Branched-chain Amino Acid Degradation Research Articles

Understanding acute metabolic decompensation in propionic and methylmalonic acidemias: a deep metabolic phenotyping approach

BackgroundPathophysiology of life-threatening acute metabolic decompensations (AMD) in propionic acidemia (PA) and isolated methylmalonic acidemia (MMA) is insufficiently understood. Here, we study the metabolomes of PA and MMA patients over time, to improve insight in which biochemical processes are at play during AMD.MethodsLongitudinal data from clinical chemistry analyses and metabolic assays over the life-course of 11 PA and 13 MMA patients were studied retrospectively. Direct-infusion high-resolution mass spectrometry was performed on 234 and 154 remnant dried blood spot and plasma samples of PA and MMA patients, respectively. In addition, a systematic literature search was performed on reported biomarkers. All results were integrated in an assessment of biochemical processes at play during AMD.ResultsWe confirmed many of the metabolite alterations reported in literature, including increases of plasma valine and isoleucine during AMD in PA patients. We revealed that plasma leucine and phenylalanine, and urinary pyruvic acid were increased during AMD in PA patients. 3-hydroxyisovaleric acid correlated positively with plasma ammonia. We found that known diagnostic biomarkers were not significantly further increased, while intermediates of the branched-chain amino acid (BCAA) degradation pathway were significantly increased during AMD.ConclusionsWe revealed that during AMD in PA and MMA, BCAA and BCAA intermediates accumulate, while known diagnostic biomarkers remain essentially unaltered. This implies that these acidic BCAA intermediates are responsible for metabolic acidosis. Based on this, we suggest to measure plasma 3-hydroxyisovaleric acid and urinary ketones or 3-hydroxybutyric acid for the biochemical follow-up of a patient’s metabolic stability.

Metabolome and proteome changes in skeletal muscle and blood of pre-weaning calves fed leucine and threonine supplemented diets

In pre-weaning calves, both leucine and threonine play important roles in growth and muscle metabolism. In this study, metabolomics, proteomics and clinical chemistry were used to assess the effects of leucine and threonine supplementation added to milk replacer on 14 newborn Holstein male calves: 7 were fed a control diet (Ctrl) and 7 were fed the Ctrl diet supplemented with 0.3% leucine and 0.3% threonine (LT) from 5.6 days of age to 53.6 days. At this time, blood and semitendinosus muscle biopsies were collected for analysis. Integrated metabolomics and proteomics showed that branched-chain amino acids (BCAA) degradation and mitochondrial oxidative metabolism (citrate cycle and respiratory chain) were the main activated pathways in muscle because of the supplementation. BCAA derivatives and metabolites related to lipid mobilization showed the major changes. The deleterious effects of activated oxidative phosphorylation were balanced by the upregulation of antioxidant proteins. An increase in protein synthesis was indicated by elevated aminoacyl-tRNA biosynthesis and increased S6 ribosomal protein phosphorylation in skeletal muscle. In conclusion, LT group showed greater BCAA availability and mitochondrial oxidative activity; as the muscle cells undergo greater aerobic metabolism, antioxidant defenses were activated to compensate for possible cell damage. Data are available via ProteomeXchange (PXD016098). SignificanceLeucine and threonine are essential amino acids for the pre-weaning calf, being of high importance for growth. In this study, we found that leucine and threonine supplementation of milk replacer to feed pre-weaning calves led to differences in the proteome, metabolome and clinical chemistry analytes in skeletal muscle and plasma, albeit no differences in productive performance were recorded. This study extends our understanding on the metabolism in dairy calves and helps optimizing their nutritional status.

Iron competition triggers antibiotic biosynthesis in Streptomyces coelicolor during coculture with Myxococcus xanthus

Microbial coculture to mimic the ecological habitat has been suggested as an approach to elucidate the effect of microbial interaction on secondary metabolite biosynthesis of Streptomyces. However, because of chemical complexity during coculture, underlying mechanisms are largely unknown. Here, we found that iron competition triggered antibiotic biosynthesis in Streptomyces coelicolor during coculture with Myxococcus xanthus. During coculture, M. xanthus enhanced the production of a siderophore, myxochelin, leading M. xanthus to dominate iron scavenging and S. coelicolor to experience iron-restricted conditions. This chemical competition, but not physical contact, activated the actinorhodin biosynthetic gene cluster and the branched-chain amino acid degradation pathway which imply the potential to produce precursors, along with activation of a novel actinorhodin export system. Furthermore, we found that iron restriction increased the expression of 21 secondary metabolite biosynthetic gene clusters (smBGCs) in other Streptomyces species. These findings suggested that the availability for key ions stimulates specific smBGCs, which had the potential to enhance secondary metabolite biosynthesis in Streptomyces.

