Organic aciduria and butyryl CoA dehydrogenase deficiency in BALB/cByJ mice

A metabolic screening program of inbred strains of mice has detected a marked organic aciduria in the BALB/cByJ strain. Gas chromatographic and mass spectrometric analysis identified large quantities ofn-butyrylglycine plus lesser quantities of ethylmalonic acid. Crosses with the nonexcreting C57BL/6J strain indicate that this condition is inherited as an autosomal recessive trait. Independently from this screening a variant with no detectable enzyme activity of butyryl CoA dehydrogenase (BCD) in liver and kidney of the BALB/cByJ strain but not other BALB/c sublines was discovered. Data from a three-point cross indicated that the null variant maps to the structural locus for the enzyme,Bcd-1, on chromosome 5. The findings indicate that a mutation at or nearBcd-1 in the BALB/cByJ strain resulted in a biochemical abnormality manifest as the BCD deficiency. It is concluded that accumulation of butyryl CoA due to a block in the oxidation of short-chain fatty acids results in an overproduction of organic metabolites leading to the observed organic aciduria. The fact that other BALB/c substrains do not exhibit this abnormality further suggests that this disorder reflects subline divergence within the BALB/c family.This work was supported by NIH Grants RR02512 and GM32592 to the University of Pennsylvania and HD23168, NS17752, and HD08536 to the Children's Hospital of Philadelphia, National Science Foundation Grant BSR 84-18828 to The Jackson Laboratory, and a Postdoctoral Fellowship from the Juvenile Diabetes Foundation International to Dr. Prochazka.     

Identification and characterization of retinoid-active short-chain dehydrogenases/reductases in Drosophila melanogaster

Background

In chordates, retinoid metabolism is an important target of short-chain dehydrogenases/reductases (SDRs). It is not known whether SDRs play a role in retinoid metabolism of protostomes, such as Drosophila melanogaster.

Methods

Drosophila genome was searched for genes encoding proteins with ∼ 50% identity to human retinol dehydrogenase 12 (RDH12). The corresponding proteins were expressed in Sf9 cells and biochemically characterized. Their phylogenetic relationships were analyzed using PHYLIP software.

Results

A total of six Drosophila SDR genes were identified. Five of these genes are clustered on chromosome 2 and one is located on chromosome X. The deduced proteins are 300 to 406 amino acids long and are associated with microsomal membranes. They recognize all-trans-retinaldehyde and all-trans-3-hydroxyretinaldehyde as substrates and prefer NADPH as a cofactor. Phylogenetically, Drosophila SDRs belong to the same branch of the SDR superfamily as human RDH12, indicating a common ancestry early in bilaterian evolution, before a protostome–deuterostome split.

Conclusions

Similarities in the substrate and cofactor specificities of Drosophila versus human SDRs suggest conservation of their function in retinoid metabolism throughout protostome and deuterostome phyla.

General significance

The discovery of Drosophila retinaldehyde reductases sheds new light on the conversion of β-carotene and zeaxantine to visual pigment and provides a better understanding of the evolutionary roots of retinoid-active SDRs.     

The functional divergence of short-chain dehydrogenases involved in tropinone reduction

Tropane alkaloids typically occur in the Solanaceae and are also found in Cochlearia officinalis, a member of the Brassicaceae. Tropinone reductases are key enzymes of tropane alkaloid metabolism. Two different tropinone reductases form one stereoisomeric product each, either tropine for esterified alkaloids or pseudotropine that is converted to calystegines. A cDNA sequence with similarity to known tropinone reductases (TR) was cloned from C. officinalis. The protein was expressed in Escherichia coli, and found to catalyze the reduction of tropinone. The enzyme is a member of the short-chain dehydrogenase enzyme family and shows broad substrate specificity. Several synthetic ketones were accepted as substrates, with higher affinity and faster enzymatic turnover than observed for tropinone. C. officinalis TR produced both the isomeric alcohols tropine and pseudotropine from tropinone using NADPH + H(+) as co-substrate. Tropinone reductases of the Solanaceae, in contrast, are strictly stereospecific and form one tropane alcohol only. The Arabidopsis thaliana homologue of C. officinalis TR showed high sequence similarity, but did not reduce tropinone. A tyrosine residue was identified in the active site of C. officinalis TR that appeared responsible for binding and orientation of tropinone. Mutagenesis of the tyrosine residue yielded an active reductase, but with complete loss of TR activity. Thus C. officinalis TR presents an example of an enzyme with relaxed substrate specificity, like short-chain dehydrogenases, that provides favorable preconditions for the evolution of novel functions in biosynthetic sequences.     

