Synthesis Of malonyl-CoA Research Articles

A Sense of Balance: Experimental Investigation and Modeling of a Malonyl-CoA Sensor in Escherichia coli.

Production of value-added chemicals in microorganisms is regarded as a viable alternative to chemical synthesis. In the past decade, several engineered pathways producing such chemicals, including plant secondary metabolites in microorganisms have been reported; upscaling their production yields, however, was often challenging. Here, we analyze a modular device designed for sensing malonyl-CoA, a common precursor for both fatty acid and flavonoid biosynthesis. The sensor can be used either for high-throughput pathway screening in synthetic biology applications or for introducing a feedback circuit to regulate production of the desired chemical. Here, we used the sensor to compare the performance of several predicted malonyl-CoA-producing pathways, and validated the utility of malonyl-CoA reductase and malonate-CoA transferase for malonyl-CoA biosynthesis. We generated a second-order dynamic linear model describing the relation of the fluorescence generated by the sensor to the biomass of the host cell representing a filter/amplifier with a gain that correlates with the level of induction. We found the time constants describing filter dynamics to be independent of the level of induction but distinctively clustered for each of the production pathways, indicating the robustness of the sensor. Moreover, by monitoring the effect of the copy-number of the production plasmid on the dose–response curve of the sensor, we managed to coarse-tune the level of pathway expression to maximize malonyl-CoA synthesis. In addition, we provide an example of the sensor’s use in analyzing the effect of inducer or substrate concentrations on production levels. The rational development of models describing sensors, supplemented with the power of high-throughput optimization provide a promising potential for engineering feedback loops regulating enzyme levels to maximize productivity yields of synthetic metabolic pathways.

Genetic analysis of the Warburg effect in yeast.

We recently discovered that the Warburg effect, defined by the dramatically enhanced metabolism of glucose to pyruvate, even in well-oxygenated cancer cells, can occur as a consequence of mutations that enhance lipid biosynthesis at the expense of respiratory capacity. Specifically, mutations in the E1 subunit of either of two respiratory enzymes, pyruvate dehydrogenase (PDC) or α-ketoglutarate dehydrogenase (KGDC), change substrate specificity from the 3-carbon α-ketoacid pyruvate, or the 5-carbon α-ketoacid α-ketoglutarate, to the 4-carbon α-ketoacid oxaloacetate (OADC). These mutations result in OADC-catalyzed synthesis of malonyl-CoA (MaCoA), the essential precursor of all fatty acids. These mutants arose as spontaneous suppressors of a yeast acc1(cs) cold-sensitive mutation encoding an altered form of AcCoA carboxylase (Acc1) that fails to produce MaCoA at the restrictive temperature (16 °C). Notably, these suppressors are respiratory defective as a result of the same nuclear mutations that suppress acc1(cs). These mutants also suppress sensitivity to Soraphen A, a potent inhibitor of Acc1 activity, at normal temperature (30 °C). To our knowledge, OADC activity has never been identified in eukaryotic cells. Our results offer a novel perspective on the Warburg effect: the reprogramming of energy metabolism in cancer cells as a consequence of mutational impairment of respiration to meet the fatty acid requirements of rapidly proliferating cells. We suggest OADC activity is a common feature of cancer cells and represents a novel target for the development of chemotherapeutics.

Role of CoA and acetyl-CoA in regulating cardiac fatty acid and glucose oxidation.

CoA (coenzyme A) and its derivatives have a critical role in regulating cardiac energy metabolism. This includes a key role as a substrate and product in the energy metabolic pathways, as well as serving as an allosteric regulator of cardiac energy metabolism. In addition, the CoA ester malonyl-CoA has an important role in regulating fatty acid oxidation, secondary to inhibiting CPT (carnitine palmitoyltransferase) 1, a key enzyme involved in mitochondrial fatty acid uptake. Alterations in malonyl-CoA synthesis by ACC (acetyl-CoA carboxylase) and degradation by MCD (malonyl-CoA decarboxylase) are important contributors to the high cardiac fatty acid oxidation rates seen in ischaemic heart disease, heart failure, obesity and diabetes. Additional control of fatty acid oxidation may also occur at the level of acetyl-CoA involvement in acetylation of mitochondrial fatty acid β-oxidative enzymes. We find that acetylation of the fatty acid β-oxidative enzymes, LCAD (long-chain acyl-CoA dehydrogenase) and β-HAD (β-hydroxyacyl-CoA dehydrogenase) is associated with an increase in activity and fatty acid oxidation in heart from obese mice with heart failure. This is associated with decreased SIRT3 (sirtuin 3) activity, an important mitochondrial deacetylase. In support of this, cardiac SIRT3 deletion increases acetylation of LCAD and β-HAD, and increases cardiac fatty acid oxidation. Acetylation of MCD is also associated with increased activity, decreases malonyl-CoA levels and an increase in fatty acid oxidation. Combined, these data suggest that malonyl-CoA and acetyl-CoA have an important role in mediating the alterations in fatty acid oxidation seen in heart failure.

