W T Labruyère

University of Amsterdam, Amsterdam, North Holland, Netherlands

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Publications (20)80.22 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Starvation elicits a complex adaptive response in an organism. No information on transcriptional regulation of metabolic adaptations is available. We, therefore, studied the gene expression profiles of brain, small intestine, kidney, liver, and skeletal muscle in mice that were subjected to 0-72 h of fasting. Functional-category enrichment, text mining, and network analyses were employed to scrutinize the overall adaptation, aiming to identify responsive pathways, processes, and networks, and their regulation. The observed transcriptomics response did not follow the accepted "carbohydrate-lipid-protein" succession of expenditure of energy substrates. Instead, these processes were activated simultaneously in different organs during the entire period. The most prominent changes occurred in lipid and steroid metabolism, especially in the liver and kidney. They were accompanied by suppression of the immune response and cell turnover, particularly in the small intestine, and by increased proteolysis in the muscle. The brain was extremely well protected from the sequels of starvation. 60% of the identified overconnected transcription factors were organ-specific, 6% were common for 4 organs, with nuclear receptors as protagonists, accounting for almost 40% of all transcriptional regulators during fasting. The common transcription factors were PPARα, HNF4α, GCRα, AR (androgen receptor), SREBP1 and -2, FOXOs, EGR1, c-JUN, c-MYC, SP1, YY1, and ETS1. Our data strongly suggest that the control of metabolism in four metabolically active organs is exerted by transcription factors that are activated by nutrient signals and serves, at least partly, to prevent irreversible brain damage.
    Journal of Biological Chemistry 03/2011; 286(18):16332-43. · 4.65 Impact Factor
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    ABSTRACT: The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a approximately 3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the approximately 4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was approximately 5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify approximately 2.5 micromol ammonia/g muscle.h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.
    Journal of Biological Chemistry 03/2010; 285(13):9516-24. · 4.65 Impact Factor
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    ABSTRACT: The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a ∼3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the ∼4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was ∼5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify ∼2.5 μmol ammonia/g muscle·h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.
    Journal of Biological Chemistry 03/2010; 285(13):9516-9524. · 4.65 Impact Factor
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    ABSTRACT: Glutamine synthetase (GS) is a key enzyme in the "glutamine-glutamate cycle" between astrocytes and neurons, but its function in vivo was thus far tested only pharmacologically. Crossing GS(fl/lacZ) or GS(fl/fl) mice with hGFAP-Cre mice resulted in prenatal excision of the GS(fl) allele in astrocytes. "GS-KO/A" mice were born without malformations, did not suffer from seizures, had a suckling reflex, and did drink immediately after birth, but then gradually failed to feed and died on postnatal day 3. Artificial feeding relieved hypoglycemia and prolonged life, identifying starvation as the immediate cause of death. Neuronal morphology and brain energy levels did not differ from controls. Within control brains, amino acid concentrations varied in a coordinate way by postnatal day 2, implying an integrated metabolic network had developed. GS deficiency caused a 14-fold decline in cortical glutamine and a sevenfold decline in cortical alanine concentration, but the rising glutamate levels were unaffected and glycine was twofold increased. Only these amino acids were uncoupled from the metabolic network. Cortical ammonia levels increased only 1.6-fold, probably reflecting reduced glutaminolysis in neurons and detoxification of ammonia to glycine. These findings identify the dramatic decrease in (cortical) glutamine concentration as the primary cause of brain dysfunction in GS-KO/A mice. The temporal dissociation between GS(fl) elimination and death, and the reciprocal changes in the cortical concentration of glutamine and alanine in GS-deficient and control neonates indicate that the phenotype of GS deficiency in the brain emerges coincidentally with the neonatal activation of the glutamine-glutamate and the associated alanine-lactate cycles.