Sowing the seeds for a career in medicine: reflections and projections

Dr. George Lister delivered the following presentation as the Lee E. Farr Lecturer on May 8, 2011, which served as the culmination of the annual Student Research Day at Yale School of Medicine. He is the Chair of Pediatrics at the University of Texas Southwestern Medical School and Pediatrician-in-Chief at Children's Medical Center of Dallas. In his lecture to the medical students, who had just completed their research theses, Dr. Lister discusses his own work on sudden infant death syndrome (SIDS), demonstrating the complexity of clinical research and proving insight into the traits required of physician scientists. Committed to medical education and recognized by several awards for his mentorship, he ends the talk by imparting valuable advice on future physicians.     

The role of short-chain fatty acids in the interplay between diet,gut microbiota,and host energy metabolism

Short-chain fatty acids (SCFAs), the end products of fermentation of dietary fibers by the anaerobic intestinal microbiota, have been shown to exert multiple beneficial effects on mammalian energy metabolism. The mechanisms underlying these effects are the subject of intensive research and encompass the complex interplay between diet, gut microbiota, and host energy metabolism. This review summarizes the role of SCFAs in host energy metabolism, starting from the production by the gut microbiota to the uptake by the host and ending with the effects on host metabolism. There are interesting leads on the underlying molecular mechanisms, but there are also many apparently contradictory results. A coherent understanding of the multilevel network in which SCFAs exert their effects is hampered by the lack of quantitative data on actual fluxes of SCFAs and metabolic processes regulated by SCFAs. In this review we address questions that, when answered, will bring us a great step forward in elucidating the role of SCFAs in mammalian energy metabolism.     

Riboflavin deficiency in cultured rat hepatoma cells: A model for studying the hepatic effects of riboflavin deficiency

Summary The acyl-CoA dehydrogenases are a family of mitochondrial flavoenzymes required for fatty acid beta-oxidation and branched-chain amino acid degradation. The hepatic activity of these enzymes, particularly the short-chain acyl-coenzyme A (CoA) dehydrogenase, is markedly decreased in riboflavin deficient rats. We now report that the in vivo effects of riboflavin deficiency on the beta-oxidation enzymes of this group are reproduced in FAO rat hepatoma cells cultured in riboflavin-deficient medium. Although it has been long known that hepatic short-chain acyl-CoA dehydrogenase activity is the most severely affected of the straight-chain specific enzymes in riboflavin deficiency, the mechanism by which its activity is decreased has not been reported. We have used this new cell culture system to characterize further this mechanism. Whole cell extracts from riboflavin-deficient and control cells were subjected to analysis by denaturing polyacrylamide gel electrophoresis. The contents of the gels were then electroblotted onto nitrocellulose filters and probed with short-chain acyl-CoA dehydrogenase-specific antiserum. The relative abundance of enzyme antigen was estimated autoradiographically. Our findings indicate that short-chain acyl-CoA dehydrogenase activity changes in parallel with its antigen, suggesting that riboflavin deprivation does not affect the activity of individual enzyme molecules. Further, no evidence of extramitochondrial enzyme precursor was found on the blots, making unlikely a significant block in the mitochondrial uptake process. These findings suggest that changes in short-chain acyl-CoA dehydrogenase activity in riboflavin deficiency result from either increased synthesis or decreased degradation of the enzyme. This work was supported by grants from the VA Medical Research Service, the Diabetes Association of Greater Cleveland, and the National Institutes of Health (HD25299), Bethesda, MD. Portions of the work presented here were presented at the 71st meeting of the Endocrine Society, Seattle, WA.     

