Thyroid hormone controls carnitine status through modifications of γ-butyrobetaine hydroxylase activity and gene expression

The carnitine system plays a key role in β-oxidation of long-chain fatty acids by permitting their transport into the mitochondrial matrix. The effects of hypothyroidism and hyperthyroidism were studied on γ-butyrobetaine hydroxylase (BBH), the enzyme responsible for carnitine biosynthesis in the rat. In rat liver, BBH activity was decreased in the hypothyroid state and increased in hyperthyroid animals. The modifications in BBH activity correlated with changes in the enzyme Vmax values. These changes were shown to be related to hepatic BBH mRNA abundance. Thyroid hormones are known to interact with lipid metabolism, in particular by increasing long-chain fatty acid oxidation through activation of carnitine-dependent fatty acid import into mitochondria. Our study showed that thyroid hormones also increased carnitine bioavailability. Received 23 October 2001; received after revision 11 January 2002; accepted 15 January 2002     

Functional interaction between TRPC1 channel and connexin-43 protein: a novel pathway underlying S1P action on skeletal myogenesis

We recently demonstrated that skeletal muscle differentiation induced by sphingosine 1-phosphate (S1P) requires gap junctions and transient receptor potential canonical 1 (TRPC1) channels. Here, we searched for the signaling pathway linking the channel activity with Cx43 expression/function, investigating the involvement of the Ca²⁺-sensitive protease, m-calpain, and its targets in S1P-induced C2C12 myoblast differentiation. Gene silencing and pharmacological inhibition of TRPC1 significantly reduced Cx43 up-regulation and Cx43/cytoskeletal interaction elicited by S1P. TRPC1-dependent functions were also required for the transient increase of m-calpain activity/expression and the subsequent decrease of PKCα levels. Remarkably, Cx43 expression in S1P-treated myoblasts was reduced by m-calpain-siRNA and enhanced by pharmacological inhibition of classical PKCs, stressing the relevance for calpain/PKCα axis in Cx43 protein remodeling. The contribution of this pathway in myogenesis was also investigated. In conclusion, these findings provide novel mechanisms by which S1P regulates myoblast differentiation and offer interesting therapeutic options to improve skeletal muscle regeneration.     

Fatty acid metabolism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of a large group of inherited diseases in humans in which one or more of the peroxisomal functions are impaired. The yeast Saccharomyces cerevisiae has been used as a model organism to study the functions of peroxisomes. Efficient oxidation of fatty acids does not only require the participation of peroxisomal enzymes but also the active involvement of other gene products. One group of important gene products in this respect includes peroxisomal membrane proteins involved in metabolite transport. This overview discusses the various aspects of fatty acid -oxidation in S. cerevisiae. Addressed are the various enzymes and their particular functions as well as the various transport mechanisms to take up fatty acids into peroxisomes or to export the -oxidation products out of the peroxisome to mitochondria for full oxidation to CO₂ and H₂O.Received 19 February 2003; received after revision 27 March 2003; accepted 27 March 2003     

Pyruvate dehydrogenase kinase 4 expression is synergistically induced by AMP-activated protein kinase and fatty acids

Organs are flexible as to which substrates they will use to maintain energy homeostasis. Under well-fed conditions, glucose is a preferred substrate for oxidation. During fasting, fatty acid oxidation will become a more important energy source. Glucose oxidation is decreased by fatty acids, a process in which the pyruvate dehydrogenase complex (PDH) and its regulator pyruvate dehydrogenase kinase 4 (PDK4) play important roles. It is currently unknown how energy status influences PDH activity. We show that AMP-activated protein kinase (AMPK) activation by hypoxia and AICAR treatment combined with fatty acid administration synergistically induce PDK4 expression. We provide evidence that AMPK activation modulates ligand-dependent activation of peroxisome proliferator-activated receptor. Finally, we show that this synergistic induction of PDK4 decreases cellular glucose oxidation. In conclusion, AMPK and fatty acids play a direct role in fuel selection in response to cellular energy status in order to spare glucose. S. M. Houten, M. Chegary: These two authors contributed equally to this work. Received 11 July 2008; received after revision 26 January 2009; accepted 02 February 2009     