Abstract 4617: BAY-ACC001, a novel ACC inhibitor, regulates fatty acids synthesis and lipid survival signaling with promising in vitro and in vivo activities in multiple preclinical tumor models.

Abstract Unlike normal cells, tumors undergo intensified de novo biogenesis of fatty acids (FAs) irrespective of the available circulating lipids. Acetyl-CoA carboxylase 1 (ACC) controls the rate limiting step in FA synthesis. ACC1 is up-regulated in a variety of human tumors and strongly associated with poorer prognosis in some tumor indications. Although targeting lipogenesis for cancer treatment appeared having strong rationale, drug discovery in this field has not been fully explored due to the lack of powerful tools for both evaluation and understanding the mode of action. Here we report the identification and the functional characterization of a highly selective ACC inhibitor BAY-ACC001, a ketoenol derivative using various pharmacological and lipidomic approaches to address the mechanism of ACC inhibition in cancer cells and its efficacy in preclinical tumor models. BAY-ACC001 inhibits human ACC1 and ACC1 with biochemical IC50s of 278 nM and 2590 nM measured by ACC1- or ACC2- mediated generation of ADP, respectively. In a cellular mechanistic assay using MCF7 tumor cells, BAY-ACC001 potently inhibited malonyl-CoA synthesis with an IC50 of 62 nM. Profiling BAY-ACC001 in a panel of 100 tumor cell lines revealed strong anti-proliferative activity in a sub-set of tumor cell lines with IC50s at low 3-digit nanomolar. Of note, in contrast to the potent activity in tumor cell lines, e.g. apoptosis induction in MCF7 breast tumor cells, ACC inhibitor showed only weak anti-proliferative effect and could not induce apoptosis in a set of non-transformed mammary epithelial cells. To elucidate the anti-tumor MoA, the levels of lipid components (∼400 lipid molecules) in MCF7 cells were analyzed using lipidomic technology. Interestingly, ACC inhibition did not lead to a simple depletion of lipid in cells, evident from a significant increase in tumor apoptosis-related lipid signaling molecules ceramides. These results suggested a bi-direction linkage between FA synthesis and the regulation of tumor cell survival. Single administration of BAY-ACC001 orally at 10 mg/kg (the maximum tolerable dose is 30 mg/kg, BID) in mice strongly reduced malonyl CoA levels in tumors. Treatment of BAY-ACC001 as single agent was efficacious in multiple tumor models, including MCF7 (breast), PC3 (prostate), HCT116 (colon) and MDA-MB-435 (melanoma) xenograft models; partial tumor remissions observed in MCF7 model. Furthermore, combination with Tamoxifen was synergistic in the MCF7 tumor model in vitro and in vivo. In conclusion, the prevalence of the exacerbated de novo FA synthesis observed in primary and metastatic tumors, the ACC-mediated novel survival signal transduction discovered in this study, and robust in vitro and in vivo anti-tumor activity of BAY-ACC001 provide a strong rationale for developing novel therapeutics targeting ACC for cancer treatment. Citation Format: Ningshu Liu, Maher Najar, Arne Scholz, Knut Eis, Ulf Bömer, Philip Lienau, Kai Thede, Dominik Mumberg, Michael Brands, Karl Ziegelbauer. BAY-ACC001, a novel ACC inhibitor, regulates fatty acids synthesis and lipid survival signaling with promising in vitro and in vivo activities in multiple preclinical tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4617. doi:10.1158/1538-7445.AM2013-4617

Mislocalization and inhibition of acetyl-CoA carboxylase 1 by a synthetic small molecule