    Glia 02/2010; 58(6):741-54. · 5.07 Impact Factor
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    ABSTRACT: Glutamine synthetase (GS) is expressed at various levels in a wide range of tissues, suggesting that a complex network of modules regulates its expression. We explored the interactions between the upstream enhancer, regulatory regions in the first intron, and the 3'-untranslated region and immediate downstream genomic sequences of the GS gene (the GS "tail"), and compared the results with those obtained previously in conjunction with the bovine growth hormone (bGH) tail. The statistical analysis of these interactions revealed that the GS tail was required for full enhancer activity of the combination of the upstream enhancer and either the middle or the 3'-intron element. The GS tail also prevented a productive interaction between the upstream enhancer and the 5'-intron element, whereas the bGH tail did not, suggesting that the 5'-intron element is a regulatory element that needs to be silenced for full GS expression. Using the CMV promoter/enhancer and transfection experiments, we established that the 2.8 kb GS mRNA polyadenylation signal is approximately 10-fold more efficient than the 1.4 kb mRNA signal. Because the steady-state levels of both mRNAs are similar, the intervening conserved elements destabilize the long mRNA. Indeed, one but not all constructs containing these elements had a shorter half life in FTO-2B cells. A construct containing only 300 bases before and 100 bases after the 2.8 kb mRNA polyadenylation site sufficed for maximal expression. A stretch of 21 adenines inside this fragment conferred, in conjunction with the upstream enhancer and the 3'-part of the first intron, sensitivity of GS expression to ambient glutamine.
    Biochimie 10/2006; 88(9):1255-64. · 3.14 Impact Factor
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    ABSTRACT: The expression of glutamine synthetase (GS) in the mammalian liver is confined to the hepatocytes surrounding the central vein and can be induced in cultures of periportal hepatocytes by co-cultivation with the rat-liver epithelial cell line RL-ET-14. We exploited these observations to identify the regulatory regions of the GS gene and the responsible signal-transduction pathway that mediates this effect. Fetal hepatocytes of wild-type or GS-transgenic mice were co-cultured with RL-ET-14 cells to induce GS expression. Small-interfering RNA was employed to silence beta-catenin expression in the fetal hepatocytes prior to co-culture. Co-cultivation of RL-ET-14 cells with fetal mouse hepatocytes induced GS expression 4.2-fold. The expression of another pericentral enzyme, ornithine aminotransferase and a periportal enzyme, carbamoylphosphate synthetase, were not affected. Co-culture of RL-ET-14 cells with transgenic fetal mouse hepatocytes demonstrated that GS expression was induced via its upstream enhancer located at -2.5 kb and that the signal mediator required a functional beta-catenin pathway. The 'RL-ET-14' factor specifically induces GS expression, working via its upstream enhancer in a beta-catenin-dependent fashion.
    Journal of Hepatology 08/2005; 43(1):126-31. · 9.86 Impact Factor
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    ABSTRACT: The pronounced increase in the protein/mRNA ratio of ammonia-metabolising enzymes in rat liver in the last prenatal week represents a clear example of a post-transcriptional level of control of gene expression. Both the underlying mechanism, namely an increase in translational efficiency of the mRNA and/or enhanced stability of the protein, and its importance for perinatal adaptation are unknown. We investigated this process in spiny mouse liver, because the comparison of rat and spiny mouse can discriminate adaptively from developmentally regulated processes in the perinatal period. We focused on glutamine synthetase (GS) because of the conveniently small size of its mRNA. Prenatally, GS enzyme activity slowly accumulated to approximately 1.3 U x g-1 liver at birth and postnatally more rapidly to 5.5 U x g-1 at 2 weeks. Both phases of enzyme accumulation obeyed exponential functions. Western-blot analysis showed that changes in GS activity reflected changes in GS protein content. GS mRNA content of the liver was 45 fmol x g-1 at 2 weeks before birth and slowly declined to approximately 25 fmol x g-1 at 2 weeks after birth. The GS protein/mRNA ratio increased 2.5-fold prenatally and sixfold postnatally. Analysis of prenatal and postnatal polysome profiles revealed no evidence of GS mRNA-containing ribonucleoprotein particles. Instead, GS mRNAs were (fully) occupied by 12 ribosomes, indicating regulation at the level of elongation. The kinetics of GS protein accumulation, in conjunction with GS mRNA content, are consistent with an approximately sixfold increase in its rate of synthesis at birth as the result of a corresponding stimulation of the rate of elongation.
