Abstract
Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15 % of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90 % reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.
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Acknowledgments
We thank the Division of Clinical Chemistry and Biochemistry of the University Children’s Hospital for determination of L-Phe from dried blood spots and routine clinical chemistry parameters, the animal facilities of the university hospital for cooperativity, and F. H. Sennhauser for continuous support. We thank Ann-Elisabeth Schwarz, Anke Bettenbrock, and Elfi Holupirek for expert technical assistance. We are grateful for financial support by the ZNZ PhD program of the University of Zurich (BT), the Swiss National Science Foundation (NB & BT), the Hartmann Müller Stiftung (BT) and Stiftung für wissenschaftliche Forschung University of Zürich (Baumgarten Stiftung; BT), and the Novartis “Stiftung für medizinisch-biologische Forschung” (BT). GMC researchers were funded by the German Federal Ministry of Education and Research Infrafrontier grant (01KX1012) and by the German Diabetes Research Center (DZD e.V.), and JB by the Helmholtz Alliance ICEMED.
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All institutional and national guidelines for the care and use of laboratory animals were followed. Animal experiments were carried out in accordance with the guidelines and policies of the State Veterinary Office of Zurich and Swiss law on animal protection, the Swiss Federal Act on Animal Protection (1978), and the Swiss Animal Protection Ordinance (1981). Animal studies presented here received approval from the Cantonal Veterinary Office, Zurich, and the Cantonal Committee for Animal Experiments, Zurich, Switzerland.
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Communicated by: Gregory Enns
Germaine Korner and Tanja Scherer contributed equally to this work.
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Supplementary Table S4
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Supplementary Fig. S1
Generation of the murine Pts-ki allele. (A) Primary amino acid sequence alignment of human and mouse PTPS, which share 82.1 % sequence identity. The human mutation PTS-p.Arg16Cys (hR16C) and the corresponding mouse mutation Pts-p.Arg15Cys (mR15C), both located in exon 1, are marked with arrows. (B) Schematic representation of genomic structure of the murine Pts wild-type allele (top), the targeting vector pMSY211 including the mR15C mutation (E1’), the p.L16L mutation to destroy the BssSI restriction site, a Pgk-DT-gene-cassette (DT) for negative selection, and a “floxed” Pgk-neo-gene-cassette (PGK neo) for positive selection (middle), and the resulting targeted mutant allele (bottom). (C) Schematic representation of the genotyping concepts for the Pts-wt, Pts-ki, and Pts-ko alleles with genomic DNA and the primer pairs a/b (Pts-ki PCR) and c/d/e (Pts-ko PCR). Pts-ki PCR: primers a and b are located upstream and downstream from exon 1 (E1), respectively. They generate a 730 bp for the wild-type/knock-out alleles and a 751 bp PCR fragment for the knock-in allele (due to additional targeting vector sequence; see C). Digestion with restriction enzyme BssSI, 3 bp downstream of the mR15C-c.43C>T mutation, leads to a 444 bp and a 286 bp fragment for the wild-type/knock-out PCR products. The PCR fragment derived from the Pts-ki allele can not be digested with BssSI because the silent p.L16L/c.48C>G mutation destroys the BssS1-recognition site. The Pts-wt and the Pts-ko alleles cannot be distinguished by the Pts-ki genotyping using primer pair a/b. Pts-ko PCR: genotyping according to our previously published method (Elzaouk et al 2003). Primer c is upstream of exon 2 (E2), primer d is specific for exon 2 and primer e is specific for the lacZ gene. The primer pair c/d results in a wild-type fragment of 287 bp and a knock-in fragment of 316 bp whereas primer pair c/e generates mutant fragment of 355 bp (due to the difference in the Pts-intron 1 sequence between the 129/Ola and C57BL/6 J mice strains; see C). (D) Conventional 2 % agarose gel representative PCR-genotyping for the Pts-ki allele (top; after BssSI digestion) and Pts-ko allele (bottom). (PPT 747 kb)
Supplementary Fig. S2
TH protein expression in brain of Pts-ki/ko mice. Western blot analysis and densitometric quantification of TH in brains from (A) newborn mice (n = 3 Pts-wt/wt, 5 Pts-ko/wt, 24 Pts-ki/wt, and 19 Pts-ki/ko) and (B) young adult animals (n = 3 Pts-wt/wt, 7 Pts-ko/wt, 12 Pts-ki/wt, and 13 Pts-ki/ko); always males and females. For details see also Materials and methods. (PPT 333 kb)
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Korner, G., Scherer, T., Adamsen, D. et al. Mildly compromised tetrahydrobiopterin cofactor biosynthesis due to Pts variants leads to unusual body fat distribution and abdominal obesity in mice. J Inherit Metab Dis 39, 309–319 (2016). https://doi.org/10.1007/s10545-015-9909-6
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DOI: https://doi.org/10.1007/s10545-015-9909-6