Biosynthesis of 1-alkenes in the defensive secretions ofTribolium confusum (Tenebrionidae); stereochemical implications

Summary The terminally unsaturated hydrocarbons of the defensive secretion ofTribolium confusum are biosynthesized from fatty acids by oxidative decarboxylation. The process involves an enantiospecific cleavage of the C–H bond of thepro-(S) C(3)–H atom and simultaneous decarboxylation of the acid into an 1-alkene and carbon dioxide via ananti-periplanar transition state geometry (anti-elimination). The stereochemistry of this biotranformation is identical in all respects with the same reaction in higher plants. The mechanism seems to be of general importance for the biosynthesis of many vinylic substructures of natural products from oxygen-containing precursors.     

Role of Drosophila alkaline ceramidase (Dacer) in Drosophila development and longevity

Ceramidases catalyze the hydrolysis of ceramides to generate sphingosine (SPH) and fatty acids, and ceramide metabolism is implicated in various biological responses in Drosophila melanogaster. Here we report the cloning, biochemical characterization, and functional analysis of a Drosophila alkaline ceramidase (Dacer). Dacer, a membrane-bound protein of 284 amino acids, shares homology with yeast and mammalian alkaline ceramidases. Overexpression of Dacer in High Five insect cells increases ceramidase activity in the alkaline pH range, indicating that Dacer is a bona fide alkaline ceramidase. Dacer mRNA is highly expressed in the midgut and at the pupal stage. An inactivation of Dacer by insertional mutagenesis increases the levels of ceramides in both Drosophila pupae and adult flies. Dacer inactivation increases Drosophila pre-adult development time, lifespan, and anti-oxidative stress capacity. Collectively, these results suggest that Dacer plays an important role in the Drosophila development and longevity by controlling the metabolism of ceramides.     

Mechanisms of lipid peroxidation: Iron catalyzed decomposition of fatty acid hydroperoxides as the basis of hydrocarbon evolution in vivo

Summary Ethane and pentane are evolved during peroxidation of tissue lipids in vivo and are believed to be derived from 3 and 6 fatty acids respectively. We present evidence supporting a scission mechanism as the means of forming these hydrocarbons during hydroperoxide decomposition. A fatty acid 6 hydroperoxide (methyl 13-hydroperoxy-6,9,11-octatrienoate), when incubated with a ferrous ion, yielded pentane as the major hydrocarbon (98%). Reaction with ferrous ion results in an alkoxy radical capable of undergoing scission to an aldehydic fatty acid and a hydrocarbon.We wish to thank Dr Ned Porter for both his generous donation of a supply of hydroperoxide and for his suggestions regarding possible reaction mechanisms. This research was supported by U.S.P.H.S. N.I.H. Grants ES00798, HL16264 and T32 GM07105.     

Regulation of glycosaminoglycan structure and atherogenesis

Cardiovascular disease is the major cause of premature death in modern society, and its impact is increasing due to rising rates of obesity and type 2diabetes. Clinical studies based on targeting metabolic abnormalities and biomarkers demonstrate significant benefits, but always an element of disease remains which is resistant to treatment. Recent evidence has strongly implicated an early interaction of atherogenic lipoproteins with vascular matrix proteoglycans as the initiating step in atherogenesis. Expert commentary has pointed to the need for vascular directed therapies to provide reductions in the residual disease component. We propose that the regulation of synthesis and thus structure of glycosaminoglycans on proteoglycans provides a potential pathway to this reduction. We review existing evidence that the vascular synthesis of glycosaminoglycan chains can be regulated in a manner which reduces lipoprotein binding and the potential application of this strategy to attenuation of the current cardiovascular disease pandemic.Received 21 October 2003; received after revision 16 December 2003; accepted 29 December 2003     

