta levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Abeta oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Abeta. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Abeta levels, restore long-term potentiation deficits in hippocampal slices from transgenic Abeta-producing mice, and reverse cognitive deficits in these mice.

Metabolic adaptation characterizes short-term resistance to weight loss induced by a low-calorie diet in overweight/obese individuals.

BACKGROUND: Low-calorie diet (LCD)-induced weight loss demonstrates response heterogeneity. Physiologically, a decrease in energy expenditure lower than what is predicted based on body composition (metabolic adaptation) and/or an impaired capacity to increase fat oxidation may hinder weight loss. Understanding the metabolic components that characterize weight loss success is important for optimizing weight loss strategies. OBJECTIVES: We tested the hypothesis that overweight/obese individuals who had lower than expected weight loss in response to a 28-d LCD would be characterized by 1) impaired fat oxidation and 2) whole-body metabolic adaptation. We also characterized the molecular mechanisms associated with weight loss success/failure. METHODS: This was a retrospective comparison of participants who met their predicted weight loss targets [overweight/obese diet sensitive (ODS), n = 23, females = 21, males = 2] and those that did not [overweight/obese diet resistant (ODR), n = 14, females = 12, males = 2] after a 28-d LCD (900-1000 kcal/d). We used whole-body (energy expenditure and fat oxidation) and tissue-specific measurements (metabolic proteins in skeletal muscle, gene expression in adipose tissue, and metabolites in serum) to detect metabolic properties and biomarkers associated with weight loss success. RESULTS: The ODR group had greater mean ± SD metabolic adaptation (-175 ± 149 kcal/d; +119%) than the ODS group (-80 ± 108 kcal/d) after the LCD (P = 0.030). Mean ± SD fat oxidation increased similarly for both groups from baseline (0.0701 ± 0.0206 g/min) to day 28 (0.0869 ± 0.0269 g/min; P < 0.001). A principal component analysis factor comprised of serum 3-hydroxybutyric acid, citrate, leucine/isoleucine, acetyl-carnitine, and 3-hydroxylbutyrlcarnitine was associated with weight loss success at day 28 (std. ß = 0.674, R2 = 0.479, P < 0.001). CONCLUSIONS: Individuals who achieved predicted weight loss targets after a 28-d LCD were characterized by reduced metabolic adaptation. Accumulation of metabolites associated with acetyl-CoA excess and enhanced ketogenesis was identified in the ODS group.This trial was registered at clinicaltrials.gov as NCT01616082.

A Metabolomic Signature of Glucagon Action in Healthy Individuals With Overweight/Obesity.

CONTEXT: Glucagon is produced and released from the pancreatic alpha-cell to regulate glucose levels during periods of fasting. The main target for glucagon action is the liver, where it activates gluconeogenesis and glycogen breakdown; however, glucagon is postulated to have other roles within the body. OBJECTIVE: We sought to identify the circulating metabolites that would serve as markers of glucagon action in humans. METHODS: In this study (NCT03139305), we performed a continuous 72-hour glucagon infusion in healthy individuals with overweight/obesity. Participants were randomized to receive glucagon 12.5 ng/kg/min (GCG 12.5), glucagon 25 ng/kg/min (GCG 25), or a placebo control. A comprehensive metabolomics analysis was then performed from plasma isolated at several time points during the infusion to identify markers of glucagon activity. RESULTS: Glucagon (GCG 12.5 and GCG 25) resulted in significant changes in the plasma metabolome as soon as 4 hours following infusion. Pathways involved in amino acid metabolism were among the most affected. Rapid and sustained reduction of a broad panel of amino acids was observed. Additionally, time-dependent changes in free fatty acids and diacylglycerol and triglyceride species were observed. CONCLUSION: These results define a distinct signature of glucagon action that is broader than the known changes in glucose levels. In particular, the robust changes in amino acid levels may prove useful to monitor changes induced by glucagon in the context of additional glucagon-like peptide-1 or gastric inhibitory polypeptide treatment, as these agents also elicit changes in glucose levels.

LXR ligand lowers LDL cholesterol in primates, is lipid neutral in hamster, and reduces atherosclerosis in mouse.

Liver X receptors (LXRs) are ligand-activated transcription factors that coordinate regulation of gene expression involved in several cellular functions but most notably cholesterol homeostasis encompassing cholesterol transport, catabolism, and absorption. WAY-252623 (LXR-623) is a highly selective and orally bioavailable synthetic modulator of LXR, which demonstrated efficacy for reducing lesion progression in the murine LDLR(-/-) atherosclerosis model with no associated increase in hepatic lipogenesis either in this model or Syrian hamsters. In nonhuman primates with normal lipid levels, WAY-252623 significantly reduced total (50-55%) and LDL-cholesterol (LDLc) (70-77%) in a time- and dose-dependent manner as well as increased expression of the target genes ABCA1/G1 in peripheral blood cells. Statistically significant decreases in LDLc were noted as early as day 7, reached a maximum by day 28, and exceeded reductions observed for simvastatin alone (20 mg/kg). Transient increases in circulating triglycerides and liver enzymes reverted to baseline levels over the course of the study. Complementary microarray analysis of duodenum and liver gene expression revealed differential activation of LXR target genes and suggested no direct activation of hepatic lipogenesis. WAY-252623 displays a unique and favorable pharmacological profile suggesting synthetic LXR ligands with these characteristics may be suitable for evaluation in patients with atherosclerotic dyslipidemia.

The gap junction modifier, GAP-134 [(2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid], improves conduction and reduces atrial fibrillation/flutter in the canine sterile pericarditis model.

