Evidence for functional and regulatory cross-talk between the tricarboxylic acid cycle 2-oxoglutarate dehydrogenase complex and 2-oxoadipate dehydrogenase on the l-lysine,l-hydroxylysine and l-tryptophan degradation pathways from studies in vitro

Herein are reported findings in vitro suggesting both functional and regulatory cross-talk between the human 2-oxoglutarate dehydrogenase complex (hOGDHc), a key regulatory enzyme within the tricarboxylic acid cycle (TCA cycle), and a novel 2-oxoadipate dehydrogenase complex (hOADHc) from the final degradation pathway of l-lysine, l-hydroxylysine and l-tryptophan. The following could be concluded from our studies by using hOGDHc and hOADHc assembled from their individually expressed components in vitro: (i) Different substrate preferences (k_cat/K_m) were displayed by the two complexes even though they share the same dihydrolipoyl succinyltransferase (hE2o) and dihydrolipoyl dehydrogenase (hE3) components; (ii) Different binding modes were in evidence for the binary hE1o-hE2o and hE1a-hE2o subcomplexes according to fluorescence titrations using site-specifically labeled hE2o-derived proteins; (iii) Similarly to hE1o, the hE1a also forms the ThDP-enamine radical from 2-oxoadipate (electron paramagnetic resonance detection) in the oxidative half reaction; (iv) Both complexes produced superoxide/H₂O₂ from O₂ in the reductive half reaction suggesting that hE1o, and hE1a (within their complexes) could both be sources of reactive oxygen species generation in mitochondria from 2-oxoglutarate and 2-oxoadipate, respectively; (v) Based on our findings, we speculate that hE2o can serve as a trans-glutarylase, in addition to being a trans-succinylase, a role suggested by others; (vi) The glutaryl-CoA produced by hOADHc inhibits hE1o, as does succinyl-CoA, suggesting a regulatory cross-talk between the two complexes on the different metabolic pathways.     

Interactions between model membranes and lignin-related compounds studied by immobilized liposome chromatography

In order to elucidate the modes of interaction between lignin precursors and membranes, we have studied the influence of temperature, lipid composition and buffer composition on the partitioning of monolignol and dilignol model substances into phospholipid bilayers. The partitioning was determined by immobilized liposome chromatography, which is an established method for studies of pharmaceutical drugs but a new approach in studies of lignin synthesis. The temperature dependence of the retention and the effect of a high ammonium sulfate concentration in the mobile phase demonstrated that the interaction involved both hydrophobic effects and polar interactions. There was also a good correlation between the partitioning and the estimated hydrophobicity, in terms of octanol/water partitioning. The partitioning behavior of the model substances suggests that passive diffusion over the cell membrane is a possible transport route for lignin precursors. This conclusion is strengthened by comparison of the present results with the partitioning of pharmaceutical drugs that are known to pass cell membranes by diffusion.     

Genome-scale analysis of the metabolic networks of oleaginous Zygomycete fungi

Microbial lipids are becoming an attractive option for the industrial production of foods and oleochemicals. To investigate the lipid physiology of the oleaginous microorganisms, at the system level, genome-scale metabolic networks of Mortierella alpina and Mucor circinelloides were constructed using bioinformatics and systems biology. As scaffolds for integrated data analysis focusing on lipid production, consensus metabolic routes governing fatty acid synthesis, and lipid storage and mobilisation were identified by comparative analysis of developed metabolic networks. Unique metabolic features were identified in individual fungi, particularly in NADPH metabolism and sterol biosynthesis, which might be related to differences in fungal lipid phenotypes. The frameworks detailing the metabolic relationship between M. alpina and M. circinelloides generated in this study is useful for further elucidation of the microbial oleaginicity, which might lead to the production improvement of microbial oils as alternative feedstocks for oleochemical industry.     

A case-control study between the gene polymorphisms of polyunsaturated fatty acids metabolic rate-limiting enzymes and coronary artery disease in a Chinese Han population

To investigate the association between the polymorphisms of fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2) and elongation of very long chain fatty acids like 2 (ELOVL2) gene and coronary artery disease (CAD) in a Chinese Han population. Three single nucleotide polymorphisms (SNPs) from these genes were genotyped using PCR-based restriction fragment length polymorphism analysis in 199 CAD cases and 192 controls of Han Chinese origin. rs174556 in the FADS1 gene showed allelic (P=0.002) and genotypic (P=0.030) association with the disease, while there was no disease association for the other two SNPs. The frequency of rs174556 minor allele (T) was significantly higher in the case group than the control group. The trans phase gene–gene interaction analysis showed that the combined genotype of rs174556 (T/T) and rs3756963 (T/T) was weakly associated with the disease (P=0.043). rs174556 in the FADS1 gene is very likely to be associated with CAD in the Chinese Han population.     

