bioRxiv preprint doi: https://doi.org/10.1101/567297; this version posted March 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Induction of the nicotinamide riboside kinase NAD+ 2 salvage pathway in skeletal muscle of H6PDH KO mice 3 4 Craig L. Doig1,2, Agnieszka E. Zelinska1, Lucy A. Oakey1,2, Rachel S. Fletcher1,2, Yasir El 5 Hassan1,2, Antje Garten1, David Cartwright1,2, Silke Heising1,2, Daniel A Tennant1,2, David 6 Watson3, Jerzy Adamski4 & Gareth G. Lavery1,2. 7 8 1Institute of Metabolism and Systems Research, 2nd Floor IBR Tower, University of 9 Birmingham, Birmingham, B15 2TT, UK. 2Centre for Endocrinology, Diabetes and 10 Metabolism, Birmingham Health Partners, Birmingham, B15 2TH. 3Strathclyde Institute of 11 Pharmacy and Medical Sciences, Hamnett Wing John Arbuthnott Building, Glasgow G4 12 0RE. 4Helmholtz Zentrum Munchen GmbH, Ingolstadter Landstrasse 1, D-85764 13 Neuherberg. Germany. 14 15 Corresponding author: 16 Gareth G Lavery, PhD 17 Wellcome Trust Senior Research Fellow 18 Institute of Metabolism and Systems Research 19 University of Birmingham 20 Edgbaston 21 Birmingham, UK 22 B15 2TT 23 Tel: +44 121 414 3719 Fax: +44 121 415 8712 24 Email: [email protected] 25 26 Disclosure statement: The authors have nothing to disclose. 27 Abbreviated title: NAD+ metabolism and ER stress in skeletal muscle myopathy 28 Word Count: 5504; 7 Figures (Inclusive of 3 Supplemental) 1 bioRxiv preprint doi: https://doi.org/10.1101/567297; this version posted March 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Abstract 2 Background: Hexose-6-Phosphate Dehydrogenase (H6PDH) is the only known generator 3 of the pyridine nucleotide NADPH from NADP+ in the Endoplasmic/Sarcoplasmic Reticulum 4 (ER/SR). However, its ability to influence cell wide pyridine biosynthesis and metabolism is 5 unclear. 6 Methods: Skeletal muscle from H6PDH Knockout mice (H6KO) and Wild type controls (WT) 7 was subject to metabolomic assessment and pathway analysis. NAD boosting was 8 performed through intraperitoneal injection of Nicotinamide Riboside (NR) (400mg/kg) twice 9 daily for 4 days. Mice were culled and skeletal muscle tissue collected the next day. Double 10 knockout mice were generated through to breeding of H6KO with NRK2 knockout mice. 11 These mice were assessed for NAD levels and ER stress response. 12 Results: H6KO skeletal muscle shows significant changes in the pathway regulating NAD+ 13 biosynthesis with the Nicotinamide Riboside Kinase 2 gene (NRK2) being the most elevated 14 gene. Assessment of the metabolic impacts of this dysregulation showed decreases in 15 mitochondrial respiration and Acylcarnitine metabolism. Pharmacologically boosting NAD+ 16 levels with NR had no significant impact over ER stress or Acetyl-CoA metabolism. A duel 17 H6-NRK2 KO mouse exhibited changes in NAD+ levels but no overt change in metabolism 18 compared to the H6KO mouse. 19 Conclusions: This work shows a relationship between ER/SR status and wider muscle cell 20 metabolism. The utility of NAD+ boosting to skeletal muscle is demonstrated however, it is 21 also evident that the extent of cellular stress and/or REDOX status may overcome any 22 beneficial impact. 23 24 Keywords: Hexose-6-Phosphate Dehydrogenase, Endoplasmic/Sarcoplasmic Reticulum, 25 Skeletal muscle, Nicotinamide Riboside, Nicotinamide Adenine Dinucleotide 26 27 28 2 bioRxiv preprint doi: https://doi.org/10.1101/567297; this version posted March 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Background 2 A complete understanding of REDOX biology within the Sarcoplasmic/Endoplasmic 3 Reticulum (SR/ER) and any influence over cellular metabolism is lacking. Hexose-6- 4 phosphate dehydrogenase (H6PDH) is the only recorded generator of NADPH (from NADP+) 5 within this cellular compartment. This exclusivity ensures its importance to not only SR/ER 6 REDOX control and coenzyme generation for pyridine nucleotides, but also implicates it into 7 wider skeletal muscle homeostasis1,2. 8 The pyridine Nicotinamide Adenine Dinucleotide (NAD+) is a fundamental contributor to 9 cellular REDOX maintenance and energetic utilisation3,4. The rate limiting nature of NAD+ to 10 biochemical reactions make it fundamental to energetic metabolism including mitochondrial 11 fatty acid catabolism and ATP generation. 