Gap junction uncoupling can alter conduction pathways and promote cardiac re-entry mechanisms that potentiate many supraventricular arrhythmias, such as atrial fibrillation (AF) and atrial flutter (AFL). Our objective was to determine whether GAP-134 [(2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid], a small dipeptide gap junction modifier, can improve conduction and ultimately prevent AF/AFL. In rat atrial strips subjected to metabolic stress, GAP-134 prevented significantly conduction velocity slowing at 10 nM compared with vehicle (p < 0.01). In the canine sterile pericarditis model, conduction time (CT; n = 5), atrial effective refractory period (AERP; n = 3), and AF/AFL duration/inducibility (n = 16) were measured 2 to 3 days postoperatively in conscious dogs. CT was significantly faster after GAP-134 infusion (average plasma concentration, 250 nM) at cycle lengths of 300 ms (66.2 +/- 1.0 versus 62.0 +/- 1.0 ms; p < 0.001) and 200 ms (64.4 +/- 0.9 versus 61.0 +/- 1.3 ms; p < 0.001). No significant changes in AERP were noted after GAP-134 infusion. The mean number of AF/AFL inductions per animal was significantly decreased after GAP-134 infusion (2.7 +/- 0.6 versus 1.6 +/- 0.8; p < 0.01), with total AF/AFL burden being decreased from 12,280 to 6063 s. Western blot experiments showed no change in connexin 43 expression. At concentrations exceeding those described in the AF/AFL experiments, GAP-134 had no effect on heart rate, blood pressure, or any electrocardiogram parameters. In conclusion, GAP-134 shows consistent efficacy on measures of conduction and AF/AFL inducibility in the canine sterile pericarditis model. These findings, along with its oral bioavailability, underscore its potential antiarrhythmic efficacy.

GAP-134 ([2S,4R]-1-[2-aminoacetyl]4-benzamidopyrrolidine-2-carboxylic acid) prevents spontaneous ventricular arrhythmias and reduces infarct size during myocardial ischemia/reperfusion injury in open-chest dogs.

The antiarrhythmic dipeptide, GAP-134, ([2S,4R]-1[2-aminoacetyl]-4-benzamido-pyrrolidine-2-carboxylic acid) was evaluated in canine ischemia/reperfusion model. In dogs subjected to 60-minute ischemia and 4-hour reperfusion, GAP-134 was administered 10 minutes before reperfusion as a bolus + intravenous (IV) infusion. The doses administered were 0.25 microg/kg bolus + 0.19 microg/kg per hour infusion; 2.5 microg/kg + 1.9 microg/kg per hour; 25 mg/kg + 19 mg/kg per hour; 75 mg/kg + 57 mg/kg per hour. Ventricular ectopy was quantified during reperfusion, including premature ventricular contractions (PVC) and ventricular tachycardia (VT). Total incidence of VT was reduced significantly with the 2 highest doses of GAP-134 (1.7 + 0.8; 2.2 + 1.4 events; P < .05) compared to controls (23.0 + 6.1). Total PVCs were reduced significantly from 11.1 + 1.6% in control animals to 2.0% + 0.7% and 1.8% + 0.8% after the 2 highest doses of GAP-134. Infarct size, expressed as percentage of left ventricle, was reduced significantly from 19.0% + 3.5% in controls to 7.9% + 1.5% and 7.1% + 0.8% (P < .05) at the 2 highest doses of GAP-134. GAP-134 is an effective antiarrhythmic agent with potential to reduce ischemia/reperfusion injury.

Metabolomics Analyses of Muscle Atrophy Induced by Hind Limb Unloading.

Acute periods of contractile inactivity cause skeletal muscle atrophy along with profound alterations in tissue metabolism. Hind limb unloading via tail suspension is a commonly used rodent model of muscle atrophy. Here, we describe a sample preparation and LC-MS/MS approach for quantifying specific panels of acylcarnitines, amino acids, and organic acids in small (~8 mg) samples of atrophied mouse soleus following a period of hind limb unloading.

Measurement of Pyridine Nucleotides in Biological Samples Using LC-MS/MS.

Pyridine nucleotides which include NAD+, NADH, NADP, and NADPH play vital roles in many different biological processes. These metabolites can be accurately quantified in a wide variety of biological samples using LC-MS/MS. The quality and precision of these measurements was enhanced using heavy isotope-labeled internal standards and carefully crafted protocols for sample processing.

Boosting NAD+ with a small molecule that activates NAMPT.

Pharmacological strategies that boost intracellular NAD+ are highly coveted for their therapeutic potential. One approach is activation of nicotinamide phosphoribosyltransferase (NAMPT) to increase production of nicotinamide mononucleotide (NMN), the predominant NAD+ precursor in mammalian cells. A high-throughput screen for NAMPT activators and hit-to-lead campaign yielded SBI-797812, a compound that is structurally similar to active-site directed NAMPT inhibitors and blocks binding of these inhibitors to NAMPT. SBI-797812 shifts the NAMPT reaction equilibrium towards NMN formation, increases NAMPT affinity for ATP, stabilizes phosphorylated NAMPT at His247, promotes consumption of the pyrophosphate by-product, and blunts feedback inhibition by NAD+. These effects of SBI-797812 turn NAMPT into a "super catalyst" that more efficiently generates NMN. Treatment of cultured cells with SBI-797812 increases intracellular NMN and NAD+. Dosing of mice with SBI-797812 elevates liver NAD+. Small molecule NAMPT activators such as SBI-797812 are a pioneering approach to raise intracellular NAD+ and realize its associated salutary effects.