Used cot mattresses as potential reservoirs of bacterial infection: nutrient availability within polyurethane foam

Aim: To evaluate possible source of nutrients for bacterial growth within polyurethane (PU) foam of used cot mattresses as determinants of bacterial population density. Methods and Results: Used infant mattresses (n = 30) were analysed for bacteria capable of degrading colloidal PU and for aqueous soluble chemical components (aromatic amines, ammonium ions, phosphates and protein). Mattress type (waterproof cover vs exposed PU foam at the infant‐head region), mattress age and previous use by another child were evaluated as factors that could influence the measured parameters. The levels of protein extracted from PU foam were (i) significantly (P = 0·0019) higher for mattresses lacking a waterproof cover at the infant‐head region and (ii) positively correlated with both culturable bacterial population densities of the PU foams and extent of growth of Staphylococcus aureus on aqueous leachates. No statistically significant (P > 0·05) associations between other measured parameters and mattress type/use factors were identified. Conclusions: Infant use of cot mattresses with exposed PU foam leads to accumulation of proteins within the PU, which can promote bacterial growth. Significance and Impact of the Study: The study provides a mechanistic explanation for increased levels of bacteria associated with exposed PU of cot mattresses. In the context of the common bacterial toxins hypothesis for the sudden infant death syndrome (SIDS), this could explain the lowered risk of SIDS associated with use of a waterproof cover above the mattress.     

Regulation of Acetyl CoA Carboxylase and Carnitine Palmitoyl Transferase‐1 in Rat Adipocytes

Objective: Acetyl CoA carboxylase (ACC) is a key enzyme in energy balance. It controls the synthesis of malonyl‐CoA, an allosteric inhibitor of carnitine palmitoyltransferase‐1 (CPT‐I). CPT‐I is the gatekeeper of free fatty acid (FFA) oxidation. To test the hypothesis that both enzymes play critical roles in regulation of FFA partitioning in adipocytes, we compared enzyme mRNA expression and specific activity from fed, fasted, and diabetic rats. Research Methods and Procedures: Direct effects of nutritional state, insulin, and FFAs on CPT‐I and ACC mRNA expression were assessed in adipocytes, liver, and cultured adipose tissue explants. We also determined FFA partitioning in adipocytes from donors exposed to different nutritional conditions. Results: CPT‐I mRNA and activity decreased in adipocytes but increased in liver in response to fasting. ACC mRNA and activity decreased in both adipocytes and liver during fasting. These changes were not caused directly by fasting‐associated changes in plasma insulin and FFA concentrations because insulin suppressed CPT‐I mRNA and did not affect ACC mRNA in vitro, whereas exogenous oleate had no effect on either. Despite the decrease in adipocyte CPT‐I mRNA and specific activity, CO₂ production from endogenous FFAs increased, suggesting increased FFA transport through CPT‐I for β‐oxidation. Discussion: Stimulation of FFA transport through CPT‐I occurs in both tissues, but CPT‐I mRNA and specific activity correlate with FFA transport in liver and not in adipocytes. We conclude that the mechanism responsible for increasing FFA oxidation in adipose tissue during fasting involves mainly allosteric regulation, whereas altered gene expression may play a central role in the liver.     

Research on and in medical education

Dr. George Lister of the University of Texas Southwestern Medical Center delivered the Lee E. Farr Lecture on Student Research Day on May 9, 2011. This day focused on the dissertation work of Yale School of Medicine MD students, whose research opportunities for prospective physicians were recently examined and critiqued by Yale's Committee to Promote Student Interest in Careers as Physician Scientists. Lister's talk served to highlight the importance of communication between the laboratory and the clinic in optimizing diagnostics and treatments, effectively affirming the validity of the Committee's objectives.     

短链脂肪酸调控骨代谢的作用及机制的研究进展

肠道菌群紊乱可导致宿主病理性骨质流失,其通过产生的代谢物从肠道扩散到体循环对骨代谢发挥重要的调控作用。短链脂肪酸（Short Chain Fatty Acids,SCFAs）是肠道细菌产生的代谢物家族中最受关注的代谢产物,近年来研究表明,SCFAs在骨代谢相关疾病的发生发展中具有重要调节作用。本文就其在骨骼系统中的作用、调节骨组织中细胞的机制及作为靶点防治骨代谢疾病骨质疏松的研究进行综述,并为此新兴且具有前景的研究领域在未来的基础研究和转化研究提供展望。     