Chromeceptin is a synthetic small molecule that inhibits insulin-induced adipogenesis of 3T3-L1 cells and impairs the function of IGF2 (insulin-like growth factor 2). The molecular target of this benzochromene derivative is MFP-2 (multifunctional protein 2). The interaction between chromeceptin and MFP-2 activates STAT6 (signal transducer and activator of transcription 6), which subsequently induces IGF inhibitory genes. It was not previously known how the binding of chromeceptin with MFP-2 blocks adipogenesis and activates STAT6. The results of the present study show that the chromeceptin-MFP-2 complex binds to and inhibits ACC1 (acetyl-CoA carboxylase 1), an enzyme important for the de novo synthesis of malonyl-CoA and fatty acids. The formation of this ternary complex removes ACC1 from the cytosol and sequesters it in peroxisomes under the guidance of Pex5p (peroxisomal-targeting signal type 1 receptor). As a result, chromeceptin impairs fatty acid synthesis from acetate where ACC1 is a rate-limiting enzyme. Overexpression of malonyl-CoA decarboxylase or siRNA (small interfering RNA) knockdown of ACC1 results in STAT6 activation, suggesting a role for malonyl-CoA in STAT6 signalling. The molecular mechanism of chromeceptin may provide a new pharmacological approach to selective inhibition of ACC1 for biological studies and pharmaceutical development.

Activation of malonyl‐CoA/fatty acid synthase axis is an early event in the effects of insulin in human skeletal muscle myotubes: Implication for obesity linked insulin resistance

Insulin simultaneously increases both glucose transport and synthesis of malonyl‐CoA by acetyl‐CoA carboxylase. Increased malonyl‐CoA concentration inhibits transport of fatty acyls into mitochondria and that shifts mitochondria from fatty acid oxidation to glucose oxidation. In the current study human myotubes were differentiated from primary myoblasts obtained from obese or athlete subjects to test effects of insulin on content of palmitoyl‐CoA, NADPH and NADH (marker for glucose consumption). Insulin initially induces the consumption of NADPH while sharply increasing the content of NADH. Analysis of long‐chain acyl‐CoAs showed that an insulin‐induced decrease in NADPH concentration is accompanied by accumulation of palmitoyl‐CoA. Accelerated synthesis of palmitate that needs two NADPH for each elongation step can explain initial insulin induced consumption of NADPH. We found increased degree of NADPH oxidation and increased synthesis of palmitoyl‐CoA in athletes. That suggests an accelerated rate of generation of malonyl‐CoA in myotubes from athletes. We hypothesize that in obesity insulin has reduced ability to increase the concentration of malonyl‐CoA in skeletal muscle and to attenuate the oxidation of fatty acids by mitochondria. As a result, the sustained oxidation of fatty acids by mitochondria in this condition generates a metabolic signal that suppresses glucose transport.

Examining Pancreatic Islet Lipotoxicity Using Two-Photon NAD(P)H Imaging

Pancreatic islet β-cells maintain blood glucose through the regulated secretion of insulin. A rise in blood glucose stimulates production of NAD(P)H resulting in a cascade of increased ATP/ADP ratio, closure of ATP-sensitive potassium channels, membrane depolarization, Ca2+-influx, and insulin secretion. During the course of Type II diabetes, the glucose-stimulated insulin response is dampened by glucose and lipid toxicity. It has recently been shown that the novel endocrine factor, FGF21, protects metabolically active tissues such as liver and white adipose tissue (WAT) by regulating fatty acid metabolism. To test the effects of FGF21 on islet β-cells, we measured the levels of Acetyl-CoA carboxylase (ACC) in response to FGF21. ACC is an enzyme involved in the synthesis of malonyl-CoA, a substrate used in fatty acid synthesis and a regulator of fatty acid oxidation. Reducing ACC expression is critical to the normal compensation of beta-cells to lipid toxicity. We show that FGF21 decreases ACC protein levels in mouse pancreatic islets. To determine whether decreased ACC protein level acts as a protective mechanism in maintaining β-cell sensitivity to glucose, we examined changes in NAD(P)H by two photon microscopy of living islets held in a microfluidic device as a direct measure of changes in the metabolism. Our data reveals that compared to control islets, FGF21-treated islets retain the glucose-stimulated NAD(P)H response in a high fat environment. We will further examine the kinetics of the islet glucose response using time-lapse NAD(P)H imaging of islets. Overall, these studies will determine the metabolic changes that occur during the β-cell response to FGF21, and enhance understanding of how this effect can protect beta-cells from high fatty acid stress.

