Martin B Lee

Publications (8)32.8 Total impact

• Article: Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development.

  Martin B Lee, Megan Kooistra, Baohua Zhang, Sandy Slow, Amanda L Fortier, Timothy A Garrow, Michael Lever, Jacquetta M Trasler, Jay M Baltz
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  ABSTRACT: Methyltransferases are an important group of enzymes with diverse roles that include epigenetic gene regulation. The universal donor of methyl groups for methyltransferases is S-adenosylmethionine (AdoMet), which in most cells is synthesized using methyl groups carried by a derivative of folic acid. Another mechanism for AdoMet synthesis uses betaine as the methyl donor via the enzyme betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), but it has been considered to be significant only in liver. Here, we show that mouse preimplantation embryos contain endogenous betaine; Bhmt mRNA is first expressed at the morula stage; BHMT is abundant at the blastocyst stage but not other preimplantation stages, and BHMT activity is similarly detectable in blastocyst homogenates but not those of two-cell or morula stage embryos. Knockdown of BHMT protein levels and reduction of enzyme activity using Bhmt-specific antisense morpholinos or a selective BHMT inhibitor resulted in decreased development of embryos to the blastocyst stage in vitro and a reduction in inner cell mass cell number in blastocysts. The detrimental effects of BHMT knockdown were fully rescued by the immediate methyl-carrying product of BHMT, methionine. A physiological role for betaine and BHMT in blastocyst viability was further indicated by increased fetal resorption following embryo transfer of BHMT knockdown blastocysts versus control. Thus, mouse blastocysts are unusual in being able to generate AdoMet not only by the ubiquitous folate-dependent mechanism but also from betaine metabolized by BHMT, likely a significant pool of methyl groups in blastocysts.
  Journal of Biological Chemistry 07/2012; 287(39):33094-103. · 4.77 Impact Factor
• Article: SIT1 is a betaine/proline transporter that is activated in mouse eggs after fertilization and functions until the 2-cell stage.

  Mohamed-Kheir Idris Anas, Martin B Lee, Chenxi Zhou, Mary-Anne Hammer, Sandy Slow, Jennifer Karmouch, X Johné Liu, Stefan Bröer, Michael Lever, Jay M Baltz
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  ABSTRACT: Betaine (N,N,N-trimethylglycine) added to culture media is known to substantially improve the development of preimplantation mouse embryos in vitro, and to be imported into 1-cell embryos by a transporter that also accepts proline. Here, we found that the betaine/proline transporter is active in preimplantation mouse embryos only for a short period of development, between the 1- and 2-cell stages. Betaine/proline transport was activated after fertilization, beginning approximately 4 hours post-egg activation and reaching a maximum by approximately 10 hours. One- and 2-cell embryos contained endogenous betaine, indicating that a likely function for the transporter in vivo is the accumulation or retention of intracellular betaine. The appearance of transport activity after egg activation was independent of protein synthesis, but was reversibly blocked by disruption of the Golgi with brefeldin A. We assessed two candidates for the betaine/proline transporter: SIT1 (IMINO; encoded by Slc6a20a) and PROT (Slc6a7). mRNA from both genes was present in eggs and 1-cell embryos. However, when exogenously expressed in Xenopus oocytes, mouse PROT did not transport betaine and had an inhibition profile different from that of the embryonic transporter. By contrast, exogenously expressed mouse SIT1 transported both betaine and proline and closely resembled the embryonic transporter. A morpholino oligonucleotide designed to block translation of SIT1, when present from the germinal vesicle stage, blocked the appearance of betaine transport activity in parthenogenotes. Thus, SIT1 is likely to be a developmentally restricted betaine transporter in mouse preimplantation embryos that is activated by fertilization.
  Development 01/2009; 135(24):4123-30. · 6.60 Impact Factor
• Article: Validation of (1)H NMR spectroscopy as an analytical tool for methylamine metabolites in urine.

