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Understanding cerebral L-lysine metabolism: the role of L-pipecolate metabolism in Gcdh-deficient mice as a model for glutaric aciduria type I

  • Original Article
  • Published:
Journal of Inherited Metabolic Disease

Abstract

Inherited deficiencies of the L-lysine catabolic pathway cause glutaric aciduria type I and pyridoxine-dependent epilepsy. Dietary modulation of cerebral L-lysine metabolism is thought to be an important therapeutic intervention for these diseases. To better understand cerebral L-lysine degradation, we studied in mice the two known catabolic routes — pipecolate and saccharopine pathways — using labeled stable L-lysine and brain peroxisomes purified according to a newly established protocol. Experiments with labeled stable L-lysine show that cerebral L-pipecolate is generated along two pathways: i) a minor proportion retrograde after ε-deamination of L-lysine along the saccharopine pathway, and ii) a major proportion anterograde after α-deamination of L-lysine along the pipecolate pathway. In line with these findings, we observed only little production of saccharopine in the murine brain. L-pipecolate oxidation was only detectable in brain peroxisomes, but L-pipecolate oxidase activity was low (7 ± 2μU/mg protein). In conclusion, L-pipecolate is a major degradation product from L-lysine in murine brain generated by α-deamination of this amino acid.

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Abbreviations

α-AAA:

α-aminoadipate

α-AASA:

α-aminoadipate semialdehyde

AADAT:

α-aminoadipate aminotransferase

AASDH:

α-aminoadipate semialdehyde dehydrogenase

AASS:

α-aminoadipate semialdehyde synthase

BBB:

blood brain barrier

Clofibrate:

2-(4-chlorophenoxy)-2-methylpropionic acid ethyl ester

D-AAO:

D-amino acid oxidase

DHTKD1:

E1 subunit of an OGDHc-like complex

GA:

glutaric acid

GA-I:

glutaric aciduria type I

GCDH:

glutaryl-CoA dehydrogenase

GC/MS:

gas chromatography mass spectrometry

HMP:

heavy mitochondrial pellet

IF:

intermediate fraction of the cerebral sucrose gradient

IF:

interphase of the liver density gradient

i.p.:

intraperitoneal

LC-MS/MS:

liquid chromatography tandem mass spectrometry

LL:

lower layer of the cerebral sucrose gradient

L-PIPOX:

L-pipecolate oxidase

MPE:

molar percentage excess

N:

nuclear pellet

PBS:

phosphate-buffered saline

PPARα:

peroxisome proliferator-activated receptor-alpha

PMP-70:

peroxisomal membrane protein-70

pPMS:

pooled post mitochondrial supernatant

pPNS:

pooled post nuclear supernatant

PYCR1:

pyrroline-5-carboxylate reductase

P6C:

Δ1-piperideine-6-carboxylate

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

SF:

suspension fraction

(s)LMP:

(suspended) light mitochondrial pellet of the liver

(s)LMP 1 or 2:

(suspended) light mitochondrial pellet 1 or 2 of the brain

TCA-cycle:

tricarboxylic acid-cycle

UL:

upper layer of the cerebral sucrose gradient

UQCRC2:

subunit of the bc 1 complex

3-OH-GA:

3-hydroxyglutaric acid

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Acknowledgments

We thank Christian Körner (Division of Inherited Metabolic Diseases, University Children’s Hospital, Heidelberg, Germany) for his kind gift of a primary antibody, Pamela M. Okun for her valuable comments on the manuscript (Center for Rare Diseases, Medical Center University of Heidelberg, Germany) and Kathrin V. Schmidt as well as Patrik Feyh (Metabolic Laboratory, University Children’s Hospital, Heidelberg, Germany) for their excellent technical assistance. We are grateful to Claus-Dieter Langhans (Metabolic Laboratory, University Children’s Hospital, Heidelberg, Germany) for critical discussion of our manuscript.

Conflict of interest

None.

Animal rights

All institutional and national guidelines for the care and use of laboratory animals were followed.

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Authors and Affiliations

  1. Division of Inherited Metabolic Diseases, University Children’s Hospital, Heidelberg, Germany

    Roland Posset, Silvana Opp, Georg F. Hoffmann, Stefan Kölker, Sven W. Sauer & Jürgen G. Okun

  2. Metabolic Unit, Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands

    Eduard A. Struys

  3. Department of Anatomy and Cell Biology II, University of Heidelberg, Heidelberg, Germany

    Alfred Völkl & Heribert Mohr

Authors
  1. Roland Posset
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  2. Silvana Opp
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  3. Eduard A. Struys
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  6. Georg F. Hoffmann
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  9. Jürgen G. Okun
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Correspondence to Jürgen G. Okun.

Additional information

Communicated by: Robert Steiner

Sven W. Sauer and Jürgen G. Okun contributed equally to the study.

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Supplemental Fig. 1

Fractionation protocol for the isolation of murine liver (a) and brain (b) peroxisomal subpopulations. For further description see materials and methods. N, nuclear pellet; pPNS, pooled post nuclear supernatant; HMP, heavy mitochondrial pellet; pPMS, pooled post mitochondrial supernatant; (s)LMP, (suspended) light mitochondrial pellet of the liver; IF + UL, intermediate fraction and upper layer of the cerebral sucrose gradient; Pellet + LL, pellet and lower layer of the cerebral sucrose gradient; SF, suspension fraction; LMP 1 or 2, light mitochondrial pellet 1 or 2 of the brain; sLMP 2, suspended LMP 2. Buffer A (5 mM MOPS, 0.25 M sucrose, 1 mM EDTA, 0.1 % (v/v) ethanol; pH 7.4); buffer B (5 mM MOPS, 0.85 M sucrose, 1 mM EDTA, 0.1 % (v/v) ethanol; pH 7.4); buffer C (5 mM MOPS, 1 mM EDTA, 0.1 % (v/v) ethanol; pH 7.4); buffer D (10 mM TRIS, 0.25 M sucrose, 1 mM EDTA, 0.1 % (v/v) ethanol; pH 7.4). (PPT 161 kb)

Supplemental Fig. 2

Distribution, purity and enrichment of liver (a) and brain (b) peroxisomes. Liver and brain gradient fractions were separated by SDS-PAGE, proteins blotted to nitrocellulose and investigated with antibodies against PMP-70, catalase, UQCRC2 and Golgi 58 K. Brain peroxisomes banded between fraction 2 to 4 and liver peroxisomes were nearly evenly distributed. The purest brain peroxisomal subpopulation is located in fraction 4, whereas mitochondria-depleted liver peroxisomal subpopulations mostly dispersed between fraction 5 to the interphase (PPTX 94 kb)

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Posset, R., Opp, S., Struys, E.A. et al. Understanding cerebral L-lysine metabolism: the role of L-pipecolate metabolism in Gcdh-deficient mice as a model for glutaric aciduria type I. J Inherit Metab Dis 38, 265–272 (2015). https://doi.org/10.1007/s10545-014-9762-z

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  • DOI: https://doi.org/10.1007/s10545-014-9762-z

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