Atypical Antipsychotics Rapidly and Inappropriately Switch Peripheral Fuel Utilization to Lipids, Impairing Metabolic Flexibility in Rodents

Patients taking atypical antipsychotics are frequented by serious metabolic (eg, hyperglycemia, obesity, and diabetes) and cardiac effects. Surprisingly, chronic treatment also appears to lower free fatty acids (FFAs). This finding is paradoxical because insulin resistance is typically associated with elevated not lower FFAs. How atypical antipsychotics bring about these converse changes in plasma glucose and FFAs is unknown. Chronic treatment with olanzapine, a prototypical, side effect prone atypical antipsychotic, lowered FFA in Sprague-Dawley rats. Olanzapine also lowered plasma FFA acutely, concomitantly impairing in vivo lipolysis and robustly elevating whole-body lipid oxidation. Increased lipid oxidation was evident from accelerated losses of triglycerides after food deprivation or lipid challenge, elevated FFA uptake into most peripheral tissues (∼2-fold) except heart, rises in long-chain 3-hydroxylated acyl-carnitines observed in diabetes, and rapid suppression of the respiratory exchange ratio (RER) during the dark cycle. Normal rises in RER following refeeding, a sign of metabolic flexibility, were severely blunted by olanzapine. Increased lipid oxidation in muscle could be explained by ∼50% lower concentrations of the negative cytoplasmic regulator of carnitine palmitoyltransferase I, malonyl-CoA. This was associated with loss of anapleurotic metabolites and citric acid cycle precursors of malonyl-CoA synthesis rather than adenosine monophosphate-activated kinase activation or direct ACC1/2 inhibition. The ability of antipsychotics to lower dark cycle RER in mice corresponded to their propensities to cause metabolic side effects. Our studies indicate that lipocentric mechanisms or altered intermediary metabolism could underlie the FFA lowering and hyperglycemia (Randle cycle) as well as some of the other side effects of atypical antipsychotics, thereby suggesting strategies for alleviating them.

Targeting Intermediary Metabolism in the Hypothalamus as a Mechanism to Regulate Appetite

The central nervous system mediates energy balance (energy intake and energy expenditure) in the body; the hypothalamus has a key role in this process. Recent evidence has demonstrated an important role for hypothalamic malonyl CoA in mediating energy balance. Malonyl CoA is generated by the carboxylation of acetyl CoA by acetyl CoA carboxylase and is then either incorporated into long-chain fatty acids by fatty acid synthase, or converted back to acetyl-CoA by malonyl CoA decarboxylase. Increased hypothalamic malonyl CoA is an indicator of energy surplus, resulting in a decrease in food intake and an increase in energy expenditure. In contrast, a decrease in hypothalamic malonyl CoA signals an energy deficit, resulting in an increased appetite and a decrease in body energy expenditure. A number of hormonal and neural orexigenic and anorexigenic signaling pathways have now been shown to be associated with changes in malonyl CoA levels in the arcuate nucleus (ARC) of the hypothalamus. Despite compelling evidence that malonyl CoA is an important mediator in the hypothalamic ARC control of food intake and regulation of energy balance, the mechanism(s) by which this occurs has not been established. Malonyl CoA inhibits carnitine palmitoyltransferase-1 (CPT-1), and it has been proposed that the substrate of CPT-1, long-chain acyl CoA(s), may act as a mediator(s) of appetite and energy balance. However, recent evidence has challenged the role of long-chain acyl CoA(s) in this process, as well as the involvement of CPT-1 in hypothalamic malonyl CoA signaling. A better understanding of how malonyl CoA regulates energy balance should provide novel approaches to targeting intermediary metabolism in the hypothalamus as a mechanism to control appetite and body weight. Here, we review the data supporting an important role for malonyl CoA in mediating hypothalamic control of energy balance, and recent evidence suggesting that targeting malonyl CoA synthesis or degradation may be a novel approach to favorably modify appetite and weight gain.