Cytochrome P450 2U1, a very peculiar member of the human P450s family

Cytochrome P450 2U1 (CYP2U1) exhibits several distinctive characteristics among the 57 human CYPs, such as its presence in almost all living organisms with a highly conserved sequence, its particular gene organization with only five exons, its major location in thymus and brain, and its protein sequence involving an unusually long N-terminal region containing 8 proline residues and an insert of about 20 amino acids containing 5 arginine residues after the transmembrane helix. Few substrates, including fatty acids, N-arachidonoylserotonin (AS), and some drugs, have been reported so far. However, its biological roles remain largely unknown, even though CYP2U1 mutations have been involved in some pathological situations, such as complicated forms of hereditary spastic paraplegia. These data together with its ability to hydroxylate some fatty acids and AS suggest its possible role in lipid metabolism.     

Post-transcriptional gene silencing of ribosomal protein S6 kinase 1 restores insulin action in leucine-treated skeletal muscle

Excessive nutrients, especially amino acids, impair insulin action on glucose metabolism in skeletal muscle. We tested the hypothesis that the branched-chain amino acid leucine reduces acute insulin action in primary myotubes via a negative feedback mechanism involving ribosomal protein S6 kinase 1 (S6K1). The effect of S6K1 on glucose metabolism was determined by applying RNA interference (siRNA). Leucine (5 mM) reduced glucose uptake and incorporation to glycogen by 13% and 22%, respectively, compared to the scramble siRNA-transfected control at the basal level. Leucine also reduced insulin-stimulated Akt phosphorylation, glucose uptake and glucose incorporation to glycogen (39%, 39% and 37%, respectively), and this reduction was restored after S6K1 silencing. Depletion of S6K1 enhanced basal glucose utilization and protected against the development of impaired insulin action, in response to excessive leucine. In conclusion, S6K1 plays an important role in the regulation of insulin action on glucose metabolism in skeletal muscle. Received 22 December 2008; received after revision 19 February 2009; accepted 23 February 2009     

Long-chain fatty acid uptake by skeletal myocytes: a confocal laser scanning microscopy study

Studies of regulation of free fatty acid (FFA) utilization by skeletal muscles have focused on plasma FFA delivery and on intracellular factors affecting FFA metabolism. The present study was conducted to directly analyse the uptake process of fatty acids into single myocytes. Cells were isolated from the rat flexor digitorum brevis muscle. Confocal laser scanning microscopy was utilized to analyse the uptake of the fluorescent fatty acid derivative 12-NBD-stearate, which is not metabolized by muscle tissue. Uptake represented a saturable function of the unbound fatty acid concentration in the medium (K _m 366 ± 118 nM, V _max 2.1 ± 0.3 AU/s) and depended on the medium sodium concentration. Reduced buffer pH increased initial uptake rates, whereas lactate (10 mM) had no effect. Membrane hyper- and depolarization decreased uptake rates. This study demonstrates for the first time kinetic data from isolated myocytes with evidence for a carrier-mediated transport mechanism for long-chain fatty acids. Received 31 March 1998; accepted 8 May 1998     

Protein structure to function: insights from computation

Computation plays an important role in functional genomics. THEMATICS is a computational method that predicts chemical and electrostatic properties of residues in enzymes and utilizes information contained in those predictions to identify active sites. The only input required is the three-dimensional structure of the query protein. The identification of residues involved in catalysis and in recognition is discussed. The two serine proteases Kex2 from Saccharomyces cerevisiae and subtilisin from Bacillus subtilis are used as examples to illustrate how the method finds the catalytic residues for both enzymes. In addition, Kex2 is specific for dibasic sites and THEMATICS finds the recognition residues for both the S1 and S2 sites of Kex2. In contrast, no such recognition sites are found for the non-specific enzyme subtilisin. The ability to identify sites that govern recognition opens the door to better understanding of specificity and to the design of highly specific inhibitors.Received 22 July 2003; received after revision 16 September 2003; accepted 20 October 2003     