Simple inhibitors of histone deacetylase activity that combine features of short-chain fatty acid and hydroxamic acid inhibitors

Butyric acid and trichostatin A (TSA) are anti-cancer compounds that cause the upregulation of genes involved in differentiation and cell cycle regulation by inhibiting histone deacetylase (HDAC) activity. In this study we have synthesized and evaluated compounds that combine the bioavailability of short-chain fatty acids, like butyric acid, with the bidentate binding ability of TSA. A series of analogs were made to examine the effects of chain length, simple aromatic cap groups, and substituted hydroxamates on the compounds' ability to inhibit rat-liver HDAC using a fluorometric assay. In keeping with previous structure-activity relationships, the most effective inhibitors consisted of longer chains and hydroxamic acid groups. It was found that 5-phenylvaleric hydroxamic acid and 4-benzoylbutyric hydroxamic acid were the most potent inhibitors with IC₅₀'s of 5 μM and 133 μM respectively.     

Fermentation of prebiotics by human colonic microbiota in vitro and short-chain fatty acids production: a critical review

Prebiotics are known for their health benefits to man, including reducing cardiovascular disease and improving gut health. This review takes a critical assessment of the impact of dietary fibres and prebiotics on the gastrointestinal microbiota in vitro. The roles of colonic organisms, slow fermentation of prebiotics, production of high butyric and propionic acids and positive modulation of the host health were taken into cognizance. Also, the short-chain fatty acids (SCFAs) molecular signalling mechanisms associated with their prebiotic substrate structural conformations and the phenotypic responses related to the gut microbes composition were discussed. Furthermore, common dietary fibres such as resistant starch, pectin, hemicelluloses, β-glucan and fructan in context of their prebiotic potentials for human health were also explained. Finally, the in vitro human colonic fermentation depends on prebiotic type and its physicochemical characteristics, which will then affect the rate of fermentation, selectivity of micro-organisms to multiply, and SCFAs concentrations and compositions.     

Effect of Randomly Interesterified Triacylglycerol Containing Medium- and Long-Chain Fatty Acids on Hepatic Fatty Acid Oxidation after a Single Administration to Rats

MLCTs, which are randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule, showed significantly higher acyl-CoA dehydrogenase activity when measured by using butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA as substrates than long-chain triacylglycerol one hour after a single administration to rats. These results suggest that not only medium-chain fatty acid oxidation, but also long-chain fatty acid oxidation were increased in the liver of rats administered with MLCT.     

生理盐水实验性干预对肉鸡盲肠微生物区系和短链脂肪酸含量的影响

目的研究生理盐水实验性干预对肉鸡盲肠微生物区系和短链脂肪酸含量的影响,以期为早期菌群干预实验的研究提供一定的理论依据。方法选取80只初出壳的小鸡,随机分为2组,分别为对照组(C组)和生理盐水组(S组)。出壳后前2 d,连续每天给S组小鸡灌服0.5 mL灭菌的生理盐水,C组不做处理。第3天,第7天于两组分别随机挑选8只鸡,测定其体重后屠宰取其盲肠内容物,采用Illumina Miseq高通量测序技术对盲肠内容物菌群结构进行测定,并用气相色谱法测定盲肠内容物中短链脂肪酸的含量。结果生理盐水实验性干预对肉鸡早期阶段的平均日增重无显著影响(P>0.05)。在门的水平上,两组肉鸡盲肠菌群占比基本相似,厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)为早期肉鸡肠道内的优势菌门。在属的水平,3日龄时S组肉鸡盲肠中拟杆菌属(Bacteroides)和蓝细菌属(Cyanobacteria_norank)的相对丰度较C组分别提高了160%和143%(P<0.05);7日龄时,两组间盲肠菌属的相对丰度无显著差异。此外,生理盐水实验性干预可极显著降低3日龄肉鸡盲肠内容物中乙酸、丁酸和异戊酸的含量(P<0.01),但7日龄时两组肉鸡盲肠内容物中短链脂肪酸的含量无显著差异(P>0.05)。结论生理盐水实验性干预会对3日龄肉鸡盲肠菌群结构及短链脂肪酸含量产生一定的影响,但这种影响不具有持续性,随着日龄的增加会逐渐消失。     