FGF21 and Pancreatic Islet Fatty Acid Metabolism

Pancreatic islet β-cells maintain blood glucose through the regulated secretion of insulin. A rise in blood glucose stimulates β-cell production of NAD(P)H and increases the ATP/ADP ratio resulting in a cascade of events including closure of ATP-sensitive potassium channels, membrane depolarization, Ca2+-influx, and insulin secretion. During the course of Type II diabetes, the glucose stimulated insulin response is dampened by glucose and lipid toxicity. It has recently been shown that the novel endocrine factor, FGF21, protects metabolically active tissues by regulating fatty acid metabolism. To test this effect in islet β-cells, we measured the levels of Acetyl-CoA carboxylase (ACC) in response to FGF21. ACC is an enzyme involved in the synthesis of malonyl-CoA, the substrate used in fatty acid synthesis and a regulator of fatty acid oxidation. We show that FGF21 causes an increase in ACC levels in βTC3 cells, a pancreatic islet β-cell line. We propose that this increase in ACC acts as a protective mechanism for maintaining β-cell sensitivity to glucose by lowering β-oxidation of fats for energy. To measure fatty acid metabolism in the islet, we will extend our biochemical studies of ACC to mouse islet tissue. Furthermore, we will examine mitochondrial metabolism of β-cells in the presence of fatty acid using two photon microscopy of NAD(P)H. More specifically, we will examine glucose-stimulated mitochondrial NAD(P)H response of β-cells under normal and high fat environments. Overall, these studies will determine whether metabolic changes in the β-cells occur under varying nutritional states and understand the effects of FGF21 regulation of ACC levels in controlling β-cell metabolism.

Fatty acid metabolism: target for metabolic syndrome

Fatty acids are a major energy source and important constituents of membrane lipids, and they serve as cellular signaling molecules that play an important role in the etiology of the metabolic syndrome. Acetyl-CoA carboxylases 1 and 2 (ACC1 and ACC2) catalyze the synthesis of malonyl-CoA, the substrate for fatty acid synthesis and the regulator of fatty acid oxidation. They are highly regulated and play important roles in the energy metabolism of fatty acids in animals, including humans. They are presently considered as an attractive target to regulate the human diseases of obesity, diabetes, cancer, and cardiovascular complications. In this review we discuss the role of fatty acid metabolism and its key players, ACC1 and ACC2, in animal evolution and physiology, as related to health and disease.

AMP-activated protein kinase and muscle insulin resistance

The Metabolic Syndrome, which includes obesity and type 2 diabetes, is reaching alarming proportions. A key factor is insulin resistance, defined as a reduced ability of insulin to stimulate glucose utilization and storage. Compelling evidence links insulin resistance with an excess fatty acid supply over energy need, resulting in lipid accumulation in non-adipose tissues. The AMPK pathway plays a key role in sensing and regulating tissue energy metabolism, influencing fuel metabolism in tissues including muscle and liver. A number of its actions could improve muscle insulin sensitivity at least partly by increasing fatty acid oxidation and diminishing synthesis of malonyl CoA, glycerolipids, ceramide and other molecules linked to insulin resistance, although the extent of these effects, particularly in the human context, is uncertain. Secondly, its activation could bypass the metabolic block associated with insulin resistance. Thirdly, it is possible that a dysregulation of the AMPK pathway may itself contribute to the metabolic derangement associated with insulin resistance. These issues are important in considering the AMPK pathway as a therapeutic target in insulin resistant states.

Biotinoyl Domain of Human Acetyl-CoA Carboxylase;Structural Insights into the Carboxyl Transfer Mechanism

