A Cellular Perspective on Brain Energy Metabolism and Functional Imaging.

The energy demands of the brain are high: they account for at least 20% of the body's energy consumption. Evolutionary studies indicate that the emergence of higher cognitive functions in humans is associated with an increased glucose utilization and expression of energy metabolism genes. Functional brain imaging techniques such as fMRI and PET, which are widely used in human neuroscience studies, detect signals that monitor energy delivery and use in register with neuronal activity. Recent technological advances in metabolic studies with cellular resolution have afforded decisive insights into the understanding of the cellular and molecular bases of the coupling between neuronal activity and energy metabolism and point at a key role of neuron-astrocyte metabolic interactions. This article reviews some of the most salient features emerging from recent studies and aims at providing an integration of brain energy metabolism across resolution scales.

NAD+-dependent post-translational modification of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein reported to be a target of several covalent modifications in many organisms. In a previous study we showed that enterohemorragic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains secrete GAPDH and that this protein binds to human plasminogen and fibrinogen. Here we report that GAPDH of these pathogens is ADP-ribosylated either in the cytoplasm or in the extracellular medium. GAPDH catalyzes its own modification which involves Cys149 at the active site. ADP-ribosylation of extracellular GAPDH may play important role in the interaction with the host as it has been proposed in other pathogens.

Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway

The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER-Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER-Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

Predicting drug metabolism: experiment and/or computation?

Drug metabolism can produce metabolites with physicochemical and pharmacological properties that differ substantially from those of the parent drug, and consequently has important implications for both drug safety and efficacy. To reduce the risk of costly clinical-stage attrition due to the metabolic characteristics of drug candidates, there is a need for efficient and reliable ways to predict drug metabolism in vitro, in silico and in vivo. In this Perspective, we provide an overview of the state of the art of experimental and computational approaches for investigating drug metabolism. We highlight the scope and limitations of these methods, and indicate strategies to harvest the synergies that result from combining measurement and prediction of drug metabolism.

Preclinical Pharmacokinetics, Pharmacodynamics and Safety of Sucroferric Oxyhydroxide.

Sucroferric oxyhydroxide (VELPHORO(®)) is a polynuclear iron-based phosphate binder recently approved for the treatment of hyperphosphataemia in patients with chronic kidney disease (CKD). As a number of the available phosphate binders do not provide the optimal combination of good efficacy, adequate tolerability and low pill burden, sucroferric oxyhydroxide constitutes a promising alternative. Among the attributes of an ideal phosphate binder is minimal absorption and, hence, low risk of systemic toxicity. Accordingly, the iron-releasing properties and absorption, distribution, metabolism and excretion (ADME) profile of sucroferric oxyhydroxide, as well as the possibility of iron accumulation and toxicity, were investigated in a series of preclinical studies. The effect of sucroferric oxyhydroxide on the progression of vascular calcification was also investigated. Sucroferric oxyhydroxide exhibited a high phosphate-binding capacity and low iron-releasing properties across the physiological pH range found in the gastrointestinal tract. In the ADME studies, uptake of (59)Fe-radiolabelled sucroferric oxyhydroxide was low in rats and dogs (<1% from a 50 mg Fe/kg bodyweight dose), with the majority of absorbed iron located in red blood cells. Long-term (up to 2 years) administration of sucroferric oxyhydroxide in rats and dogs was associated with modest increases in tissue iron levels and no iron toxicity. Moreoever, in uraemic rats, sucroferric oxyhydroxide was associated with reduced progression of vascular calcification compared with calcium carbonate. In conclusion, sucroferric oxyhydroxide offers a new option for the treatment of hyperphosphataemia, with a high phosphate-binding capacity, minimal iron release, and low potential for iron accumulation and toxicity.

Catecholamine metabolism in paraganglioma and pheochromocytoma: similar tumors in different sites?

Pheochromocytoma (PHEO) and paraganglioma (PGL) are catecholamine-producing neuroendocrine tumors that arise respectively inside or outside the adrenal medulla. Several reports have shown that adrenal glucocorticoids (GC) play an important regulatory role on the genes encoding the main enzymes involved in catecholamine (CAT) synthesis i.e. tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). To assess the influence of tumor location on CAT metabolism, 66 tissue samples (53 PHEO, 13 PGL) and 73 plasma samples (50 PHEO, 23 PGL) were studied. Western blot and qPCR were performed for TH, DBH and PNMT expression. We found a significantly lower intra-tumoral concentration of CAT and metanephrines (MNs) in PGL along with a downregulation of TH and PNMT at both mRNA and protein level compared with PHEO. However, when PHEO were partitioned into noradrenergic (NorAd) and mixed tumors based on an intra-tumoral CAT ratio (NE/E >90%), PGL and NorAd PHEO sustained similar TH, DBH and PNMT gene and protein expression. CAT concentration and composition were also similar between NorAd PHEO and PGL, excluding the use of CAT or MNs to discriminate between PGL and PHEO on the basis of biochemical tests. We observed an increase of TH mRNA concentration without correlation with TH protein expression in primary cell culture of PHEO and PGL incubated with dexamethasone during 24 hours; no changes were monitored for PNMT and DBH at both mRNA and protein level in PHEO and PGL. Altogether, these results indicate that long term CAT synthesis is not driven by the close environment where the tumor develops and suggest that GC alone is not sufficient to regulate CAT synthesis pathway in PHEO/PGL.