Associations of polymorphisms in the promoter I of bovine acetyl‐CoA carboxylase‐α gene with beef fatty acid composition

The objectives of this study were to identify single nucleotide polymorphisms (SNPs) in the promoter I (PI) region of the bovine acetyl‐CoA carboxylase‐α (ACACA) gene and to evaluate the extent to which they were associated with lipid‐related traits. Eight novel SNPs were identified, which were AJ276223:g.2064T>A (SNP1), g.2155C>T (SNP2), g.2203G>T (SNP3), g.2268T>C (SNP4), g.2274G>A (SNP5), g.2340A>G (SNP6), g.2350T>C (SNP7) and g.2370A>G (SNP8). Complete linkage disequilibrium was observed among SNP1, 2, 4, 5, 6 and 8. Phenotypic data were collected from 573 cross‐bred steers with six sire breeds, including Hereford, Angus, Brangus, Beefmaster, Bonsmara and Romosinuano. The genotypes of SNP1/2/4/5/6/8 were significantly associated with adjusted backfat thickness. The genotypes of SNP3 were significantly associated with triacylglycerol (TAG) content and fatty acid composition of longissimus dorsi muscle (LM) in Brangus‐, Romosinuano‐ and Bonsmara‐sired cattle. Cattle with g.2203GG genotype had greater concentrations of TAG, total lipid, total saturated fatty acid and total monounsaturated fatty acid than did cattle with g.2203GT genotype. The genotypes of SNP7 were significantly associated with fatty acid composition of LM. Cattle with genotype g.2350TC had greater amounts of several fatty acids in LM than did cattle with genotype g.2350CC. Our results suggested that the SNPs in the PI region of ACACA gene are associated with variations in the fatty acid contents in LM.     

Metabolic engineering of Escherichia coli for production of fatty acid short-chain esters through combination of the fatty acid and 2-keto acid pathways

Fatty acid short-chain esters (FASEs) are biodiesels that are renewable, nontoxic, and biodegradable biofuels. A novel approach for the biosynthesis of FASEs has been developed using metabolically-engineered E. coli through combination of the fatty acid and 2-keto acid pathways. Several genetic engineering strategies were also developed to increase fatty acyl-CoA availability to improve FASEs production. Fed-batch cultivation of the engineered E. coli resulted in a titer of 1008 mg/L FASEs. Since the fatty acid and 2-keto acid pathways are native microbial synthesis pathways, this strategy can be implemented in a variety of microorganisms to produce various FASEs from cheap and readily-available, renewable, raw materials such as sugars and cellulose in the future.     

Cortisol levels and control of inflammatory responses to toxic shock syndrome toxin-1 (TSST-1): the prevalence of night-time deaths in sudden infant death syndrome (SIDS)

There is evidence that inflammatory responses have been induced in the tissues and body fluids of many SIDS infants. We suggested that some of these deaths are due to uncontrolled inflammatory responses to infectious agents and possibly cigarette smoke. The majority of SIDS deaths occur during the 2-4 month age range when infants have decreasing levels of maternal antibodies to infectious agents. Most deaths occur during the early hours of the morning. Adults are more susceptible to inflammatory responses at night due to lower levels of cortisol associated with circadian rhythm patterns. Infants develop these patterns between the ages of 7 weeks and 4 months, at which time their night-time cortisol levels drop dramatically. The objective of this study was to use an in vitro model system to assess the effects of different cortisol levels on proinflammatory cytokine production in response to the staphylococcal toxic shock syndrome toxin-1 (TSST-1) which has been identified in a significant number of SIDS infants. Levels of cortisol present in infants at night and during the day before and after the development of the circadian rhythm pattern were examined. Human buffy coats (n = 9) were stimulated with TSST-1 and responses assessed over 72 hours by a bioassay for tumour necrosis factor-alpha (TNF-alpha) and an enzyme linked immunosorbent assay (ELISA) for interleukin-6 (IL-6). Cortisol levels present in an infant at night after development of circadian rhythm (< or = 5 microg dl(-1)), did not significantly increase or decrease production of either TNF-alpha or IL-6. Concentrations of cortisol greater than 5 microg dl(-1) usually found in infants during the day or at night prior to the physiological change significantly decreased production of TNF-alpha at 12 hours and of IL-6 at 12 and 16 hours. Only cortisol levels greater than 5 microg dl(-1) significantly decreased production of the pro-inflammatory cytokines by human buffy coats stimulated with TSST-1. If the switch to the circadian rhythm pattern occurs in an infant when maternal antibodies are still present or after they have developed their own active immunity, the infant could neutralise common viruses, toxins or bacteria: however, if this switch occurs in an infant when antibody levels are low, this could be a window of vulnerability during which infants are at an increased risk of death if uncontrolled inflammatory responses are induced by infectious agents or their products.     