Acetyl-CoA carboxylase (ACC) catalyzes the first step in fatty acid biosynthesis: the synthesis of malonyl-CoA from acetyl-CoA. As essential regulators of fatty acid biosynthesis and metabolism, ACCs are regarded as therapeutic targets for the treatment of metabolic diseases such as obesity, In ACC, the biotinoyl domain performs a critical function by transferring an activated carboxyl group from the biotin carboxylase domain to the carboxyl transferase domain, followed by carboxyl transfer to malonyl-CoA. Despite the intensive research on this enzyme, only the bacterial and yeast ACC structures are currently available, To explore the mechanism of ACC holoenzyme function, we determined the structure of the biotinoyl domain of human ACC2 and analyze its characteristics using NMR spectroscopy. The 3D structure of the hACC2 biotinoyl domain has a similar folding topology to the previously determined domains from E. coli and P. Shermanii, however, the 'thumb' structure is absent in the hACC2 biotinoyl domain. Observations of the NMR signals upon the biotinylation indicate that the biotin group of hACC2 does not affect the structure of the biotinoyl domain, while the biotin group for E. coli ACC interacts directly with the thumb residues that are not present in the hACC2 structure. These results imply that, in the E. coli ACC reaction, the biotin moiety carrying the carboxyl group from BC to CT can pause at the thumb of the BCCP domain. The human biotinoyl domain, however, lacks the thumb structure and does not have additional non-covalent interactions with the biotin moiety; thus, the flexible motion of the biotinylated lysine residue must underlie the "swinging arm" motion. This study provides insight into the mechanism of ACC holoenzyme function and supports the "swinging arm" model in human ACCs.

Ligand Specificity of Group I Biotin Protein Ligase of Mycobacterium tuberculosis

BackgroundFatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC) the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP) provides the co-factor for catalytic activity of ACC.Methodology/Principal FindingsBPL/BirA (Biotin Protein Ligase), and its substrate, biotin carboxyl carrier protein (BCCP) of Mycobacterium tuberculosis (Mt) were cloned and expressed in E. coli BL21. In contrast to EcBirA and PhBPL, the ∼29.5 kDa MtBPL exists as a monomer in native, biotin and bio-5′AMP liganded forms. This was confirmed by molecular weight profiling by gel filtration on Superdex S-200 and Dynamic Light Scattering (DLS). Computational docking of biotin and bio-5′AMP to MtBPL show that adenylation alters the contact residues for biotin. MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5′AMP. Docking simulations also suggest that bio-5′AMP hydrogen bonds to the conserved ‘GRGRRG’ sequence but not biotin. The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot. The Km for BCCP was ∼5.2 µM and ∼420 nM for biotin. MtBPL has low affinity (Kb = 1.06×10−6 M) for biotin relative to EcBirA but their Km are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5′AMP by EcBirA is channeled for its repressor activity.Conclusions/SignificanceThese studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis.

Ampkα Phosphorylation In The M. Vastus Lateralis Following Sprint Exercise In Humans

AMP-activated protein kinase (AMPK) is a key regulator of muscle energy metabolism in skeletal muscle. When active AMPK phosporylates, i.e., inactivates, Acetyl-CoA carboxylase reducing the synthesis of malonyl CoA. This releases the inhibition caused by malonyl CoA on fatty acid oxidation. Since exhaustive sprint exercise causes a marked reduction of muscle glycogen content we hypothesised that during the recovery period following sprint exercise in humans AMPKα will be phosphorylated. PURPOSE: The aim of this study was to determine if a short duration exhaustive sprint exercise (Wingate test) elicits phosphorylation of AMPKα in human skeletal muscle and to determine the degree of AMPKα activation during the recovery period. METHODS: Eleven healthy physical education students, five women ((mean ± SEM), age: 25 ± 1.4 years; height: 162 ± 3.2 cm; body mass: 56 ± 3.5 kg and percentage of body fat 26.3 ± 1.6) and six men (age: 23 ± 0.7 years; height: 176 ± 4.1 cm; body mass: 78 ± 4.6 kg and percentage of body fat 16.9 ± 2.8) were subjected to a 30-s Wingate test. Immediately before the start of the Wingate test, at the end of test, and at 30 min, 2h and 4h into the recovery period, muscle biopsies were taken from de middle portion of the musculus vastus lateralis. Biopsies were processed for the assessment of the AMPKα phosphorylation by Western blot. Lean body mass and fat mass were determined by dual energy x-ray absorptiometry (DXA). RESULTS: AMPKα phosphorylation immediately after the end of the Wingate test remained unchanged (2.00 ± 0.01 and 2.15 ± 0.14 arbitrary units (A.U.) pre and posttest, respectively, p=0.275). However, thirty min latter AMPKα phosphorylation was increased to 2.64 ± 0.14 A.U. (p<0.01). Two and four hours after the Wingate test AMPKα phosphorylation was reduced to values comparable to those observed prior to the sprint (1.78 ± 0.11 A.U. at 2h, p=0.083; and 1.94 ± 0.15 A.U. at 4h, p=0.749) after exercise). The response was similar in both genders. CONCLUSION: Skeletal muscle AMPKα phosphorylation is transiently increased during the recovery process following an all-out 30-s sprint (Wingate test) in humans. Supported by grants from Ministerio de Educación y Ciencia, Spain (BFU2006-13784), Gobierno de Canarias, Spain (PI2005/177) and Universidad de Las Palmas de Gran Canaria, Spain (UNI2006/05).

