Brain energy metabolism and neurotransmission at near-freezing temperatures: in vivo (1)H MRS study of a hibernating mammal

The brain of a hibernating mammal withstands physiological extremes that would result in cerebral damage and death in a non-hibernating species such as humans. To examine the possibility that this neuroprotection results from alterations in cerebral metabolism, we used in vivo(1)H NMR spectroscopy at high field (9.4 T) to measure the concentration of 18 metabolites (neurochemical profile) in the brain of 13-lined ground squirrels (Spermophilus tridecemlineatus) before, during, and after hibernation. Resolved in vivo(1)H NMR spectra were obtained even at low temperature in torpid hibernators ( approximately 7 degrees C). The phosphocreatine-to-creatine ratio was increased during torpor (+143%) indicating energy storage, and remained increased to a lesser extent during interbout arousal (IBA) (+83%). The total gamma-aminobutyric acid concentration was increased during torpor (+135%) and quickly returned to baseline during IBA. Glutamine (Gln) was decreased (-54%) during torpor but quickly returned to normal levels during IBA and after terminal arousal in the spring. Glutamate (Glu) was also decreased during torpor (-17%), but remained decreased during IBA (-20% compared with fall), and returned to normal level in the spring. Our observation that Glu and Gln levels are depressed in the brain of hibernators suggests that the balance between anaplerosis and loss of Glu and Gln (because of glutamatergic neurotransmission or other mechanisms) is altered in hibernation

Interaction between the biotin carboxyl carrier domain and the biotin carboxylase domain in pyruvate carboxylase from rhizobium etli

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family.Adam D. Lietzan, Ann L. Menefee, Tonya N. Zeczycki, Sudhanshu Kumar, Paul V. Attwood, John C. Wallace, W. Wallace Cleland and Martin St. Mauric

Structure, mechanism and regulation of pyruvate carboxylase

Published on the Internet 15 July 2008PC (pyruvate carboxylase) is a biotin-containing enzyme that catalyses the HCO3 −- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was discovered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC frommost organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the biotin carboxylase, carboxyltransferase and biotin carboxyl carrier domains, and also a unique allosteric effector domain. In the presence of the allosteric effector, acetyl- CoA, the biotin moiety transfers the carboxy group between the biotin carboxylase domain active site on one polypeptide chain and the carboxyltransferase active site on the adjacent antiparallel polypeptide chain. In addition, the bona fide role of PC in the nongluconeogenic tissues has been studied using a combination of classical biochemistry and genetic approaches. The first cloning of the promoter of the PC gene in mammals and subsequent transcriptional studies reveal some key cognate transcription factors regulating tissue-specific expression. The present review summarizes these advances and also offers some prospects in terms of future directions for the study of this important enzyme.Sarawut Jitrapakdee, Martin St Maurice, Ivan Rayment, W. Wallace Cleland, John C. Wallace and Paul V. Attwoo

Contribution of Hypothalamic–Pituitary–Adrenal Activity and Environmental Stress to Vulnerability for Smoking in Adolescents

Although tobacco smoking, which has been linked to depression, is a major public health problem, little is known about the neurobiological factors that confer vulnerability to smoking in youngsters and the effects of adolescent smoking on the course of depression. This study examined whether hypothalamic-pituitary-adrenal (HPA) activity and stressful life experiences are related to smoking behavior in depressed and non-depressed adolescents, and whether smoking predicts a worsening course of depression. Smoking history and stressful experiences were assessed in 151 adolescents (48 with no personal or family history of psychiatric disorder, 48 with no psychiatric history, but at high risk for depression by virtue of parental depression, and 55 with current major depressive disorder). Evening salivary cortisol and nocturnal urinary-free cortisol were measured for three consecutive evenings. The participants were then followed at regular intervals for up to 5 years to assess smoking history, clinical course of depression and stressful experiences during the follow-up period. Increased evening/night-time cortisol levels were associated with both initiation and persistence of smoking during follow-up. Stressful life experiences further increased the risk for smoking in depressed as well as non-depressed youth. Smoking was also associated with a higher frequency of depressive episodes during follow-up. A model that included stressful experiences and cortisol levels reduced the contribution of smoking per se to depression. High evening/night-time cortisol level appears to be a vulnerability marker for smoking in adolescents, with stressful experiences further increasing the risk for smoking in vulnerable youth. High evening/night-time cortisol levels and stressful experiences accounted, at least partially, for the association between depressive illness and smoking behavior