    European Journal of Biochemistry 07/1999; 262(3):803-9. · 3.58 Impact Factor
  • Contributions to nephrology 02/1997; 121:25-30. · 1.49 Impact Factor
  • Contributions To Nephrology - CONTRIB NEPHROL. 01/1997; 121:25-30.
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    ABSTRACT: In previous studies of the glutamine synthetase gene, the promoter and two enhancer elements, one in the upstream region and one within the first intron, were identified. To analyze the role of the far-upstream enhancer element in the regulation of the expression of the glutamine synthetase gene, two classes of transgenic mice were generated. In GSK mice, the basal promoter directs the expression of the chloramphenicol acetyltransferase reporter gene. In GSL mice reporter gene expression is driven, in addition, by the upstream regulatory region, including the far-upstream enhancer. Whereas chloramphenicol acetyltransferase expression was barely detectable in GSK mice, high levels were detected in GSL mice. By comparing chloramphenicol acetyltransferase expression with that of endogenous glutamine synthetase in GSL mice, three groups of organs were distinguished in which the effects of the upstream regulatory region on the expression of glutamine synthetase were quantitatively different. The chloramphenicol acetyltransferase mRNA in the GSL mice was shown to be localized in the pericentral hepatocytes of the liver. The developmental changes in chloramphenicol acetyltransferase enzyme activity in the liver were similar to those in endogenous glutamine synthetase. These results show that the upstream region is a major determinant for three characteristics of glutamine synthetase expression: its organ specificity, its pericentral expression pattern in the liver, and its developmental appearance in the liver.
    Journal of Biological Chemistry 12/1995; 270(47):28251-6. · 4.65 Impact Factor
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    ABSTRACT: Carbamoylphosphate synthetase I (CbmPS) is first expressed in rat hepatocytes shortly before birth. After birth, expression of CbmPS gradually becomes confined to the hepatocytes surrounding the portal veins. To obtain insight into the spatiotemporal regulation of its expression, the rat CbmPS gene was isolated and characterized. The gene is 110 kb in length and contains 38 exons. The basal promoter comprises the first 161 nucleotides upstream of the transcription-initiation site. Determination of the state of methylation of the 5' portion of the gene identified a CCGG sequence at -6.3 kb that is selectively demethylated in adult tissues which express CbmPS. This site remains methylated before birth, however, despite recruitment of all hepatocytes for CbmPS synthesis, indicating that its demethylation is a consequence of rather than a condition for expression of CbmPS. Transient expression assays revealed that the region surrounding the CCGG site at 6.3 kb functions as an enhancer. In FTO-2B hepatoma cells and Rat-1 fibroblasts, this enhancer is constitutively active when tested in front of the basal viral thymidine kinase promoter. When tested in front of the basal CbmPS promoter in hepatoma cells, however, the activity of this enhancer is dependent on the presence of glucocorticoids. In Rat-1 fibroblasts, the presence of both glucocorticoids and cyclic AMP is required for full activity, suggesting that the hepatocyte-specific expression of CbmPS is related to tissue-specific differences in the sensitivity to cyclic AMP. Matrix-attachment regions (MAR) are present upstream and downstream of the CbmPS gene. The downstream MAR defines the 3' boundary of the gene. The upstream MAR is located midway between the basal promoter and the enhancer, and may function as a hinge point to facilitate the positioning of the enhancer in the vicinity of the basal promoter.
