Avinash Abhyankar Department of Clinical Sciences, Malmö Medical Genetics Unit Lund University

1. Novel congenic strain of the Goto-Kakizaki rat reveals nuclear-mitochondrial interactions in type 2 diabetes Avinash Abhyankar Department of Clinical Sciences, Malmö Medical Genetics Unit Lund University

2. Background QTL x Cross: P=4.9x10-5 Linkage on chromosome 9 for fasting insulin

3. Generation of C9B F344 GK

4. Intra-peritoneal glucose challenge *P<0.05, **P<0.01, ***P<0.001 ** ** ** * ** * C9B has impaired insulin response to glucose stimulation at 45 days age

5. Intra-peritoneal glucose challenge *P<0.05, **P<0.01, ***P<0.001 *

6. Insulin secretion from islets *P<0.05, **P<0.01, ***P<0.001 ** C9B has impaired insulin response to glucose stimulation (20 mM)

7. Mitochondrial function *P<0.05, **P<0.01, ***P<0.001 ** C9B consumes less oxygen as compared to F344

8. Mitochondrial genes *P<0.05, **P<0.01, ***P<0.001 * • Downstream targets • NRF1 • NRF2 • mtSSB • TFAM • COX IV • COX I ** * * C9B shows up-regulation of PGC1a and down-regulation of mitochondrially encoded genes

9. Prioritizing genes Single Marker vsMultiple Marker QTL Analysis

10. Prioritizing genes • Literature • Homology • Conserved Domains • Gene ontology • GEO • 36 genes • 23 known • 13 predicted/unknown

11. Prioritizing genes

12. Prioritizing genes - SNPs F344 GK

13. Prioritizing genes - SNPs

14. Prioritizing genes - SNPs

15. Prioritizing genes - SNPs IL1R1 IL1R2 SLC9A2 SLC9A4 MGAT4A MRPS9

16. Liver Gene Expression *P<0.05, **P<0.01, ***P<0.001 * *

17. Liver Gene Expression *P<0.05, **P<0.01, ***P<0.001 * * *

18. WAT Gene Expression *P<0.05, **P<0.01, ***P<0.001 * **

19. EDL Gene Expression *P<0.05, **P<0.01, ***P<0.001

20. Islet Gene Expression *P<0.05, **P<0.01, ***P<0.001 *

21. Islet Gene Expression *P<0.05, **P<0.01, ***P<0.001 * *

22. mtDNA sequencing Complete mitochondrial genome (~16310 bp) of 13 commonly used laboratory rat strains including 3 wild strains from Sweden, Japan and India sequenced

23. mtDNA sequencing Cyt b: 1 substitution COX I: 1 substitution COX II: 1 substitution ND2: 4 substitutions 1 Deletion ND4: 3 substitutions ND6: 1 substitution ATP6: 1 substitution

24. mtDNA sequencing

25. Ka/Ks analysis

26. Ka/Ks analysis GK, SHR, BBDR, BBDP, MHS, WK F344, BN, DA, B, L4 ND2 Gene

27. ?Co-evolution::gDNA SNPs Four regions identified in the rat genome which show cross specific effects

28. ?Co-evolution::gDNA SNPs

29. ?Co-evolution::mtDNA mtDNA::D-loop

30. ?Co-evolution::mtDNA mtDNA::D-loop

31. ?Co-evolution::Conplastics Studying conplastic animals to understand co-evolution F344 GK

32. ?Co-evolution::Conplastics IPGTT - Glucose *P<0.05, **P<0.01, ***P<0.001 * **

33. ?Co-evolution::Conplastics

34. ?Co-evolution::Conplastics *P<0.05, **P<0.01, ***P<0.001 * * *

35. Conclusions • We have identified a locus on rat chromosome 9 which shows parent-origin-specific phenotypic differences. • The identified locus harbours gene(s) interacting with the mitochondria and affecting insulin homeostasis

36. To-do… • Sequencing project • Phylogeny reconstruction with all 30 mitochondrial genomes for gDNA and mtDNA • Recalculate tests for evolution with the new dataset • C9B project • Expression studies on isolated islets • Islet histology • Gene copy number detection for candidate genes • Liver/Muscle glycogen estimation • Conplastics project • Insulin measurements • Oxygen consumption and membrane potential • Expression of genes related to energy metabolism

37. Prof. Holger Luthman Hee-Bok Park Charlotte Granhall Karin Berger Anders Rosengren Anders Långberg Ramunas Jakubka Thank you!