Hugues Henry

Endogenous synthesis and transport of creatine in the rat brain: an in situ hybridization study

Creatine is synthesized from arginine by L-arginine:glycine amidinotransferase (AGAT) and S-adenosyl-L-methionine:N-guanidinoacetate methyltransferase (GAMT) and can be taken up by cells by creatine transporters (CRT). While creatine is mainly synthesized by the liver and the kidney, most of other tissues, including the brain, also express AGAT and GAMT. There is evidence that the permeability of the blood-brain barrier (BBB) for creatine is limited, suggesting that the brain is dependent on its own creatine synthesis. In order to better understand creatine synthesis and transport in the central nervous system (CNS), we studied the regional distribution of cells expressing AGAT, GAMT and the creatine transporter CRT1 in the adult rat brain by non-radioisotopic in situ hybridization. AGAT and GAMT presented an ubiquitous neuronal and glial expression, whereas CRT1 was present in neurons and oligodendrocytes throughout the brain, but not in astrocytes. This indicates that all cells in the CNS can synthesize creatine from arginine. The absence of expression of CRT1 in astrocytes and particularly in those contacting capillary endothelial cells (BBB) reinforces the idea that under normal conditions the creatine used by the brain is synthesized mainly in the CNS. Furthermore, the expression of CRT1 by neurons and oligodendrocytes indicates that creatine trafficking is possible in those brain areas of main creatine consumption.

Creatine synthesis and transport during rat embryogenesis: Spatiotemporal expression of AGAT, GAMT and CT1

BackgroundCreatine (Cr) is synthesized by a two-step mechanism involving arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), and is taken up by cells through a specific Cr transporter, CT1. Recently, genetic defects of this pathway have been described, that lead to Cr deficiency, neurological symptoms in early infancy and severe neurodevelopmental delay. To investigate the involvement of Cr synthesis and uptake pathways during embryonic development, we determined the spatiotemporal expression of AGAT, GAMT and CT1 during the rat embryogenesis, at the mRNA and protein level.ResultsWe show that AGAT and GAMT are expressed in hepatic primordium as soon as 12.5 days, then progressively acquire their adult pattern of expression, with high levels of AGAT in kidney and pancreas, and high levels of GAMT in liver and pancreas. AGAT and CT1 are prominent in CNS, skeletal muscles and intestine, where they appear earlier than GAMT. High levels of CT1 are found in epithelia.ConclusionOur results suggest that de novo synthesis of Cr by AGAT and GAMT, as well as cellular Cr uptake by CT1, are essential during embryonic development. This work provides new clues on how creatine can be provided to developing tissues, and suggests that Cr deficiencies might induce irreversible damages already in utero, particularly on the nervous system.

Systems Genetics of Metabolism: The Use of the BXD Murine Reference Panel for Multiscalar Integration of Traits

Metabolic homeostasis is achieved by complex molecular and cellular networks that differ significantly among individuals and are difficult to model with genetically engineered lines of mice optimized to study single gene function. Here, we systematically acquired metabolic phenotypes by using the EUMODIC EMPReSS protocols across a large panel of isogenic but diverse strains of mice (BXD type) to study the genetic control of metabolism. We generated and analyzed 140 classical phenotypes and deposited these in an open-access web service for systems genetics (www.genenetwork.org). Heritability, influence of sex, and genetic modifiers of traits were examined singly and jointly by using quantitative-trait locus (QTL) and expression QTL-mapping methods. Traits and networks were linked to loci encompassing both known variants and novel candidate genes, including alkaline phosphatase (ALPL), here linked to hypophosphatasia. The assembled and curated phenotypes provide key resources and exemplars that can be used to dissect complex metabolic traits and disorders.

Cloning and Characterization of Helicobacter pyloriSuccinyl CoA:Acetoacetate CoA-transferase, a Novel Prokaryotic Member of the CoA-transferase Family

Sequencing of a fragment of Helicobacter pylori genome led to the identification of two open reading frames showing striking homology with Coenzyme A (CoA) transferases, enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The genes were present in all H. pyloristrains tested by polymerase chain reaction or slot blotting but not inCampylobacter jejuni. Genes for the putative A and B subunits of H. pylori CoA-transferase were introduced into the bacterial expression vector pKK223-3 and expressed inEscherichia coli JM105 cells. Amino acid sequence comparisons, combined with measurements of enzyme activities using different CoA donors and acceptors, identified the H. pylori CoA-transferase as a succinyl CoA:acetoacetate CoA-transferase. This activity was consistently observed in differentH. pylori strains. Antibodies raised against either recombinant A or B subunits recognized two distinct subunits ofM r ∼26,000 and 24,000 that are both necessary for H. pylori CoA-transferase function. The lack of α-ketoglutarate dehydrogenase and of succinyl CoA synthetase activities indicates that the generation of succinyl CoA is not mediated by the tricarboxylic acid cycle in H. pylori. We postulate the existence of an alternative pathway where the CoA-transferase is essential for energy metabolism.