    European Journal of Biochemistry 04/1995; 228(2):351-61. · 3.58 Impact Factor
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    ABSTRACT: Introduction of foreign molecules, such as DNA, RNA, or proteins, into living cells is a powerful means to test the biological functions of these molecules. One of the techniques by which foreign molecules can be introduced into living cells is electroporation (for reviews, see refs. 1,2). Compared to other techniques for the introduction of molecules into living cells, electroporation does not seem to cause prolonged alterations in the biological structure and function of the target cells. Nevertheless, the pores that are induced by the electroporation technique (3–5) can be visualized by rapid-freezing electron microscopy techniques (6) and allow a direct contact between the cytoplasm of the cells and the electroporation medium. The components of the electroporation medium can, therefore, penetrate into the cell interior as long as the pores exist (7). In this perspective, it is obvious that cells are exquisitely sensitive to the composition of the electroporation medium (8–10) and that an electroporation medium that resembles the composition of the cytoplasm would probably enhance the viability of the cells after electroporation.
    Methods in molecular biology (Clifton, N.J.) 02/1995; 48:185-97. · 1.29 Impact Factor
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    Nucleic Acids Research 11/1993; 21(21):4987-8. · 8.81 Impact Factor
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    ABSTRACT: Hepatic glutamine synthetase (GS) shows a unique expression pattern limited to a few hepatocytes surrounding the terminal hepatic veins. Starting from the genomic clone of the rat GS gene, lambda GS1 [Van de Zande, L. P. G. W., Labruyère, W. T., Arnberg, A. C., Wilson, R. H., Van den Bogaert, A. J. W., Das, A. T., Frijters, C., Charles, R., Moorman, A. F. M. & Lamers, W. H. (1990) Gene (Amst.) 87, 225-232] additional genomic clones containing up to 9 kb of 5'flanking region were isolated in order to characterize cis-acting elements involved in the regulation of GS expression. Sequence analysis of the 5'flanking region up to -2520 bp revealed a putative AP2-binding site at -223 bp and a second GC box at -2343 bp in addition to the canonical TATA, CCAAT and GC boxes found proximal to the transcription-start site. A possible negative glucocorticoid-responsive element (GRE) and regions with very weak similarity to a GRE and to a known silencer element were noted at -506 bp, -406 bp and at -798 bp, respectively. Within the sequenced part of the 5'flanking region no known regulatory elements associated with liver-specific gene expression were found except for a putative HNF3-binding site at -896 bp. Functional analysis by transient transfection assays using constructs with the pSSCAT or the pXP1 vector revealed that the elements present within the first 153 bp and particularly the first 368 bp of upstream sequence constitute an active promoter the activity of which is decreased by additional sequences up to -2148 bp. The presence of dexamethasone led to a 2-4-fold increase in the promoter activity of all these constructs. Using the heterologous truncated thymidine-kinase-gene promoter of the plasmid pT81-luc a strong enhancer element was located between -2520 bp and -2148 bp. Its activity was not affected by dexamethasone but was negatively influenced by flanking sequences in both directions. This enhancer was also effective with the homologous GS promoter (-153 to +59 bp) and the heterologous full thymidine-kinase-gene promoter (pT109luc). No further enhancers were found up to -6200 bp. Using the same approach, a second enhancer was found between +259 bp and +950 bp within the first intron. Deoxyribonuclease-I hypersensitivity studies confirmed the presence of a hypersensitive site between +350 bp and +550 bp and suggested a second site between +850 bp and +1200 bp.(ABSTRACT TRUNCATED AT 400 WORDS)
    European Journal of Biochemistry 05/1993; 213(3):1067-73. · 3.58 Impact Factor
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    Nucleic Acids Research 12/1990; 18(21):6464. · 8.81 Impact Factor
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    ABSTRACT: The expression patterns of the mRNAs for the ammonia-metabolizing enzymes carbamoylphosphate synthetase (CPS), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in developing pre- and neonatal rat liver by in situ hybridization. In the period of 11 to 14 embryonic days (ED) the concentrations of GS and GDH mRNA increases rapidly in the liver, whereas a substantial rise of CPS mRNA in the liver does not occur until ED 18. Hepatocyte heterogeneity related to the vascular architecture can first be observed at ED 18 for GS mRNA, at ED 20 for GDH mRNA and three days after birth for CPS mRNA. The adult phenotype is gradually established during the second neonatal week, i.e. GS mRNA becomes confined to a pericentral compartment of one to two hepatocytes thickness, CPS mRNA to a large periportal compartment being no longer expressed in the pericentral compartment and GDH mRNA is expressed over the entire porto-central distance, decreasing in concentration going from central to portal. Comparison of the observed mRNA distribution patterns in the perinatal liver, with published data on the distribution of the respective proteins, points to the occurrence of posttranslational, in addition to pretranslational control mechanisms in the period of ontogenesis of hepatocyte heterogeneity. Interestingly, during development all three mRNAS are expressed outside the liver to a considerable extent and in a highly specific way, indicating that several organs are involved in the developmentally regulated expression of the mRNAs for the ammonia-metabolizing enzymes, that were hitherto not recognized as such.
