Whole-body metabolic fate of branched-chain amino acids.

Oxidation of branched-chain amino acids (BCAAs) is tightly regulated in mammals. We review here the distribution and regulation of whole-body BCAA oxidation. Phosphorylation and dephosphorylation of the rate-limiting enzyme, branched-chain α-ketoacid dehydrogenase complex directly regulates BCAA oxidation, and various other indirect mechanisms of regulation also exist. Most tissues throughout the body are capable of BCAA oxidation, and the flux of oxidative BCAA disposal in each tissue is influenced by three key factors: 1. tissue-specific preference for BCAA oxidation relative to other fuels, 2. the overall oxidative activity of mitochondria within a tissue, and 3. total tissue mass. Perturbations in BCAA oxidation have been implicated in many disease contexts, underscoring the importance of BCAA homeostasis in overall health.

Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132.

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based ß-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure-activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

Quantification of nutrient fluxes during acute exercise in mice.

Despite the known metabolic benefits of exercise, an integrated metabolic understanding of exercise is lacking. Here, we use in vivo steady-state isotope-labeled infusions to quantify fuel flux and oxidation during exercise in fasted, fed, and exhausted female mice, revealing several novel findings. Exercise strongly promoted glucose fluxes from liver glycogen, lactate, and glycerol, distinct from humans. Several organs spared glucose, a process that broke down in exhausted mice despite concomitant hypoglycemia. Proteolysis increased markedly, also divergent from humans. Fatty acid oxidation dominated during fasted exercise. Ketone production and oxidation rose rapidly, seemingly driven by a hepatic bottleneck caused by gluconeogenesis-induced cataplerotic stress. Altered fuel consumption was observed in organs not directly involved in muscle contraction, including the pancreas and brown fat. Several futile cycles surprisingly persisted during exercise, despite their energy cost. In sum, we provide a comprehensive, integrated, holistic, and quantitative accounting of metabolism during exercise in an intact organism.

Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

Off-target depletion of plasma tryptophan by allosteric inhibitors of BCKDK.

The activation of branched chain amino acid (BCAA) catabolism has garnered interest as a potential therapeutic approach to improve insulin sensitivity, enhance recovery from heart failure, and blunt tumor growth. Evidence for this interest relies in part on BT2, a small molecule that promotes BCAA oxidation and is protective in mouse models of these pathologies. BT2 and other analogs allosterically inhibit branched chain ketoacid dehydrogenase kinase (BCKDK) to promote BCAA oxidation, which is presumed to underlie the salutary effects of BT2. Potential "off-target" effects of BT2 have not been considered, however. We therefore tested for metabolic off-target effects of BT2 in Bckdk-/- animals. As expected, BT2 failed to activate BCAA oxidation in these animals. Surprisingly, however, BT2 strongly reduced plasma tryptophan levels and promoted catabolism of tryptophan to kynurenine in both control and Bckdk-/- mice. Mechanistic studies revealed that none of the principal tryptophan catabolic or kynurenine-producing/consuming enzymes (TDO, IDO1, IDO2, or KATs) were required for BT2-mediated lowering of plasma tryptophan. Instead, using equilibrium dialysis assays and mice lacking albumin, we show that BT2 avidly binds plasma albumin and displaces tryptophan, releasing it for catabolism. These data confirm that BT2 activates BCAA oxidation via inhibition of BCKDK but also reveal a robust off-target effect on tryptophan metabolism via displacement from serum albumin. The data highlight a potential confounding effect for pharmaceutical compounds that compete for binding with albumin-bound tryptophan.

Cardiac NAD+ depletion in mice promotes hypertrophic cardiomyopathy and arrhythmias prior to impaired bioenergetics.

Nicotinamide adenine dinucleotide (NAD+) is an essential co-factor in metabolic reactions and co-substrate for signaling enzymes. Failing human hearts display decreased expression of the major NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (Nampt) and lower NAD+ levels, and supplementation with NAD+ precursors is protective in preclinical models. Here we show that Nampt loss in adult cardiomyocytes caused depletion of NAD+ along with marked metabolic derangements, hypertrophic remodeling and sudden cardiac deaths, despite unchanged ejection fraction, endurance and mitochondrial respiratory capacity. These effects were directly attributable to NAD+ loss as all were ameliorated by restoring cardiac NAD+ levels with the NAD+ precursor nicotinamide riboside (NR). Electrocardiograms revealed that loss of myocardial Nampt caused a shortening of QT intervals with spontaneous lethal arrhythmias causing sudden cardiac death. Thus, changes in NAD+ concentration can have a profound influence on cardiac physiology even at levels sufficient to maintain energetics.