Solution Structure and Backbone Dynamics of the Biotinylation Domain of Helicobacter pylori Biotin-carboxyl Carrier Protein

Acetyl-CoA carboxylase (ACC) is an excellent candidate for antibiotics drug target, which mediates malonyl-CoA synthesis from acetyl-CoA through acetylation process. It is also involved in the committed step of fatty acid synthesis which is essential for living organisms. We have determined the three dimensional structure of C terminal domain of HP0371, biotin-carboxyl carrier protein of H. pyroli, in solution state using heteronuclear multi-dimensional NMR spectroscopy. The structure of HP0371 shows a flatten b-sheet fold which is similar with that of E. coli. However, the sequence and structure of protruding thumb are different with that of E. coli and the thumb shows different basis of structural rigidity based on backbone dynamics data.

Biotinoyl domain of human acetyl‐CoA carboxylase: Structural insights into the carboxyl transfer mechanism

Acetyl-CoA carboxylase (ACC) catalyzes the first step in fatty acid biosynthesis: the synthesis of malonyl-CoA from acetyl-CoA. As essential regulators of fatty acid biosynthesis and metabolism, ACCs are regarded as therapeutic targets for the treatment of metabolic diseases such as obesity. In ACC, the biotinoyl domain performs a critical function by transferring an activated carboxyl group from the biotin carboxylase domain to the carboxyl transferase domain, followed by carboxyl transfer to malonyl-CoA. Despite the intensive research on this enzyme, only the bacterial and yeast ACC structures are currently available. To explore the mechanism of ACC holoenzyme function, we determined the structure of the biotinoyl domain of human ACC2 and analyzed its characteristics and interaction with the biotin ligase, BirA using NMR spectroscopy. The 3D structure of the hACC2 biotinoyl domain has a similar folding topology to the earlier determined domains from E. coli and P. shermanii. However, the local structures near the biotinylation sites have notable differences that include the geometry of the consensus "Met-Lys-Met" (MKM) motif and the absence of "thumb" structure in the hACC2 biotinoyl domain. Observations of the NMR signals upon the biotinylation indicate that the biotin group of hACC2 does not affect the structure of the biotinoyl domain, while the biotin group for E. coli ACC interacts directly with the thumb residues that are not present in the hACC2 structure. These results imply that, in the E. coli ACC reaction, the biotin moiety carrying the carboxyl group from BC to CT can pause at the thumb of the BCCP domain. The human biotinoyl domain, however, lacks the thumb structure and does not have additional noncovalent interactions with the biotin moiety; thus, the flexible motion of the biotinylated lysine residue must underlie the "swinging arm" motion. The chemical shift perturbation and the cross saturation experiments of the human ACC2 holo-biotinoyl upon the addition of the biotin ligase (BirA) showed the interaction surface near the MKM motif, the two glutamic acids (Glu 926, Glu 953), and the positively charged residues (several lysine and arginine residues). This study provides insight into the mechanism of ACC holoenzyme function and supports the swinging arm model in human ACCs.