Role of CYP2E1-mediated metabolism in the acute and vestibular toxicities of nineteen nitriles in the mouse

Allylnitrile, cis-crotononitrile, and 3,3 -iminodipropionitrile are known to cause vestibular toxicity in rodents, and evidence is available indicating that cis-2-pentenenitrile shares this effect. We evaluated nineteen nitriles for vestibular toxicity in wild type (129S1) and CYP2E1-null mice, including all the above, several neurotoxic nitriles, and structurally similar nitriles. A new acute toxicity test protocol was developed to facilitate evaluation of the vestibular toxicity by a specific behavioral test battery at doses up to sub-lethal levels while using a limited number of animals. A mean number of 8.5±0.3 animals per nitrile, strain and sex was necessary to obtain evidence of vestibular toxicity and optionally an estimation of the lethal dose. For several but not all nitriles, lethal doses significantly increased in CYP2E1-null mice. The protocol revealed the vestibular toxicity of five nitriles, including previously identified ototoxic compounds and one nitrile (trans-crotononitrile) known to have a different profile of neurotoxic effects in the rat. In all five cases, both sexes were affected and no decrease in susceptibility was apparent in CYP2E1-null mice respect to 129S1 mice. Fourteen nitriles caused no vestibular toxicity, including six nitriles tested in CYP2E1-null mice at doses significantly larger than the maximal doses that can be tested in wild type animals. We conclude that only a subset of low molecular weight nitriles is toxic to the vestibular system, that species-dependent differences exist in this vestibular toxicity, and that CYP2E1-mediated metabolism is not involved in this effect of nitriles although it has a role in the acute lethality of some of these compounds

BS-SEM evaluation of the tisular interactions between cortical bone and calcium-phosphate covered titanium implants

The improvement of the reliability of the contact between the osseous tissues and the implant materials has been tested by recovering the metallic implants with ceramic materials, usually calcium phosphates. In our study, the calcium phosphate recovering layers were deposited by means of a pulsed-laser deposition technique. Our aim was to to evaluate the tissue interactions established between cortical bone and titanium implants covered by five different layers, ranging from amorphous calcium phosphate to crystalline hydroxyapatite, obtained by altering the parameters of the laser ablation process. The surgical protocol of the study consisted in the simultaneous implantation of the five types of implants in both the tibial dyaphisis of three Beagle dogs, sacrificed respectively one, two and three months after the last surgical procedures. After the sacrifice, the samples were submitted to a scheduled procedure of embedding in plastic polymers without prior decalcification, in order to perform the ultrastructural studies: scanning microscopy with secondary and backscattered electrons (BS-SEM). Our observations show that both in terms of the calcified tissues appearing as a response to the presence of the different coatings and of time of recovering, the implants coated with crystalline calcium phosphate layers by laser ablation present a better result than the amorphous-calcium-phosphate-coated implants. Moreover, the constant presence of chondroid tissue, related with the mechanical induction by forces applied on the recovering area, strongly suggests that the mechanisms implied in osteointegration are related to endomembranous, rather than endochondral ossification processes

A radiochemical method for carbamoyl-phosphate synthetase I: application to rats fed a hyperproteic diet

A method for the measurement of carbamoyl-phosphate synthetase I activity in animal tissues has been developed using the livers of rats under normal and hyperproteic diets. The method is based on the incorporation of 14C-ammonium bicarbonate to carbamoyl-phosphate in the presence of ATP-Mg and N-acetyl-glutamate. The reaction is stopped by chilling, lowering the pH and adding ethanol. Excess bicarbonate is flushed out under a gentle stream of cold CO2. The only label remaining in the medium was that incorporated into carbamoyl-phosphate, since all 14C-CO2 from bicarbonate was eliminated. The method is rapid and requires only a low pressure supply of CO2 to remove the excess substrate. The reaction is linear up to 10 min using homogenate dilutions of 1:20 to 1:200 (w/v). Rat liver activity was in the range of 89±8 nkat/g. Hyperproteic diet resulted in a significant 1.4-fold increase. The design of the method allows for the processing of multiple samples at the same time, and incubation medium manipulation is unnecessary, since the plastic incubation vial and its contents are finally counted together.