Ammonium-Induced Impairment of Axonal Growth Is Prevented through Glial Creatine

Hyperammonemia in neonates and infants affects brain development and causes mental retardation. We report that ammonium impaired cholinergic axonal growth and altered localization and phosphorylation of intermediate neurofilament protein in rat reaggregated brain cell primary cultures. This effect was restricted to the phase of early maturation but did not occur after synaptogenesis. Exposure to NH4Cl decreased intracellular creatine, phosphocreatine, and ADP. We demonstrate that creatine cotreatment protected axons from ammonium toxic effects, although this did not restore high-energy phosphates. The protection by creatine was glial cell-dependent. Our findings suggest that the means to efficiently sustain CNS creatine concentration in hyperammonemic neonates and infants should be assessed to prevent impairment of axonogenesis and irreversible brain damage.

Creatine transporters: A reappraisal

Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na+/Cl−-dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain.In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (α-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the α-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and α-ketoglutarate dehydrogenase (α-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our α-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is also supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of ~ 60 kDa was enriched This latter is most likely representing the genuine plasma membrane CrT.Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehydrogenases, further research is required to characterise on a biochemical/biophysical level the CrT polypeptides, e.g. to determine whether the ~ 60 kDa polypeptide is indeed a bona-fide CrT and to identify the mitochondrial transporter that is able to facilitate Cr-uptake into these organelles. Therefore, the anti-CrT antibodies available so far should only be used with these precautions in mind. This holds especially true for quantitation of CrT polypeptides by Western blots, e.g. when trying to answer whether CrTs are up- or down-regulated by certain experimental interventions or under pathological conditions.In conclusion, we still hold to the scheme that besides the high-affinity and high-efficiency plasmalemma CrT there exists an additional low affinity high Km Cr uptake mechanism in mitochondria. However, the exact biochemical nature of this mitochondrial creatine transport, still remains elusive. Finally, similar to the creatine kinase (CK) isoenzymes, which are specifically located at different cellular compartments, also the substrates of CK are compartmentalized in cytosolic and mitochondrial pools. This is in line with 14C-Cr-isotope tracer studies and a number of [31P]-NMR magnetization transfer studies, as well as with recent [1H]-NMR spectroscopy data.

AGAT, GAMT and SLC6A8 distribution in the central nervous system, in relation to creatine deficiency syndromes: a review.

SummaryCreatine deficiency syndromes, either due to AGAT, GAMT or SLC6A8 deficiencies, lead to a complete absence, or a very strong decrease, of creatine within the brain, as measured by magnetic resonance spectroscopy. While the mammalian central nervous system (CNS) expresses AGAT, GAMT and SLC6A8, the lack of SLC6A8 in astrocytes around the blood–brain barrier limits the brain capacity to import creatine from the periphery, and suggests that the CNS has to rely mainly on endogenous creatine synthesis through AGAT and GAMT expression. This seems contradictory with SLC6A8 deficiency, which, despite AGAT and GAMT expression, also leads to creatine deficiency in the CNS. We present novel data showing that in cortical grey matter, AGAT and GAMT are expressed in a dissociated way: e.g. only a few cells co-express both genes. This suggests that to allow synthesis of creatine within the CNS, at least for a significant part of it, guanidinoacetate must be transported from AGAT- to GAMT-expressing cells, possibly through SLC6A8. This would explain the creatine deficiency observed in SLC6A8-deficient patients. By bringing together creatine deficiency syndromes, AGAT, GAMT and SLC6A8 distribution in CNS, as well as a synthetic view on creatine and guanidinoacetate levels in the brain, this review presents a comprehensive framework, including new hypotheses, on brain creatine metabolism and transport, both in normal conditions and in case of creatine deficiency.