Lower Circulating Branched-Chain Amino Acid Concentrations Among Vegetarians are Associated with Changes in Gut Microbial Composition and Function.

Vegetarian diets confer health benefits to many cardiometabolic diseases, although whether and how gut microbiota in vegetarians contributes to host metabolism remains unclear. Thus, the aim is to explore the possible links between the gut microbiota and circulating gut microbiota-host co-metabolites among vegetarians and omnivores. Fecal and serum samples from 36 adults following a vegan, lacto-ovo vegetarian, or omnivorous diet are collected. A 16S rRNA gene, metagenome, metatranscriptome, and metabolome integrated multi-omics approach is adopted to profile fecal microbial composition and functionality and circulating gut microbiota-host co-metabolites. 16S rRNA gene and metagenomic sequencing suggest a significant difference in gut microbial composition between the two vegetarian groups and the omnivorous group at the family, genus, and species level. Metabolomic analysis reveals that circulating branched-chain amino acids (BCAAs)-valine, leucine, and isoleucine-are significantly lower in the two vegetarian groups than those in the omnivorous group. In line with the lower concentrations of BCAAs, metatranscriptomic analysis shows that the gut microbial pathway for the degradation of BCAAs is significantly upregulated among vegetarians compared with the omnivores. The results indicate that gut microbiota plays an important role in the modulation of circulating BCAAs among vegetarians.

Branched-chain amino acid catabolism of Thermoanaerobacter pseudoethanolicus reveals potential route to branched-chain alcohol formation

The fermentation of branched-chain amino acids (BCAAs) to branched-chain fatty acids (BCFAs) and branched-chain alcohols (BCOHs) is described using Thermoanaerobacter pseudoethanolicus. BCAAs were not degraded without an electron scavenging system but were degraded to a mixture of their BCFA (major) and BCOH (minor) when thiosulfate was added to the culture. Various environmental parameters were investigated using isoleucine as the substrate which ultimately demonstrated that at higher liquid-gas phase ratios the formation of 2-methyl-1-butanol from isoleucine achieved a maximal titer of 3.4mM at a 1:1 liquid-gas ratio suggesting that higher partial pressure of hydrogen influences the BCOH/BCFA ratio but did not increase further with higher L-G phase ratios. Alternately, increasing the thiosulfate concentration decreased the BCOH to BCFA ratio. Kinetic monitoring of BCAA degradation revealed that the formation of BCOHs occurs slowly after the onset of BCFA formation. 13C2-labeled studies of leucine confirmed the production of a mixture of 3-methyl-1-butyrate and 3-methyl-1-butanol, while experiments involving 13C1-labeled 3-methyl-1-butyrate in fermentations containing leucine demonstrated that the carboxylic acid is reduced to the corresponding alcohol. Thus, the role of carboxylic acid reduction is likely of importance in the production of BCOH formation during the degradation of BCAA such as leucine.

Plasma metabolomics profiles suggest beneficial effects of a low–glycemic load dietary pattern on inflammation and energy metabolism

Low-glycemic load dietary patterns, characterized by consumption of whole grains, legumes, fruits, and vegetables, are associated with reduced risk of several chronic diseases. Using samples from a randomized, controlled, crossover feeding trial, we evaluated the effects on metabolic profiles of a low-glycemic whole-grain dietary pattern (WG) compared with a dietary pattern high in refined grains and added sugars (RG) for 28 d. LC-MS-based targeted metabolomics analysis was performed on fasting plasma samples from 80 healthy participants (n= 40 men, n = 40 women) aged 18-45 y. Linear mixed models were used to evaluate differences in response between diets for individual metabolites. Kyoto Encyclopedia of Genes and Genomes (KEGG)-defined pathways and 2 novel data-driven analyses were conducted to consider differences at the pathway level. There were 121 metabolites with detectable signal in >98% of all plasma samples. Eighteen metabolites were significantly different between diets at day 28 [false discovery rate (FDR)<0.05]. Inositol, hydroxyphenylpyruvate, citrulline, ornithine, 13-hydroxyoctadecadienoic acid, glutamine, and oxaloacetate were higher after the WG diet than after the RG diet, whereas melatonin, betaine, creatine, acetylcholine, aspartate, hydroxyproline, methylhistidine, tryptophan, cystamine, carnitine, and trimethylamine were lower. Analyses using KEGG-defined pathways revealed statistically significant differences in tryptophan metabolism between diets, with kynurenine and melatonin positively associated with serum C-reactive protein concentrations. Novel data-driven methods at the metabolite and network levels found correlations among metabolites involved in branched-chain amino acid (BCAA) degradation, trimethylamine-N-oxide production, and β oxidation of fatty acids (FDR<0.1) that differed between diets, with more favorable metabolic profiles detected after the WG diet. Higher BCAAs and trimethylamine were positively associated with homeostasis model assessment-insulin resistance. These exploratory metabolomics results support beneficial effects of a low-glycemic load dietary pattern characterized by whole grains, legumes, fruits, and vegetables, compared with a diet high in refined grains and added sugars on inflammation and energy metabolism pathways. This trial was registered at clinicaltrials.gov as NCT00622661.