Systems biology of the metabolic network regulated by the Akt pathway

Cancer has been proposed as an example of systems biology disease or network disease. Accordingly, tumor cells differ from their normal counterparts more in terms of intracellular network dynamics than single markers. Here we shall focus on a recently recognized hallmark of cancer, the deregulation of cellular energetics. The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been confirmed as an essential step toward cell transformation. We will consider how the effects of Akt activation are connected with cell metabolism; more precisely, we will review existing metabolic models and discuss the current knowledge available to construct a kinetic model of the most relevant metabolic processes regulated by the PI3K/Akt pathway. The model will enable a systems biology approach to predict the metabolic targets that may inhibit cell growth under hyper activation of Akt.     

Hypothalamic lipotoxicity and the metabolic syndrome

Ectopic accumulation of lipids in peripheral tissues, such as pancreatic β cells, liver, heart and skeletal muscle, leads to lipotoxicity, a process that contributes substantially to the pathophysiology of insulin resistance, type 2 diabetes, steatotic liver disease and heart failure. Current evidence has demonstrated that hypothalamic sensing of circulating lipids and modulation of hypothalamic endogenous fatty acid and lipid metabolism are two bona fide mechanisms modulating energy homeostasis at the whole body level. Key enzymes, such as AMP-activated protein kinase (AMPK) and fatty acid synthase (FAS), as well as intermediate metabolites, such as malonyl-CoA and long-chain fatty acids-CoA (LCFAs-CoA), play a major role in this neuronal network, integrating peripheral signals with classical neuropeptide-based mechanisms. However, one key question to be addressed is whether impairment of lipid metabolism and accumulation of specific lipid species in the hypothalamus, leading to lipotoxicity, have deleterious effects on hypothalamic neurons. In this review, we summarize what is known about hypothalamic lipid metabolism with focus on the events associated to lipotoxicity, such as endoplasmic reticulum (ER) stress in the hypothalamus. A better understanding of these molecular mechanisms will help to identify new drug targets for the treatment of obesity and metabolic syndrome.     

Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N¹-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced ³H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the “reprogramming” of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.     

Marked change in the balance between CYP27A1 and CYP46A1 mediated elimination of cholesterol during differentiation of human neuronal cells

Cholesterol metabolism in the brain is distinct from that in other tissues due to the fact that cholesterol itself is unable to pass across the blood-brain barrier. Elimination of brain cholesterol is mainly dependent on a neuronal-specific cytochrome P450, CYP46A1, catalyzing the conversion of cholesterol into 24(S)-hydroxycholesterol (24OHC), which is able to pass the blood-brain barrier. A suitable model for studying this elimination from human neuronal cells has not been described previously. It is shown here that differentiated Ntera2/clone D1 (NT2) cells express the key genes involved in brain cholesterol homeostasis including CYP46A1, and that the expression profiles of the genes observed during neuronal differentiation are those expected to occur in vivo. Thus there was a decrease in the mRNA levels corresponding to cholesterol synthesis enzymes and a marked increase in the mRNA level of CYP46A1. The latter increase was associated with increased levels of CYP46A1 protein and increased production of 24OHC. The magnitude of the secretion of 24OHC from the differentiated NT2 cells into the medium was similar to that expected to occur under in vivo conditions. An alternative to elimination of cholesterol by the CYP46A1 mechanism is elimination by CYP27A1, and the product of this enzyme, 27-hydroxycholesterol (27OHC), is also known to pass the blood-brain barrier. The CYP27A1 protein level decreased during the differentiation of the NT2 cells in parallel with decreased production of 27OHC. The ratio between 24OHC and 27OHC in the medium from the cultured cells increased, by a factor of 13, during the differentiation process. The results suggest that progenitor cells eliminate cholesterol in the form of 27OHC while neurogenesis induces a change to the CYP46A1 dependent pathway. Furthermore this study demonstrates that differentiated NT2 cells are suitable for studies of cholesterol homeostasis in human neurons.     