Creatine deficiency syndromes and the importance of creatine synthesis in the brain.

Creatine deficiency syndromes, due to deficiencies in AGAT, GAMT (creatine synthesis pathway) or SLC6A8 (creatine transporter), lead to complete absence or very strong decrease of creatine in CNS as measured by magnetic resonance spectroscopy. Brain is the main organ affected in creatine-deficient patients, who show severe neurodevelopmental delay and present neurological symptoms in early infancy. AGAT- and GAMT-deficient patients can be treated by oral creatine supplementation which improves their neurological status, while this treatment is inefficient on SLC6A8-deficient patients. While it has long been thought that most, if not all, of brain creatine was of peripheral origin, the past years have brought evidence that creatine can cross blood–brain barrier, however, only with poor efficiency, and that CNS must ensure parts of its creatine needs by its own endogenous synthesis. Moreover, we showed very recently that in many brain structures, including cortex and basal ganglia, AGAT and GAMT, while found in every brain cell types, are not co-expressed but are rather expressed in a dissociated way. This suggests that to allow creatine synthesis in these structures, guanidinoacetate must be transported from AGAT- to GAMT-expressing cells, most probably through SLC6A8. This new understanding of creatine metabolism and transport in CNS will not only allow a better comprehension of brain consequences of creatine deficiency syndromes, but will also contribute to better decipher creatine roles in CNS, not only in energy as ATP regeneration and buffering, but also in its recently suggested functions as neurotransmitter or osmolyte.

Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry--a method comparison and validation

RATIONALE The aim of the work was to develop and validate a method for the quantification of vitamin D metabolites in serum using ultra-high-pressure liquid chromatography coupled to mass spectrometry (LC/MS), and to validate a high-resolution mass spectrometry (LC/HRMS) approach against a tandem mass spectrometry (LC/MS/MS) approach using a large clinical sample set. METHODS A fast, accurate and reliable method for the quantification of the vitamin D metabolites, 25-hydroxyvitamin D2 (25OH-D2) and 25-hydroxyvitamin D3 (25OH-D3), in human serum was developed and validated. The C3 epimer of 25OH-D3 (3-epi-25OH-D3) was also separated from 25OH-D3. The samples were rapidly prepared via a protein precipitation step followed by solid-phase extraction (SPE) using an HLB μelution plate. Quantification was performed using both LC/MS/MS and LC/HRMS systems. RESULTS Recovery, matrix effect, inter- and intra-day reproducibility were assessed. Lower limits of quantification (LLOQs) were determined for both 25OH-D2 and 25OH-D3 for the LC/MS/MS approach (6.2 and 3.4 µg/L, respectively) and the LC/HRMS approach (2.1 and 1.7 µg/L, respectively). A Passing & Bablok fit was determined between both approaches for 25OH-D3 on 662 clinical samples (1.11 + 1.06x). It was also shown that results can be affected by the inclusion of the isomer 3-epi-25OH-D3. CONCLUSIONS Quantification of the relevant vitamin D metabolites was successfully developed and validated here. It was shown that LC/HRMS is an accurate, powerful and easy to use approach for quantification within clinical laboratories. Finally, the results here suggest that it is important to separate 3-epi-25OH-D3 from 25OH-D3.

Dissociation of AGAT, GAMT and SLC6A8 in CNS: Relevance to creatine deficiency syndromes

AGAT and GAMT, the two enzymes of the creatine synthesis pathway, are well expressed within CNS, suggesting autonomous brain creatine synthesis. This contradicts SLC6A8 deficiency, which causes creatine deficiency despite CNS expression of AGAT and GAMT. We hypothesized that AGAT and GAMT were not co-expressed by brain cells, and that guanidinoacetate must be transported between cells to allow creatine synthesis. We finely analyzed the cell-to-cell co-expression of AGAT, GAMT and SLC6A8 in various regions of rat CNS, and showed that in most structures, cells co-expressing AGAT+GAMT (equipped for autonomous creatine synthesis) were in low proportions (<20%). Using reaggregating brain cell cultures, we also showed that brain cells take up guanidinoacetate and convert it to creatine. Guanidinoacetate uptake was competed by creatine. This suggests that in most brain regions, guanidinoacetate is transported from AGAT- to GAMT-expressing cells through SLC6A8 to allow creatine synthesis, thereby explaining creatine deficiency in SLC6A8-deficient CNS.