P2‐166: BRANCHED‐CHAIN AMINO ACIDS (BCAAS): A NEW PLAYER IN ALZHEIMER'S DISEASE

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that is the third leading cause of mortality in the US. AD brain is mainly characterized by accumulated plaques and tangles and diminished neurotransmitters. AD is strongly associated with type 2 diabetes (T2D). Emerging studies suggest that branched-chain amino acids (BCAAs), the essential amino acids we need to obtain from food, are involved in the pathogenesis of insulin resistance and T2D. In support of this concept, BCAA degradation is impaired in obese and/or diabetic individuals, and BCAA supplementation leads to insulin resistance and perturbed glycemic control. It is currently unknown if similar defective BCAA metabolism exists in AD patients. Since BCAAs along with aromatic amino acids are critical for production and maintenance of brain neurotransmitters, here we tested if BCAA metabolism in liver – an organ with the highest BCAA degradation activity – is impaired in AD. Eight month-old wildtype or AD transgenic mice were fed a standard chow diet until sacrifice. Serum BCAA levels were measured by BCAA assay, and proteins and genes related to BCAA metabolism in liver were determined by western blot and RT-qPCR, respectively. Serum BCAA profile of healthy or AD individuals were assessed by metabolomics analysis. The activity of branched-chain α-keto acid dehydrogenase (BCKDH), the rate-limiting enzyme in BCAA degradation pathway, in liver was significantly suppressed in AD mice compared to wildtypes as evidenced by the protein expression and its phosphorylated, inactive, state. This is supported by increased hepatic BCKDH Kinase at both protein and gene levels in AD mice. Serum BCAAs and/or their metabolites were higher in both AD mice and humans compared to healthy controls, indicating impaired BCAA metabolism. Our findings suggest that hepatic BCAA catabolism is impaired in AD mice. This may lead to high plasma BCAAs and their metabolites that can potentially contribute to the imbalance of brain neurotransmitters and development of AD or related dementia. A longitudinal assessment of BCAA metabolism will allow us to determine if they play a predictive and/or causal role in the development of AD.

Abstract 5270: A potential role of branched chain amino acid metabolism in breast cancer cell invasiveness

Abstract Carcinomas have two broadly distinct modes of invasion: collective and individual. Collective invasion is characterized as groups of cells that migrate while retaining cell-cell contacts. Individual invasion occurs through mechanisms such as the acquiring of mesenchymal-like features by cancer cells. While these different modes of invasion have been well studied at the genomic and transcriptional levels, their metabolic alterations remain much less understood. To address this deficiency, we performed untargeted NMR metabolomics, in conjunction with RNA-seq, on two variants of MCF7, a widely used breast cancer cell line. We report herein several findings. First, the two MCF7 variants, one parental (referred to as MCF-7 hereafter) and another having undergone many passages (referred to as Augusta hereafter), differ morphologically, with MCF-7 being more epithelial and Augusta being more mesenchymal-like. Importantly, fibroblast co-culture experiments indicate that MCF-7 cells resemble collective invasion, while Augusta cells resemble individual invasion. Furthermore, both scratch-assay and gene set enrichment analysis of RNA-seq data indicate that Augusta cells are more invasive. Second, our NMR metabolomics analysis reveals that branched chain amino acids (BCAAs) represent a significant metabolic difference between MCF-7 and Augusta cells. Partial least squares discriminant analysis of the metabolomics data identified BCAAs as essential distinguishing features between the two cell lines. Specifically, compared to MCF-7 cells, BCAAs are significantly depleted inside Augusta cells, consistent with the upregulation of several key BCAA degradation genes. We hypothesize that Augusta cells can more efficiently utilize BCAAs, and are performing 13C-labeled BCAA flux analysis to test this hypothesis. Previous studies have shown that an aberrantly expressed BCAA metabolism gene sustains growth and enhances invasiveness of breast cancer cells. However, increased catabolism of BCAAs has not been reported in literature on this subject. Our simple cell line model allows for a deeper molecular understanding of BCAA metabolism in breast cancer cell invasion. Citation Format: Maxwell B. Colonna, Arthur S. Edison, Shaying Zhao. A potential role of branched chain amino acid metabolism in breast cancer cell invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5270.