High vulnerability of the heart and liver to 3‐hydroxypalmitic acid–induced disruption of mitochondrial functions in intact cell systems

Patients affected by long‐chain 3‐hydroxyacyl‐CoA dehydrogenase (LCHAD) deficiency predominantly present severe liver and cardiac dysfunction, as well as neurological symptoms during metabolic crises, whose pathogenesis is still poorly known. In this study, we demonstrate for the first time that pathological concentrations of 3‐hydroxypalmitic acid (3HPA), the long‐chain hydroxyl fatty acid (LCHFA) that most accumulates in LCHAD deficiency, significantly decreased adenosine triphosphate‐linked and uncoupled mitochondrial respiration in intact cell systems consisting of heart fibers, cardiomyocytes, and hepatocytes, but less intense in diced forebrain. 3HPA also significantly reduced mitochondrial Ca²⁺ retention capacity and membrane potential in Ca²⁺‐loaded mitochondria more markedly in the heart and the liver, with mild or no effects in the brain, supporting a higher susceptibility of the heart and the liver to the toxic effects of this fatty acid. It is postulated that disruption of mitochondrial energy and Ca²⁺ homeostasis caused by the accumulation of LCHFA may contribute toward the severe cardiac and hepatic clinical manifestations observed in the affected patients.     

Effects of extrusion of corn on growth performance,nutrient digestibility and short-chain fatty acid profiles in the hindgut of weaned piglets

Abstract

The experiment was conducted to investigate the effects of different inclusion levels of extruded corn on growth performance, nutrient digestibility and short-chain fatty acids profiles in the hindgut of weaned piglets. Ninety weaning piglets (28 d of age; 8.21 ± 0.69 kg BW) were allotted to one of five dietary treatments only substituted for unprocessed corn at varying levels (0, 1/4, 2/4, 3/4, 4/4) with extruded corn in a 28-d experiment. On day 28, 30 piglets were killed to measure concentrations and molar ratios of short-chain fatty acids in the hindgut. From day 0 – 14, digestibility of DM, N and GE was increased linearly and diarrhea frequency was decreased due to the increasing inclusion levels of extruded corn. From day 14 – 28, digestibility of GE and N was enhanced linearly due to the increasing proportion of extruded corn. On day 28, as proportion of extruded corn increased, the level of butyrate in the caecum and colon was increased linearly and the relative proportion of propionate was reduced. Results indicated that increasing proportions of extruded corn had no effect on growth performance, but significantly improved digestibility of N and energy at day 10 of trial and may be advantageous to the intestinal health of piglets.     

Oxidative and reductive acetyl CoA/carbon monoxide dehydrogenase pathway in Desulfobacterium autotrophicum

It has been proposed that in some anaerobic facultatively autotrophic bacteria the acetyl CoA/CO dehydrogenase pathway is operating both in the reductive and in the oxidative direction, depending on the growth conditions. One of these anaerobes, the Gram-negative sulfate-reducing cubacterium Desulfobacterium autotrophicum, was examined for enzymes of the proposed pathway. All the required enzyme activities were present in sufficient amounts both in autotrophically and in heterotrophically grown cells, provided that the cellular tetrahydropterin rather than tetrahydrofolate was used as cosubstrate in some of the enzyme assays. The question arises whether two sets of enzymes are operating in the reductive and oxidative direction, respectively. The key enzyme of this pathway, CO dehydrogenase, which was reasonably oxygen stable, was analysed by native polyacrylamide gel electrophoresis and anaerobic activity staining. Extracts from heterotrophically grown cells exhibited five enzyme activity bands. Extracts from autotrophically grown cells showed the same pattern but an additional activity band appeared.