Metabolism and signaling activities of nuclear lipids

Apart from the lipids present in the nuclear envelope, the nucleus also contains lipids which are located further inside and are resistant to treatment with nonionic detergents. Evidence is being accumulated on the importance of internal nuclear lipid metabolism. Nuclear lipid metabolism gives rise to several lipid second messengers that function within the nucleus. Moreover, it is beginning to emerge that nuclear lipids not only act as precursors of bioactive second messengers but may be directly involved in regulation of nuclear structure and gene expression. Over the last 10years, especially the role of the inositol lipid cycle in nuclear signal transduction has been extensively studied. This cycle is activated following a variety of stimuli and is regulated independently from the inositide cycle located at the plasma membrane. However, the nucleus contain other lipids, such as phosphatidylcholine, sphingomyelin, fatty acids and eicosanoids. There are numerous reports which suggest that these classes of nuclear lipids may play roles in the nucleus as important as those of phosphoinositides. This review aims at highlighting the most important aspects regarding the metabolism and signaling activities of nuclear phosphatidylcholine, sphingomyelin, fatty acids and eicosanoids.Received 7 November 2003; received after revision 18 December 2003; accepted 29 December 2003     

Pancreastatin,a chromogranin A-derived peptide,inhibits leptin and enhances UCP-2 expression in isolated rat adipocytes

Leptin, the ob gene product, is an adipocyte-secreted hormone that centrally regulates weight by decreasing caloric intake and increasing energy expenditure. Expression of leptin is regulated by dietary status, insulin, glucocorticoids and catecholamines. Pancreastatin (PST), a chromogranin A-derived peptide, correlates with catecholamine levels, and may play a role in the physiology of stress, modulating endocrine secretion and metabolism. Thus, PST has been found to exert a lipolytic and anti-insulin effect in white adipocytes. The aim of the present work was to investigate a possible role of PST modulating the expression of key genes involved in lipid storage and metabolism: leptin, PPAR-2, UCP-1 and UCP-2. We incubated isolated rat epididymal adipocytes with 100 nM PST for 16 and 24 h. Leptin, UCP-2 and UCP-1 mRNA levels were assessed by RT-PCR, followed by Southern blot. Leptin secretion was also measured by ELISA. PST inhibited leptin expression and secretion at 16-h incubation, but this effect was no longer observed after 24 h. On the other hand, PST stimulated the expression of UCP-2 after 16 h. However, the effect was still significant after 24 h. The inhibitory effect of PST on leptin expression and secretion and the stimulation of UCP-2 expression were prevented by blocking PKC. UCP-1 and PPR-2 expression did not change after PST stimulation. Leptin differentially regulates the expression of key genes in the rat adipocyte, upregulating the expression of UCP-2 and inhibiting the expression and secretion of leptin by a mechanism that involves PKC activity. These effects may contribute to the metabolic action of catecholamines in physiological and pathophysiological conditions with increased sympathetic activity.Received 5 September 2003; received after revision 6 October 2003; accepted 14 October 2003     

Alpha 1-adrenergic stimulation of ketogenesis and fatty acid oxidation is associated with inhibition of lipogenesis in rat hepatocytes