732-P: Insulin Sensitivity Nonresponders after Long-Term Intensive Exercise Training Are Characterized by Perturbed Amino Acid Metabolism and Mitochondrial Dysfunction

Exercise may effectively improve insulin sensitivity, although some individuals do not reap this benefit. Yet, mechanisms underlying variations in training-improved insulin sensitivity remain unknown. We investigated men with or without dysglycemia subjected to 12 w of high-intensity endurance and strength exercise, and classified them as nonresponders (n=5) or responders (n=21) based on glucose infusion rate (GIR) from euglycemic hyperinsulinemic clamping. We explored variables related to variation in training-improved GIR. VO2max, muscle strength, fat depots, adipose tissue (AT) and skeletal muscle (SkM) lipidomics, mRNA sequencing, and blood amino acid profiles were evaluated pre- and post-training. By design, nonresponders did not improve their GIR, whereas responders exhibited a pronounced increase (42%). There was no difference in attendance to training sessions between the groups, or any correlation between attendance rate and training-improved GIR. Both groups improved VO2max, leg press, pull down and chest press strength, but responders increased significantly more in VO2max and chest press strength. Nonresponders exhibited higher baseline plasma concentrations of leucine, isoleucine, valine, homocysteine and cysteine, more intraperitoneal AT and impaired insulin signaling, branched-chain amino acids and methionine and cysteine metabolism in AT, and higher SkM triacylglycerol, free fatty acids and cholesterol levels and impaired SkM oxidative phosphorylation, branched-chain amino acids degradation and tricarboxylic acid cycle. Failing to improve insulin sensitivity after long-term intensive training may relate to perturbed amino acids metabolism and mitochondrial dysfunction. Initial plasma levels of branched-chain and sulphur amino acids may determine individuals not obtaining glucometabolic improvements after training. Disclosure S. Lee: None. H.L. Gulseth: Consultant; Self; Novo Nordisk A/S. Speaker’s Bureau; Self; Merck Sharp &amp; Dohme Corp., Sanofi-Aventis. C.A. Drevon: Board Member; Self; Vitas AS. Consultant; Self; Vitas AS. K.I. Birkeland: Research Support; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lifecare, Inc., Merck Sharp &amp; Dohme Corp., Roche Diabetes Care. Other Relationship; Self; Norwegian Diabetes Association.

Branched-chain Amino Acid Metabolism Is Impaired in Mice and Humans with Alzheimer’s Disease (OR27-04-19)

ObjectivesAlzheimer’s disease (AD) is an irreversible neurodegenerative disorder that is the third leading cause of mortality in the US. AD brain is mainly characterized by accumulated plaques and tangles and diminished neurotransmitters. AD is strongly associated with type 2 diabetes (T2D). Emerging studies suggest that branched-chain amino acids (BCAAs), the essential amino acids we need to obtain from food, are involved in the pathogenesis of insulin resistance and T2D. In support of this concept, BCAA degradation is impaired in obese and/or diabetic individuals, and BCAA supplementation leads to insulin resistance and perturbed glycemic control. It is currently unknown if similar defective BCAA metabolism exists in AD patients. Since BCAAs along with aromatic amino acids are critical for production and maintenance of brain neurotransmitters, here we tested if BCAA metabolism in liver – an organ with the highest BCAA degradation activity – is impaired in AD. MethodsEight month-old wildtype or AD transgenic mice were fed a standard chow diet until sacrifice. Serum BCAA levels were measured by BCAA assay, and proteins and genes related to BCAA metabolism in liver were determined by western blot and RT-qPCR, respectively. Serum BCAA profile of healthy or AD individuals were assessed by metabolomics analysis. ResultsThe activity of branched-chain α-keto acid dehydrogenase (BCKDH), the rate-limiting enzyme in BCAA degradation pathway, in liver was significantly suppressed in AD mice compared to wildtypes as evidenced by the protein expression and its phosphorylated, inactive, state. This is supported by increased hepatic BCKDH Kinase at both protein and gene levels in AD mice. Serum BCAAs and/or their metabolites were higher in both AD mice and humans compared to healthy controls, indicating impaired BCAA metabolism. ConclusionsOur findings suggest that hepatic BCAA catabolism is impaired in AD mice. This may lead to high plasma BCAAs and their metabolites that can potentially contribute to the imbalance of brain neurotransmitters and development of AD or related dementia. A longitudinal assessment of BCAA metabolism will allow us to determine if they play a predictive, diagnostic, and/or causal role in the development of AD. Funding SourcesNIH DK099463, Wylie Briscoe Fund, Texas Tech University.