A novel murine CTP:phosphoethanolamine cytidylyltransferase splice variant is a post-translational repressor and an indicator that both cytidylyltransferase domains are required for activity

CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) has an important regulatory function in biosynthesis of the membrane phospholipid phosphatidylethanolamine. We previously determined that the full-length Pcyt2α and its splice variant Pcyt2β are the main active isoforms of this enzyme. Here we report that mouse Pcyt2 could be spliced at Introns 7 and 8 to produce a unique third isoform, Pcyt2γ, in which the second cytidylyltransferase domain at the C-terminus becomes deleted. Pcyt2γ is ubiquitously expressed in embryonic and adult mouse tissues, and is the most abundant in the kidney, skeletal muscle and testis. Pcyt2γ splicing mechanism dominates over Pcyt2β exon-skipping mechanism in most examined tissues. Although Pcyt2γ maintains the N-terminal cytidylyltransferase domain as most cytidylyltransferases, the lack of the C-terminal cytidylyltransferase domain causes a complete loss of catalytic activity. However, Pcyt2γ interacts with the active isoform, Pcyt2α, and significantly reduces Pcyt2α homodimerization and activity. The inactive N-domain (H35Y, H35A) and C-domain (H244Y, H244A) mutants of Pcyt2α also reduce Pcyt2α homodimerization and activity. This study revealed the importance of both cytidylyltransferase ³⁵HYGH and ²⁴⁴HIGH motifs for the activity of murine Pcyt2α and established that the naturally occurring splice variant Pcyt2γ has a function to restrain the enzyme activity through the formation of unproductive enzyme complexes.     

The pro-tumorigenic effects of metabolic alterations in glioblastoma including brain tumor initiating cells

De-regulated cellular energetics is an emerging hallmark of cancer with alterations to glycolysis, oxidative phosphorylation, the pentose phosphate pathway, lipid oxidation and synthesis and amino acid metabolism. Understanding and targeting of metabolic reprogramming in cancers may yield new treatment options, but metabolic heterogeneity and plasticity complicate this strategy. One highly heterogeneous cancer for which current treatments ultimately fail is the deadly brain tumor glioblastoma. Therapeutic resistance, within glioblastoma and other solid tumors, is thought to be linked to subsets of tumor initiating cells, also known as cancer stem cells. Recent profiling of glioblastoma and brain tumor initiating cells reveals changes in metabolism, as compiled here, that may be more broadly applicable. We will summarize the profound role for metabolism in tumor progression and therapeutic resistance and discuss current approaches to target glioma metabolism to improve standard of care.     

Assessing bioenergetic function in response to oxidative stress by metabolic profiling

It is now clear that mitochondria are an important target for oxidative stress in a broad range of pathologies, including cardiovascular disease, diabetes, neurodegeneration, and cancer. Methods for assessing the impact of reactive species on isolated mitochondria are well established but constrained by the need for large amounts of material to prepare intact mitochondria for polarographic measurements. With the availability of high-resolution polarography and fluorescence techniques for the measurement of oxygen concentration in solution, measurements of mitochondrial function in intact cells can be made. Recently, the development of extracellular flux methods to monitor changes in oxygen concentration and pH in cultures of adherent cells in multiple-sample wells simultaneously has greatly enhanced the ability to measure bioenergetic function in response to oxidative stress. Here we describe these methods in detail using representative cell types from renal, cardiovascular, nervous, and tumorigenic model systems while illustrating the application of three protocols to analyze the bioenergetic response of cells to oxidative stress.     

Complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: A review

Plasma membranes are complex entities common to all living cells. The basic principle of their organization appears very simple, but they are actually of high complexity and represent very dynamic structures. The interactions between bioactive molecules and lipids are important for numerous processes, from drug bioavailability to viral fusion. The cell membrane is a carefully balanced environment and any change inflicted upon its structure by a bioactive molecule must be considered in conjunction with the overall effect that this may have on the function and integrity of the membrane. Conceptually, understanding the molecular mechanisms by which bioactive molecules interact with cell membranes is of fundamental importance.     