12 Pharmacologic augmentation of NAD+ through the use of precursors such as Nicotinamide 13 Riboside (NR) has shown restorative potential in NAD+ based therapies using a variety of 14 cell types and animal models with supportive human studies5-10. Recent studies back this, 15 showing a loss of NAD+ salvage function leads to progressive muscle degeneration and 16 boosting of the nicotinamide salvaging pathways liberates skeletal muscles from some of the 17 deleterious impacts of ageing, muscular dystrophy and high-fat diet phenotypes10-13. The 18 phosphorylation of NR into nicotinamide mononucelotide (NMN) occurs through two 19 nicotinamide riboside kinases (NRKs) NRK1 and NRK2. In many tissues NRK1 is the 20 dominant and rate limiting source of NR phosphorylation14. However, in skeletal muscle 21 NRK2 is highly expressed and recent data highlights its roles within myocyte physiology8. 22 Biosynthetic salvage of nicotinamide mediated by NRK2 has only recently been identified as 23 having relevance to skeletal muscle biology and its function within atrophic models, ER 24 stress and REDOX dysfunction are unstudied. 25 Furthermore, maintenance of the Sarcoplasmic Reticulum (skeletal muscle only) SR/ER 26 REDOX pyridine pools and its contribution to whole cell NAD+ levels is unclear. Hexose-6- 27 phosphate dehydrogenase (H6PD) converts glucose 6-phosphate (G6P) to 6- 28 Phosphoglucogonate (6-PG) and 6-Phosphglucogonatelactonase (6-PGL). Within the 3 bioRxiv preprint doi: https://doi.org/10.1101/567297; this version posted March 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 SR/ER H6PD generates the rate limiting cofactor for intracellular glucocorticoid activation by 2 11beta-hydroxysteroid dehydrogenase type 1 (HSD11b1)15. The knockout of H6PDH 3 produces profound metabolic dysfunction, with glycogen storage shifts in liver accompanied 4 by improved glucose sensitivity16. Adipose analysis of whole body H6PD KO mice revealed 5 reduced adiposity and changes in lipolytic rates, suggesting that absence of H6PDH 6 switches the direction with which HSD11b1 acts upon glucocorticoids17. Thus the action of 7 H6PDH upon metabolism are multifaceted and tissue-specific. Skeletal muscle from H6PDH 8 KO mice exhibits changes in REDOX balance of the ER/SR and inducing Unfolded Protein 9 Response. This in-turn induces myofibre loss, vacuole formation and fibre type switching. 10 Permutations extend to the cytosolic and nuclear environment shifting metabolic activity of 11 purine biosyntheisis18. Given the importance of the SR/ER to skeletal muscle and H6PD 12 being the only source of NADPH within the SR/ER there is potential for SR/ER pyridine 13 metabolism may be shifted in ways that are not yet attributed to H6PD function. In this study, 14 we examined H6PD as a regulator skeletal muscle metabolism through the conversion of 15 NADP+ to NADP(H), and how this impacts upon cellular processes extending beyond the 16 SR/ER confined environment. These data show that H6PDH not only has implications for the 17 biosynthetic processes governing NAD+ generation but also impacts wider cellular pathways 18 such as lipid homeostasis and mitochondrial fatty acid oxidation. 19 Here we explore the relationship between H6PDH and NRK2, attempting to boost skeletal 20 muscle NAD+ bioavailability with acute NR supplementation. In order to further understand 21 dependence of SR/ER actions upon NRK2 mediated NAD+ biosynthesis we generated a 22 whole body double-knockout of H6PD and NRK2, exploring how ablation of NRK2 within 23 H6PDH defunct skeletal muscle alters metabolic function and mitochondrial activity. Our 24 work reveals previously unconsidered aspects of NAD+ metabolism showing an ER sensing 25 mechanism that up-regulates a skeletal muscle NAD+ biosynthesis pathway in response to 26 altered NADPH availability. 27 4 bioRxiv preprint doi: https://doi.org/10.1101/567297; this version posted March 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Materials and Methods 2 Animal care, Mouse strains, storage 3 C57/BL5J mice were group housed in a standard temperature (22oC) and humidity- 4 controlled environment with 12:12-hour light:dark cycle. Nesting material was provided and 5 mice had ad libitum access to water and standard chow. Mice were sacrificed using 6 schedule one cervical dislocation and tissues were immediately. Collections were all 7 performed at 10-11am. Intraperitoneal injections of Nicotinamide Riboside (400mg/kg) were 8 given twice daily. (10am and 3pm) for 4 days before cervical dislocation and skeletal muscle 9 bed collection on Day 5 (10am), tissues were flash frozen, fixed or for high resolution 10 respirometry stored in BIOPS buffer. Heterozygotes of H6PD KO and NRK2 KO were breed 11 to generate H6-NRK2 KO mice.