Evidence for the Accumulation of 3‐Hydroxypropionate and β‐Alanine in Arabidopsis thaliana Seedlings via Isoleucine Degradation

Amino acids serve several cellular processes outside of proteinogenesis that influence the growth and development of the plant, as well as the generation of metabolic energy. Here, we examine the contribution of branched‐chain amino acid degradation to the production of β‐alanine in the model organism, Arabidopsis thaliana. β‐alanine is a key intermediate in the synthesis of the essential molecules vitamin B5 and coenzyme A (CoA). Despite the importance, the current knowledge surrounding β‐alanine lacks strong evidence for the primary biosynthetic pathways in higher plants. Previous studies found that polyamines, uracil, and propionate can all serve as sources for β‐alanine. Given that the catabolism of branched‐chain amino acids (BCAAs) valine and isoleucine result in the production of propionyl‐CoA (a derivative of propionate), we hypothesized that these BCAAs could also serve as a source of β‐alanine. Propionyl‐CoA is typically converted to acetyl‐CoA via β‐oxidation, however, one of the intermediates, malonate semialdehyde, can react to form β‐alanine. To trace the production of β‐alanine from propionyl‐CoA and the BCAA isoleucine, we treated wild‐type and transgenic seedlings with isotopically‐labeled precursors and analyzed extracts using 13C‐NMR and GC‐MS. We observed the production of both β‐alanine and a key intermediate, 3‐hydroxypropionate (3HP) in samples treated with propionate and isoleucine. Our data shows that the fed 13C‐isotopes were efficiently incorporated into and recycled through propionyl‐CoA metabolism, resulting in the production of 13C‐labeled β‐alanine. These findings suggest that while there is evidence for the production of β‐alanine via polyamines, uracil, and propionate in other plants; isoleucine, and possibly valine, can also serve as a precursor to this metabolically important molecule.Support or Funding InformationClare Boothe Luce Undergraduate Scholars program funded by the Henry Luce Foundation (Grant #9601)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative Proteomic Analysis Reveals the Upregulation of Ketogenesis in Cardiomyocytes Differentiated from Induced Pluripotent Stem Cells.

Diverse metabolic pathways, such as the tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation, regulate the differentiation of induced pluripotent stem cells (iPSCs) to cells of specific lineages and organs. Here, the protein dynamics during cardiac differentiation of human iPSCs into cardiomyocytes (CMs) are characterized. The differentiation is induced by N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide, a Wnt signaling inhibitor, and confirmed by the mRNA and protein expression of cTnT and MLC2A in CMs. For comparative proteomics, cells from three stages, namely, hiPSCs, cardiac progenitor cells, and CMs, are prepared using the three-plex tandem mass tag labeling approach. In total, 3970 proteins in triplicate analysis are identified. As the result, the upregulation of proteins associated with branched chain amino acid degradation and ketogenesis by the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis are observed. The levels of 3-hydroxymethyl-3-methylglutaryl-CoA lyase, 3-hydroxymethyl-3-methylglutaryl-CoA synthase 2, and 3-hydroxybutyrate dehydrogenase 1, involved in ketone body metabolism, are determined using western blotting, and the level of acetoacetate, the final product of ketogenesis, is higher in CMs. Taken together, these observations indicate that proteins required for the production of diverse energy sources are naturally self-expressed during cardiomyogenic differentiation. Furthermore, acetoacetate concentration might act as a regulator of this differentiation.

Circadian regulation in human white adipose tissue revealed by transcriptome and metabolic network analysis

Studying circadian rhythms in most human tissues is hampered by difficulty in collecting serial samples. Here we reveal circadian rhythms in the transcriptome and metabolic pathways of human white adipose tissue. Subcutaneous adipose tissue was taken from seven healthy males under highly controlled ‘constant routine’ conditions. Five biopsies per participant were taken at six-hourly intervals for microarray analysis and in silico integrative metabolic modelling. We identified 837 transcripts exhibiting circadian expression profiles (2% of 41619 transcript targeting probes on the array), with clear separation of transcripts peaking in the morning (258 probes) and evening (579 probes). There was only partial overlap of our rhythmic transcripts with published animal adipose and human blood transcriptome data. Morning-peaking transcripts associated with regulation of gene expression, nitrogen compound metabolism, and nucleic acid biology; evening-peaking transcripts associated with organic acid metabolism, cofactor metabolism and redox activity. In silico pathway analysis further indicated circadian regulation of lipid and nucleic acid metabolism; it also predicted circadian variation in key metabolic pathways such as the citric acid cycle and branched chain amino acid degradation. In summary, in vivo circadian rhythms exist in multiple adipose metabolic pathways, including those involved in lipid metabolism, and core aspects of cellular biochemistry.