Complete mitochondrial genomes from four subspecies of common chaffinch (Fringilla coelebs): New inferences about mitochondrial rate heterogeneity,neutral theory,and phylogenetic relationships within the order Passeriformes

We describe whole mitochondrial genome sequences from four subspecies of the common chaffinch (Fringilla coelebs), and compare them to 31 publicly available mitochondrial genome sequences from other Passeriformes. Rates and patterns of mitochondrial gene evolution are analyzed at different taxonomic levels within this avian order, and evidence is adduced for and against the nearly neutral theory of molecular evolution and the role of positive selection in shaping genetic variation of this small but critical genome. We find evidence of mitochondrial rate heterogeneity in birds as in other vertebrates, likely due to differences in mutational pressure across the genome. Unlike in gadine fish and some of the human mitochondrial work we do not observe strong support for the nearly neutral theory of molecular evolution; instead evidence from molecular clocks, distribution of dN/dS ratios at different levels of the taxonomic hierarchy and in different lineages, McDonald–Kreitman tests within Fringillidae, and site-specific tests of selection within Passeriformes, all point to a role for positive selection, especially for the complex I NADH dehydrogenase genes. The protein-coding mitogenome phylogeny of the order Passeriformes is broadly consistent with previously-reported molecular findings, but provides support for a sister relationship between the superfamilies Muscicapoidea and Passeroidea on a short basal internode of the Passerida where relationships have been difficult to resolve. An unexpected placement of the Paridae (represented by Hume's groundpecker) within the Muscicapoidea was observed. Consistent with other molecular studies the mtDNA phylogeny reveals paraphyly within the Muscicapoidea and a sister relationship of Fringilla with Carduelis rather than Emberiza.     

Pharmacological modulation of NMDA receptor activity and the advent of negative and positive allosteric modulators

The NMDA receptor (NMDAR) family of l-glutamate receptors are well known to have diverse roles in CNS function as well as in various neuropathological and psychiatric conditions. Until recently, the types of agents available to pharmacologically regulate NMDAR function have been quite limited in terms of mechanism of action and subtype selectivity. This has changed significantly in the past two years. The purpose of this review is to summarize the many drug classes now available for modulating NMDAR activity. Previously, this included competitive antagonists at the l-glutamate and glycine binding sites, high and low affinity channel blockers, and GluN2B-selective N-terminal domain binding site antagonists. More recently, we and others have identified new classes of NMDAR agents that are either positive or negative allosteric modulators (PAMs and NAMs, respectively). These compounds include the pan potentiator UBP646, the GluN2A-selective potentiator/GluN2C and GluN2D inhibitor UBP512, the GluN2D-selective potentiator UBP551, the GluN2C/GluN2D-selective potentiator CIQ as well as the new NMDAR-NAMs such as the pan-inhibitor UBP618, the GluN2C/GluN2D-selective inhibitor QZN46 and the GluN2A inhibitors UBP608 and TCN201. These new agents do not bind within the l-glutamate or glycine binding sites, the ion channel pore or the N-terminal regulatory domain. Collectively, these new allosteric modulators appear to be acting at multiple novel sites on the NMDAR complex. Importantly, these agents display improved subtype-selectivity and as NMDAR PAMs and NAMs, they represent a new generation of potential NMDAR therapeutics.     

Lipid droplet proteins and metabolic diseases

Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage which are composed of a neutral lipid core bounded by a protein decorated phospholipid monolayer. Although lipid storage is their most obvious function, LDs are far from inert as they participate in maintaining lipid homeostasis through lipid synthesis, metabolism, and transportation. Furthermore, they are involved in cell signaling and other molecular events closely associated with human disease such as dyslipidemia, obesity, lipodystrophy, diabetes, fatty liver, atherosclerosis, and others. The last decade has seen a great increase in the attention paid to LD biology. Regardless, many fundamental features of LD biology remain obscure. In this review, we will discuss key aspects of LD biology including their biogenesis, growth and regression. We will also summarize the current knowledge about the role LDs play in human disease, especially from the perspective of the dynamics of the associated proteins. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Lipid peroxidation and neurodegenerative disease

Lipid peroxidation is a complex process involving the interaction of oxygen-derived free radicals with polyunsaturated fatty acids, resulting in a variety of highly reactive electrophilic aldehydes. Since 1975, lipid peroxidation has been extensively studied in a variety of organisms. As neurodegenerative diseases became better understood, research establishing a link between this form of oxidative damage, neurodegeneration, and disease has provided a wealth of knowledge to the scientific community. With the advent of proteomics in 1995, the identification of biomarkers for neurodegenerative disorders became of paramount importance to better understand disease pathogenesis and develop potential therapeutic strategies. This review focuses on the relationship between lipid peroxidation and neurodegenerative diseases. It also demonstrates how findings in current research support the common themes of altered energy metabolism and mitochondrial dysfunction in neurodegenerative disorders.