  Martin B Lee, Malina K Storer, John W Blunt, Michael Lever
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  ABSTRACT: Methylamines have many metabolic roles and there is an increasing demand for their measurement. Glycine betaine is an important osmolyte, and a reservoir for methyl groups. Proline betaine and trigonelline are important dietary betaines. Trimethylamine, derived from gut flora, is normally converted to trimethylamine oxide but in 'fish odour syndrome' is excreted as TMA. These compounds are all suitable for quantification by (1)H NMR spectroscopy as they all have methyl protons. Urine samples are acidified and (1)H NMR spectra are obtained using presaturation for water suppression. Peak integrals or heights are compared to an internal standard of acetonitrile. Inter- and intra-assay CV's were <5% for TMAO and creatinine, and <10% for the other analytes. Responses were linear from 50 to 1000 microM for all metabolites, and recoveries were > or =97%. Limits of detection using NMR are slightly higher than alternative HPLC assays (15-25 microM). However, sensitivity is adequate for the detection of raised levels in urine, and sample analysis was complete in less than 5 min. (1)H NMR spectroscopy is a convenient, rapid and economical option for the determination of betaines and related compounds in urine in a single analysis.
  Clinica Chimica Acta 04/2006; 365(1-2):264-9. · 2.54 Impact Factor
• Article: The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification.

  Martin B Lee, Lioudmila A Lebedeva, Miyuki Suzawa, Subhagya A Wadekar, Marion Desclozeaux, Holly A Ingraham
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  ABSTRACT: Structural analysis of nuclear receptor subfamily V orphan nuclear receptors suggests that ligand-independent mechanisms must regulate this subclass of receptors. Here, we report that steroidogenic factor 1 (SF-1) and liver receptor homolog 1 are repressed via posttranslational SUMO modification at conserved lysines within the hinge domain. Indeed, mutating these lysines or adding the SUMO isopeptidase SENP1 dramatically increased both native and Gal4-chimera receptor activities. The mechanism by which SUMO conjugation attenuates SF-1 activity was found to be largely histone deacetylase independent and was unaffected by the AF2 corepressor Dax1. Instead, our data suggest that SUMO-mediated repression involves direct interaction of the DEAD-box protein DP103 with sumoylated SF-1. Of potential E3-SUMO ligase candidates, PIASy and PIASxalpha strongly promoted SF-1 sumoylation, and addition of DP103 enhanced both PIAS-dependent receptor sumoylation and SF-1 relocalization to discrete nuclear bodies. Taken together, we propose that DEAD-box RNA helicases are directly coupled to transcriptional repression by protein sumoylation.
  Molecular and Cellular Biology 04/2005; 25(5):1879-90. · 5.53 Impact Factor
• Article: Dimethylglycine supplementation does not affect plasma homocysteine concentrations in pre-dialysis chronic renal failure patients.

  Sandy Slow, David O McGregor, Michael Lever, Martin B Lee, Peter M George, Stephen T Chambers
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  ABSTRACT: To determine whether daily dimethylglycine supplementation affects plasma homocysteine concentrations. A randomized, blinded, crossover design was used. Seven pre-dialysis chronic renal failure patients consumed 400 mg of dimethylglycine or placebo daily for 28 days. Fasting blood samples and 12-h urine samples were collected at baseline and at the end of each treatment period for analysis. No significant differences were observed in plasma homocysteine (P = 0.624), glycine betaine (P = 0.452) and methionine (P = 0.457) concentrations between dimethylglycine and placebo treatments. Daily supplementation with dimethylglycine does not affect plasma homocysteine.
  Clinical Biochemistry 12/2004; 37(11):974-6. · 2.08 Impact Factor
• Article: A nuclear-magnetic-resonance-based assay for betaine-homocysteine methyltransferase activity.