Characteristic Amino Acid and Energy Metabolism in AML Stem Cells As Revealed By Quantitative Multiplex Proteomics

Acute Myeloid Leukemia (AML) is a rapidly progressing hematologic malignancy characterized by the accumulation of clonal myeloid progenitor cells arrested in their ability to differentiate into mature blood cells. While classic chemotherapy regimens lead to remission in the majority of patients, relapse rates are very high. Relapse and also refractoriness are caused by the hierarchical organization of AML with a minor fraction of leukemic stem cells (LSC) at the apex generating leukemic progeny, which make up the majority of leukemic cells. LSC harbor self-renewal activity, relative quiescence, resistance to apoptosis, and increased drug efflux that likely render them less susceptible to conventional therapies aimed at the bulk proliferative disease. From a clinical perspective, the leukemic stem cell model implies that in order to eradicate the disease and achieve long-term remissions, treatment courses must eliminate the LSC population.To identify putative therapeutic target structures and/or pathways selectively aiming at LSC we performed high-resolution proteomic analyses of LSC and non-LSC populations of primary AML patient samples and healthy control subjects. To identify functional LSCs, we fractionated AML patient samples according to CD34 and CD38 surface expression by fluorescent-activated cell sorting and transplanted the resulting four populations per sample into immunocompromized NSG mice. Leukemic populations, which were able to initiate the AML in mice were labeled LSCs, while engraftment failures were considered non-LSCs. Six AML patients containing both LSC and non-LSC fractions were chosen for proteomic analysis. As healthy controls, CD34+CD38- hematopoietic stem and progenitor cells (HSPC) populations obtained from elderly patients without hematologic conditions undergoing hip replacement surgery were analyzed. We performed in-depth quantitative multiplex proteomic analysis employing tandem mass tag labeling and high-resolution mass spectrometry and identified more than 7,200 proteins. Our analysis revealed between 1,097 and 1,937 differentially expressed proteins when comparing LSC with non-LSC populations for each AML sample. Gene set enrichment analyses (GSEA) showed a significant enrichment for DNA replication, cell cycle, ribosome biogenesis, and protein translation in non-LSC populations, in agreement with a more quiescent state of LSC.Next to Branched Chain Amino Acid degradation, which we have recently shown to control the activity of alpha-ketoglutarate dependent dioxygenases such as TET2 and EGLN1 in AML stem cells (Raffel et al., (2017). Nature 551(7680), 384-388) other metabolic processes including lipid metabolism and oxidative phosphorylation were enriched in LSC. When compared to healthy HSPCs, especially oxidative phosphorylation, RNA processing and cell adhesion were over-represented in LSC. We will present proteins differentially expressed and pathways enriched in LSCs versus non-LSCs. These may help to understand the mechanism of LSC self-renewal and function and simultaneously represent putative novel targets to eliminate leukemic stem cells in clinical settings. DisclosuresNo relevant conflicts of interest to declare.

Label-Free Quantitative Proteomics in a Methylmalonyl-CoA Mutase-Silenced Neuroblastoma Cell Line.

Methylmalonic acidemias (MMAs) are inborn errors of metabolism due to the deficient activity of methylmalonyl-CoA mutase (MUT). MUT catalyzes the formation of succinyl-CoA from methylmalonyl-CoA, produced from propionyl-CoA catabolism and derived from odd chain fatty acids β-oxidation, cholesterol, and branched-chain amino acids degradation. Increased methylmalonyl-CoA levels allow for the presymptomatic diagnosis of the disease, even though no approved therapies exist. MMA patients show hyperammonemia, ketoacidosis, lethargy, respiratory distress, cognitive impairment, and hepatomegaly. The long-term consequences concern neurologic damage and terminal kidney failure, with little chance of survival. The cellular pathways affected by MUT deficiency were investigated using a quantitative proteomics approach on a cellular model of MUT knockdown. Currently, a consistent reduction of the MUT protein expression was obtained in the neuroblastoma cell line (SH-SY5Y) by using small-interfering RNA (siRNA) directed against an MUT transcript (MUT siRNA). The MUT absence did not affect the cell viability and apoptotic process in SH-SY5Y. In the present study, we evaluate and quantify the alterations in the protein expression profile as a consequence of MUT-silencing by a mass spectrometry-based label-free quantitative analysis, using two different quantitative strategies. Both quantitative methods allowed us to observe that the expression of the proteins involved in mitochondrial oxido-reductive homeostasis balance was affected by MUT deficiency. The alterated functional mitochondrial activity was observed in siRNA_MUT cells cultured with a propionate-supplemented medium. Finally, alterations in the levels of proteins involved in the metabolic pathways, like carbohydrate metabolism and lipid metabolism, were found.