Comprehensive quantification of metabolic flux during acute cold stress in mice.

Cold-induced thermogenesis (CIT) is widely studied as a potential avenue to treat obesity, but a thorough understanding of the metabolic changes driving CIT is lacking. Here, we present a comprehensive and quantitative analysis of the metabolic response to acute cold exposure, leveraging metabolomic profiling and minimally perturbative isotope tracing studies in unanesthetized mice. During cold exposure, brown adipose tissue (BAT) primarily fueled the tricarboxylic acid (TCA) cycle with fat in fasted mice and glucose in fed mice, underscoring BAT's metabolic flexibility. BAT minimally used branched-chain amino acids or ketones, which were instead avidly consumed by muscle during cold exposure. Surprisingly, isotopic labeling analyses revealed that BAT uses glucose largely for TCA anaplerosis via pyruvate carboxylation. Finally, we find that cold-induced hepatic gluconeogenesis is critical for CIT during fasting, demonstrating a key functional role for glucose metabolism. Together, these findings provide a detailed map of the metabolic rewiring driving acute CIT.

Branched-chain amino acid catabolism in muscle affects systemic BCAA levels but not insulin resistance.

Elevated levels of plasma branched-chain amino acids (BCAAs) have been associated with insulin resistance and type 2 diabetes since the 1960s. Pharmacological activation of branched-chain α-ketoacid dehydrogenase (BCKDH), the rate-limiting enzyme of BCAA oxidation, lowers plasma BCAAs and improves insulin sensitivity. Here we show that modulation of BCKDH in skeletal muscle, but not liver, affects fasting plasma BCAAs in male mice. However, despite lowering BCAAs, increased BCAA oxidation in skeletal muscle does not improve insulin sensitivity. Our data indicate that skeletal muscle controls plasma BCAAs, that lowering fasting plasma BCAAs is insufficient to improve insulin sensitivity and that neither skeletal muscle nor liver account for the improved insulin sensitivity seen with pharmacological activation of BCKDH. These findings suggest potential concerted contributions of multiple tissues in the modulation of BCAA metabolism to alter insulin sensitivity.

A randomized placebo-controlled clinical trial for pharmacological activation of BCAA catabolism in patients with type 2 diabetes.

Elevations in plasma branched-chain amino acid (BCAA) levels associate with insulin resistance and type 2 diabetes (T2D). Pre-clinical models suggest that lowering BCAA levels improve glucose tolerance, but data in humans are lacking. Here, we used sodium phenylbutyrate (NaPB), an accelerator of BCAA catabolism, as tool to lower plasma BCAA levels in patients with T2D, and evaluate its effect on metabolic health. This trial (NetherlandsTrialRegister: NTR7426) had a randomized, placebo-controlled, double-blind cross-over design and was performed in the Maastricht University Medical Center (MUMC+), the Netherlands, between February 2019 and February 2020. Patients were eligible for the trial if they were 40-75years, BMI of 25-38 kg/m², relatively well-controlled T2D (HbA1C < 8.5%) and treated with oral glucose-lowering medication. Eighteen participants were randomly assigned to receive either NaPB 4.8 g/m²/day and placebo for 2 weeks via controlled randomization and sixteen participants completed the study. The primary outcome was peripheral insulin sensitivity. Secondary outcomes were ex vivo muscle mitochondrial oxidative capacity, substrate oxidation and ectopic fat accumulation. Fasting blood samples were collected to determine levels of BCAA, their catabolic intermediates, insulin, triglycerides, free fatty acids (FFA) and glucose. NaPB led to a robust 27% improvement in peripheral insulin sensitivity compared to placebo (ΔRd:13.2 ± 1.8 vs. 9.6 ± 1.8 µmol/kg/min, p = 0.02). This was paralleled by an improvement in pyruvate-driven muscle mitochondrial oxidative capacity and whole-body insulin-stimulated carbohydrate oxidation, and a reduction in plasma BCAA and glucose levels. No effects were observed on levels of insulin, triglycerides and FFA, neither did fat accumulation in muscle and liver change. No adverse events were reported. These data establish the proof-of-concept in humans that modulating the BCAA oxidative pathway may represent a potential treatment strategy for patients with T2D.

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.