The effect of norepinephrine on fatty acid synthesis (3H2O incorporation into fatty acids), on fatty acid oxidation to CO2 and on ketogenesis was studied in isolated hepatocytes of fed rats. After incubation with norepinephrine (50 microM), lipogenesis was lower (5.7 +/- 1.1 nmoles 3H2O incorporated into fatty acids/mg dry weight/30 min) than in controls (7.5 +/- 1.7; n = 6, p less than 0.02). In contrast, (1-14C) palmitate conversion into total ketone bodies was increased to 10.9 +/- 1.8 nmoles/mg/30 min with norepinephrine, vs 8.5 +/- 1.6 in controls (p less than 0.05), and more (1-14C) palmitate was converted to 14CO2 with norepinephrine than in controls (1.48 +/- 0.10 nmoles/mg/30 min vs 1.06 +/- 0.11, p less than 0.05). The inhibitory effect of norepinephrine on lipogenesis was abolished by addition of the alpha 1-receptor blocker prazosin, but not by alpha 2 or beta-blockers. The results demonstrate that the ketogenic effect of norepinephrine is coupled with an inhibitory effect on lipogenesis which may be explained by diminished activity of acetyl-CoA carboxylase, diminished formation of malonyl-CoA and decreased activity of carnitine palmitoyl transferase I.     

Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury

Stroke and neurotrauma mediate neuronal death through a series of events that involve multiple interdependent molecular pathways. It has been suggested that these pathways are triggered following elevations in extracellular excitatory amino acids, primarily glutamate [1]. This report outlines mechanisms involving glutamate-mediated excitotoxicity with specific focus on (i) the role of Ca²⁺ in neurotoxicity, (ii) The concept of source specificity of neurotoxicity, (iii) the role of the ionotropic N-methyl-D-aspartate (NMDA)-subtype glutamate receptor and its associated submembrane molecules that may give rise to signaling specificity in excitotoxicity and (iv) the role of glutamate-mediated free-radical generation and associated cell death pathways. We also highlight a novel, peptide-based approach for uncoupling NMDA receptors from excitotoxicity in the rat central nervous system subjected to focal ischemia, thereby reducing stroke infarct volume and improving neurological functioning.Received 11 August 2003; received after revision 15 September 2003; accepted 17 September 2003     

Functions of fatty acid binding proteins

Summary Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14–15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABP_pm). These proteins have not been fully characterized to date, but strong evidence suggests that they function in the transport of long-chain fatty acids across the plasma membrane.     

Sphingosine 1-phosphate induces differentiation of adipose tissue-derived mesenchymal stem cells towards smooth muscle cells

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which regulates multiple biological parameters in a number of cell types, including stem cells. Here we report, for the first time, that S1P dose-dependently stimulates differentiation of adipose tissue-derived mesenchymal stem cells (ASMC) towards smooth muscle cells. Indeed, S1P not only induced the expression of smooth muscle cell-specific proteins such as α-smooth muscle actin (αSMA) and transgelin, but also profoundly affected ASMC morphology by enhancing cytoskeletal F-actin assembly, which incorporated αSMA. More importantly, S1P challenge was responsible for the functional appearance of Ca²⁺ currents, characteristic of differentiated excitable cells such as smooth muscle cells. By employing various agonists and antagonists to inhibit S1P receptor subtypes, S1P₂ turned out to be critical for the pro-differentiating effect of S1P, while S1P₃ appeared to play a secondary role. This study individuates an important role of S1P in AMSC which can be exploited to favour vascular regeneration. Received 06 March 2009; accepted 17 March 2009     

Enrichment of ligands with molecular dockings and subsequent characterization for human alcohol dehydrogenase 3

Alcohol dehydrogenase 3 (ADH3) has been assigned a role in nitric oxide homeostasis due to its function as an S-nitrosoglutathione reductase. As altered S-nitrosoglutathione levels are often associated with disease, compounds that modulate ADH3 activity might be of therapeutic interest. We performed a virtual screening with molecular dockings of more than 40,000 compounds into the active site of human ADH3. A novel knowledge-based scoring method was used to rank compounds, and several compounds that were not known to interact with ADH3 were tested in vitro. Two of these showed substrate activity (9-decen-1-ol and dodecyltetraglycol), where calculated binding scoring energies correlated well with the logarithm of the k _cat/K _m values for the substrates. Two compounds showed inhibition capacity (deoxycholic acid and doxorubicin), and with these data three different lines for specific inhibitors for ADH3 are suggested: fatty acids, glutathione analogs, and cholic acids.