Berberine alleviates insulin resistance by reducing peripheral branched-chain amino acids.

Increased circulating branched-chain amino acids (BCAAs) have been involved in the pathogenesis of obesity and insulin resistance (IR). However, evidence relating berberine (BBR), gut microbiota, BCAAs, and IR is limited. Here, we showed that BBR could effectively rectify steatohepatitis and glucose intolerance in high-fat diet (HFD)-fed mice. BBR reorganized gut microbiota populations under both the normal chow diet (NCD) and HFD. Particularly, BBR noticeably decreased the relative abundance of BCAA-producing bacteria, including order Clostridiales; families Streptococcaceae, Clostridiaceae, and Prevotellaceae; and genera Streptococcus and Prevotella. Compared with the HFD group, predictive metagenomics indicated a reduction in the proportion of gut microbiota genes involved in BCAA biosynthesis but the enrichment genes for BCAA degradation and transport by BBR treatment. Accordingly, the elevated serum BCAAs of HFD group were significantly decreased by BBR. Furthermore, the Western blotting results implied that BBR could promote the BCAA catabolism in the liver and epididymal white adipose tissues of HFD-fed mice by activation of the multienzyme branched-chain α-ketoacid dehydrogenase complex (BCKDC), whereas by inhibition of the phosphorylation state of BCKDHA (E1α subunit) and branched-chain α-ketoacid dehydrogenase kinase (BCKDK). The ex vivo assay further confirmed that BBR could increase BCAA catabolism in both AML12 hepatocytes and 3T3-L1 adipocytes. Finally, data from healthy subjects and diabetics confirmed that BBR could improve glycemic control and modulate circulating BCAAs. Together, our findings clarified BBR improving IR associated not only with gut microbiota alteration in BCAA biosynthesis but also with BCAA catabolism in liver and adipose tissues.

Molecular Atlas of Postnatal Mouse Heart Development.

BackgroundThe molecular mechanisms mediating postnatal loss of cardiac regeneration in mammals are not fully understood. We aimed to provide an integrated resource of mRNA, protein, and metabolite changes in the neonatal heart for identification of metabolism‐related mechanisms associated with cardiac regeneration.Methods and ResultsMouse ventricular tissue samples taken on postnatal day 1 (P01), P04, P09, and P23 were analyzed with RNA sequencing and global proteomics and metabolomics. Gene ontology analysis, KEGG pathway analysis, and fuzzy c‐means clustering were used to identify up‐ or downregulated biological processes and metabolic pathways on all 3 levels, and Ingenuity pathway analysis (Qiagen) was used to identify upstream regulators. Differential expression was observed for 8547 mRNAs and for 1199 of 2285 quantified proteins. Furthermore, 151 metabolites with significant changes were identified. Differentially regulated metabolic pathways include branched chain amino acid degradation (upregulated at P23), fatty acid metabolism (upregulated at P04 and P09; downregulated at P23) as well as the HMGCS (HMG‐CoA [hydroxymethylglutaryl‐coenzyme A] synthase)–mediated mevalonate pathway and ketogenesis (transiently activated). Pharmacological inhibition of HMGCS in primary neonatal cardiomyocytes reduced the percentage of BrdU‐positive cardiomyocytes, providing evidence that the mevalonate and ketogenesis routes may participate in regulating the cardiomyocyte cell cycle.ConclusionsThis study is the first systems‐level resource combining data from genomewide transcriptomics with global quantitative proteomics and untargeted metabolomics analyses in the mouse heart throughout the early postnatal period. These integrated data of molecular changes associated with the loss of cardiac regeneration may open up new possibilities for the development of regenerative therapies.

Glucose promotes cell growth by suppressing branched-chain amino acid degradation.