Role of malonyl-CoA in heart disease and the hypothalamic control of obesity

Obesity is an important contributor to the risk of developing insulin resistance, diabetes, and heart disease. Alterations in tissue levels of malonyl-CoA have the potential to impact on the severity of a number of these disorders. This review will focus on the emerging role of malonyl-CoA as a key "metabolic effector" of both obesity and cardiac fatty acid oxidation. In addition to being a substrate for fatty acid biosynthesis, malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase (CPT) 1, a key enzyme involved in mitochondrial fatty acid uptake. A decrease in myocardial malonyl-CoA levels and an increase in CPT1 activity contribute to an increase in cardiac fatty acid oxidation. An increase in malonyl-CoA degradation due to increased malonyl-CoA decarboxylase (MCD) activity may be one mechanism responsible for this decrease in malonyl-CoA. Another mechanism involves the inhibition of acetyl-CoA carboxylase (ACC) synthesis of malonyl-CoA, due to AMP-activated protein kinase (AMPK) phosphorylation of ACC. Recent studies have demonstrated a role of malonyl-CoA in the hypothalamus as a regulator of food intake. Increases in hypothalamic malonyl-CoA and inhibition of CPT1 are associated with a decrease in food intake in mice and rats, while a decrease in hypothalamic malonyl-CoA increases food intake and weight gain. The exact mechanism(s) responsible for these effects of malonyl-CoA are not clear, but have been proposed to be due to an increase in the levels of long chain acyl CoA, which occurs as a result of malonyl-CoA inhibition of CPT1. Both hypothalamic and cardiac studies have demonstrated that control of malonyl-CoA levels has an important impact on obesity and heart disease. Targeting enzymes that control malonyl-CoA levels may be an important therapeutic approach to treating heart disease and obesity.

Expression of genes regulating Malonyl‐CoA in human skeletal muscle

In humans and animal models, increased intramuscular lipid (IML) stores have been implicated in insulin resistance. Malonyl-CoA plays a critical role in cellular lipid metabolism both by serving as a precursor in the synthesis of lipids and by inhibiting lipid oxidation. In muscle, Malonyl-CoA acts primarily as a negative allosteric regulator of carnitine palmitoyl transferase-1 (CPT1) activity, thereby blocking the transport of long chain fatty acyl CoAs into the mitochondria for oxidation. In muscle, increased malonyl-CoA, decreased muscle CPT1 activity, and increased IML have all been reported in obesity. In order to determine whether malonyl-CoA synthesis might be under transcriptional as well as biochemical regulation, we measured mRNA content of several key genes that contribute to the cellular metabolism of malonyl-CoA in muscle biopsies from lean to morbidly obese subjects. Employing quantitative real-time PCR, we determined that expression of mitochondrial acetyl-CoA carboxylase 2 (ACC2) was increased by 50% with obesity (P < 0.05). In both lean and obese subjects, expression of mitochondrial ACC2 was 20-fold greater than that of cytoplasmic ACC1, consistent with their hypothesized roles in synthesizing malonyl-CoA from acetyl-CoA for CPT1 regulation and lipogenesis, respectively. In addition, in both lean and obese subjects, expression of malonyl-CoA decarboxylase was approximately 40-fold greater than fatty acid synthase, consistent with degradation, rather than lipogenesis, being the primary fate of malonyl-CoA in human muscle. No other genes showed signs of increased mRNA content with obesity, suggesting that there may be selective transcriptional regulation of malonyl-CoA metabolism in human obesity.

Inhibition of oleate oxidation in rat heart by acetate and propionate: A mass isotopomer study

We investigated the impact of acetate and propionate on the oxidation of oleate in rat hearts perfused with 0.4 mM [1-13C]oleate + various concentrations of [1,2-13C2]acetate or propionate. We assayed the contributions of oleate and acetate to the acetyl moiety of citrate (a probe of mitochondrial acetyl-CoA) and to malonyl-CoA. We found that acetate, even at low concentration, markedly inhibits the oxidation of [1-13C]oleate, without changes in [malonyl-CoA]. Also, 2 mM propionate (which does not generate acetyl-CoA in heart) decreased the contribution of 0.4 mM [1-13C]oleate to mitochondrial acetyl-CoA (from 67 to 35%), with a surprising 50% decrease in [malonyl-CoA]. In the presence of [1-13C]oleate and [1,2-13C2]acetate, the M2 labeling of malonyl-CoA was lower than that of the acetyl of citrate. This is not compatible with a cytosolic site of acetate activation in the heart. In contrast, the M1 labeling of malonyl-CoA was higher than that of the acetyl of citrate. This reflects the contribution of peroxisomal oxidation of [1-13C]oleate to the supply of acetyl-CoA used for malonyl-CoA synthesis. The profound inhibition of fatty acid oxidation by acetate may contribute to the accumulation of long-chain fatty acyl-CoAs and carnitines in the heart, and to the cardiomyopathies observed in alcoholics and hemodialyzed patients. (Supported by NIH Roadmap grant R33DK070291, and grant 2PO1AG015885)