    The Histochemical Journal 10/1990; 22(9):457-68.
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    ABSTRACT: From a rat genomic library in phage λCharon4A, a complete glutamine synthetase-encoding gene was isolated. The gene is 9.5–10 kb long, consists of seven exons, and codes for two mRNA species of 1375 nucleotides (nt) and 2787 nt, respectively. For both mRNAs, full-lenght cDNAs containing a short poly(A) tract were identified. The sequences of the entire mRNA and of the exon-intron transitions were determined. The smaller mRNA is identical to the 5′ 1375 nt of the long mRNA and contains the entire protein-coding region. The position of the transcription start point was mapped. Within the first 118 bp of promoter sequence, a (T)ATAA-box, a CCAAT-box and an SP1-binding site were identified.
    Gene 04/1990; · 2.20 Impact Factor
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    ABSTRACT: The distribution pattern of creatine kinase (E.C 2.7.3.2) isozymes in prenatal rat heart and skeletal muscle was studied by immunohistochemistry. Between embryonic day (ED) 12-18, creatine kinase M (CK-M) is heterogeneously expressed in the heart: a pronounced staining of CK-M is first observed in the outflow tract and the trabeculae of the right ventricle (ED12-14), and subsequently in the venous valves, the interatrial septum and the sinoatrial node. From ED18 onwards, a homogeneous expression of CK-M is observed due to an increase in isozyme concentration in the remaining part of the myocardium. By contrast, the developmental appearance of creatine kinase B (CK-B) occurs almost homogeneously throughout the heart between ED11-14. Thereafter, a decrease of the CK-B is first observed in the inflow tract (in particular in the sinoatrial node), in the inner part of those atrial walls that are adjacent to the atrioventricular junction, and temporarily in a band in the upper part of the interventricular septum. From ED18, a selective disappearance of CK-B is found in the papillary muscle of the left ventricle. At birth, a considerable amount of CK-B remains present in the ventricular walls. Although some of the stage-dependent regional differences in expression of the creatine kinase isozymes, in particular those of the M-subunit, are shared by other mammalian and avian species, their significance for the developmental changes in the physiology of the heart is speculative at present.
    Anatomy and Embryology 02/1990; 182(2):195-203. · 1.42 Impact Factor
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    Nucleic Acids Research 09/1988; 16(15):7726. · 8.81 Impact Factor
  • Physical Review B - PHYS REV B. 01/1988; 16(15):7726-7726.

Publication Stats

273 Citations
80.22 Total Impact Points

Institutions

  • 1988–2010
    • University of Amsterdam
      • • Faculty of Medicine AMC
      • • Department of Anatomy and Embryology
      Amsterdam, North Holland, Netherlands
  • 1993
    • University of Tuebingen
      Tübingen, Baden-Württemberg, Germany
  • 1990
    • University of Groningen
      Groningen, Groningen, Netherlands
    • Academisch Medisch Centrum Universiteit van Amsterdam
      • Department of Anatomy, Embryology and Physiology
      Amsterdam, North Holland, Netherlands