Glucose and branched-chain amino acids (BCAAs) are essential nutrients and key determinants of cell growth and stress responses. High BCAA level inhibits glucose metabolism but reciprocal regulation of BCAA metabolism by glucose has not been demonstrated. Here we show that glucose suppresses BCAA catabolism in cardiomyocytes to promote hypertrophic response. High glucose inhibits CREB stimulated KLF15 transcription resulting in downregulation of enzymes in the BCAA catabolism pathway. Accumulation of BCAA through the glucose-KLF15-BCAA degradation axis is required for the activation of mTOR signaling during the hypertrophic growth of cardiomyocytes. Restoration of KLF15 prevents cardiac hypertrophy in response to pressure overload in wildtype mice but not in mutant mice deficient of BCAA degradation gene. Thus, regulation of KLF15 transcription by glucose is critical for the glucose-BCAA circuit which controls a cascade of obligatory metabolic responses previously unrecognized for cell growth.

Genetically-Regulated Mechanisms of Insulin Resistance in Adipose Tissue

Insulin resistance (IR) is a harbinger of type 2 diabetes (T2D) and partly determined by genetic factors. However, genetically-regulated mechanisms of IR remain poorly understood. Using gene expression, genotype, and insulin sensitivity (SI and Matsuda index from FSIVGT and OGTT) data from an African American cohort (AAGMEx, N=256), we performed transcript-wide association and expression quantitative trait loci (eQTL) analyses to identify IR-associated cis-regulated transcripts (cis-eGenes) in adipose tissue. Data from a European ancestry cohort (METSIM, N=770) was used for testing replication of Matsuda index-associated cis-eGenes. In vitro genetic silencing studies were performed in a human adipocyte model to define the molecular mechanism of selected genes. Expression level of 2724 transcripts in adipose were negatively (1338) or positively (1386) associated (p&amp;lt;0.001) with Matsuda index in both cohorts. In the AAGMEx cohort, we identified cis-eQTLs (FDR&amp;lt;1%) for 786 Matsuda index-associated transcripts. Cis-eQTLs for 510 Matsuda index-associated genes were identified in both the AAGMEx and METSIM cohorts. These genes were enriched for mitochondrial dysfunction, phagosome maturation, and branched-chain amino acid degradation pathways. The enoyl-CoA hydratase domain-containing 3 (ECHDC3) was the top ranked Matsuda index-associated cis-eGene. ECHDC3 expression level was positively correlated with Matsuda index (r=0.47, p=5.3x10-15) and rs200943982 was the top ECHDC3 cis-eSNP (p=1.94x10-9) in AAGMEx. The shRNA-mediated silencing of ECHDC3 in SGBS adipocytes reduced insulin-stimulated Akt phosphorylation. RNA-seq analysis identified 691 differentially-expressed genes in ECHDC3 knockdown cells, which were enriched in γ-linolenate and cholesterol biosynthesis, LXR/RXR activation pathways, and known IR genes. In summary, variation-based genomic analysis with concurrent in vitro genetic silencing studies identified novel genetic regulatory mechanisms of IR. Disclosure N.K. Sharma: None. C.C. Key: None. M.E. Comeau: None. C.D. Langefeld: None. J.S. Parks: None. S.K. Das: None.

Proteomics of the Rat Myocardium during Development of Type 2 Diabetes Mellitus Reveals Progressive Alterations in Major Metabolic Pathways.

Congestive heart failure and poor clinical outcome after myocardial infarction are known complications in patients with type-2 diabetes mellitus (T2DM). Protein alterations may be involved in the mechanisms underlying these disarrays in the diabetic heart. Here we map proteins involved in intracellular metabolic pathways in the Zucker diabetic fatty rat heart as T2DM develops using MS based proteomics. The prediabetic state only induced minor pathway changes, whereas onset and late T2DM caused pronounced perturbations. Two actin-associated proteins, ARPC2 and TPM3, were up-regulated at the prediabetic state indicating increased actin dynamics. All differentially regulated proteins involved in fatty acid metabolism, both peroxisomal and mitochondrial, were up-regulated at late T2DM, whereas enzymes of branched chain amino acid degradation were all down-regulated. At both onset and late T2DM, two members of the serine protease inhibitor superfamily, SERPINA3K and SERPINA3L, were down-regulated. Furthermore, we found alterations in proteins involved in clearance of advanced glycation end-products and lipotoxicity, DCXR and CBR1, at both onset and late T2DM. These proteins deserve elucidation with regard to their role in T2DM pathogenesis and their respective role in the deterioration of the diabetic heart. Data are available via ProteomeXchange with identifiers PXD009538, PXD009554, and PXD009555.