  Martin B Lee, John W Blunt, Michael Lever, Peter M George
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  ABSTRACT: Betaine-homocysteine methyltransferase (BHMT) activity can be measured directly and kinetically by (1)H-nuclear magnetic resonance spectroscopy. The disappearance of substrates and the formation of products are monitored simultaneously. Alternative substrates, separately and when mixed with glycine betaine, can also be monitored. Each assay can be completed in 1h. Using 2mM glycine betaine and homocysteine as substrates in 20 mM phosphate buffer (pH 7.5) and measuring the production of N,N-dimethylglycine, the CV is 6.3% (n=6) and the detection limit is 6 nkatal. An endpoint assay for BHMT activity was also developed, by measuring the N,N-dimethylglycine produced after incubation with 2 mM glycine betaine and homocysteine (CV=5.3%, n = 6) with a detection limit of 2 nkatal. These assays were used to show that the natural betaines trigonelline, proline betaine, arsenobetaine, and l-carnitine are neither substrates nor significant inhibitors of rat liver BHMT, that the thetins dimethylthetin and dimethylsulfoniopropionate are substrates and inhibit methyl transfer from glycine betaine, and that the K(m) for glycine betaine is 0.19+/-0.03 mM with a V(max) of 17+/-0.7 nMol min(-1) mg(-1).
  Analytical Biochemistry 08/2004; 330(2):199-205. · 3.00 Impact Factor
• Article: Betaine analogues alter homocysteine metabolism in rats.

  Sandy Slow, Michael Lever, Martin B Lee, Peter M George, Stephen T Chambers
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  ABSTRACT: Glycine betaine supplementation lowers homocysteine levels in homocystinuria and in chronic renal failure patients through methylation catalysed by betaine-homocysteine methyltransferase (BHMT). The aim of this study was to determine the effect of glycine betaine analogues on homocysteine metabolism in Lewis rats. Glycine betaine, proline betaine, trigonelline, dimethylsulfoniopropionate (DMSP) or dimethylthetin (1.5 mmoles) was subcutaneously administered to rats fed a low betaine diet. The effect of each betaine on total plasma homocysteine and urinary and plasma betaine concentrations was monitored for 24h following administration. Baseline plasma homocysteine was 8.5 +/- micromol/l (S.E.M., n=44) and compared to controls concentrations decreased following glycine betaine (0.8+/-0.4 micromol/l, P = 0.064), DMSP (1.0+/-0.5 micromol/l, P = 0.041) and dimethylthetin (1.5 +/- 0.7micromol/l, P = 0.033) treatment, while concentrations increased following proline betaine (2.24 +/-0.7micromol/l, P = 0.002) and trigonelline (1.6 +/-0.3 micromol/l, P < 0.001) treatment. The effect of glycine betaine, DMSP and dimethylthetin on circulating homocysteine concentrations was thought to be mediated by BHMT in vivo. This hypothesis was supported by the finding that circulating glycine betaine concentrations increased following DMSP and dimethylthetin treatment. Proline betaine and trigonelline appeared to be poor BHMT substrates, being largely excreted in the urine unchanged, yet increased circulating homocysteine levels. This suggests they are inhibitors of BHMT. Urinary excretion of glycine betaine increased following treatment with all betaines, suggesting that the resorption of glycine betaine in the kidney was inhibited. The study shows that glycine betaine analogues have multiple effects on homocysteine metabolism (250).
  The International Journal of Biochemistry & Cell Biology 05/2004; 36(5):870-80. · 4.63 Impact Factor
• Article: Requirement of the orphan nuclear receptor SF-1 in terminal differentiation of ventromedial hypothalamic neurons.

  Phu V Tran, Martin B Lee, Oscar Marín, Baoji Xu, Kevin R Jones, Louis F Reichardt, John R Rubenstein, Holly A Ingraham
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  ABSTRACT: The ventromedial hypothalamic nucleus (VMN) is known to mediate autonomic responses in feeding and reproductive behaviors. To date, the most definitive molecular marker for the VMN is the orphan nuclear receptor steroidogenic factor-1 (SF-1). However, it is unclear whether SF-1 functions in the VMN as it does in peripheral endocrine organ development where loss of SF-1 results in organ agenesis due to apoptosis. Here, we provide evidence that SF-1 has a distinct role in later stages of VMN development by demonstrating the persistence of VMN precursors, the misexpression of an early marker (NKX2-1) concomitant with the absence of a late marker (BDNF neurotrophin), and the complete loss of projections to the bed nucleus of stria terminalis and the amygdala in sf-1 null mice. Our findings demonstrate that SF-1 is required for terminal differentiation of the VMN and suggest that transcriptional targets of SF-1 mediate normal circuitry between the hypothalamus and limbic structures in the telencephalon.
  Molecular and Cellular Neuroscience 05/2003; 22(4):441-53. · 3.66 Impact Factor