Stephan Broer Australian National University. Canberra.
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El lunes y martes 20 y 21 de noviembre coordinamos un simposio internacional en la Fundación Ramón Areces, sobre los defectos del transporte de aminoácidos.
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Similaire à Stephan Broer Australian National University. Canberra.
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Stephan Broer Australian National University. Canberra.
- 1. Prof. Stefan Broer Research School of Biology SLC6A19 – from genes and genetic disorders to function and drug discovery Research School of Biology
- 3. Overview of presentation • Genes and genetic disorders • Function • Protein restriction • Structure and Drug discovery
- 4. Research School of Biology Gl c AA0 Amino acids Energy Gut Pancreas Portal vein Insulin Protein AA AA Protein Branched-chain Amino acids AA+ AA-
- 6. 5p15.33 5p13.1 5q11.2 5q14.3 5q34 5q35.3 5p15.33 5p15.32 5p15.31 5p15.2 5p15.1 D5S406 D5S392 D5S1970 KIAA1909 FLJ44869 SDHA PDCD6 AHRR SEC6L1 SLC9A3 (NHE-3) TPPP ZDHHC11 BRD9 TRIP13 NKD2 SLC12A7 (NKCC) SLC6A19 (B0AT1) SLC6A18 (B0AT3) TERT CRR9 SLC6A3 Nozaki et al., 2001 Biochem. Biophys. Res. Comm. Seow et al., 2004 Nat. Genetics
- 7. GlyT1 GlyT2 ATB0,+ ProT GAT3 GAT1 GAT2 GAT4 TauT CT1 DAT NET SERT SIT1a SIT1b B0AT1B0AT3 NTT4 B0AT2 NTT5 Amino acids β/γ-Amino acids Monoamines Novel amino acid transporters Broer et al. 2006 Kowalczuk et al. 2005 Broer et al., 2004 Vanslambrouck et al., 2010
- 8. Overview of presentation • Genes and genetic disorders • Function • Protein restriction • Structure and Drug discovery
- 9. 30 40 50 60 70 80 -60 -50 -40 -30 -20 -10 0 Current(nA) Time (min) Leu Glu Arg Leu Gln Phe Ala His Trp Leu Na+ AA B0-like transport activity Research School of Biology Broer et al., 2004 JBC
- 10. Mutations in the human Hartnup transporterni wt G66R G93R D173N R178X R240X R240Q L242P V252I R328C E405K E501K D517G 0 20 40 60 80 100 120 140 160 180 Transportactivity(%) c wt G66R G93R R178X G284R R328C E405K D517G 0 20 40 60 80 100 Leucineuptake(percentofwildtype)
- 11. Angiotensin Converting Enzyme 2 Collectrin Angiotensin(II) Angiotensin(1-7) Kidney Intestine Kidney The collectrin ko-mouse has Hartnup disorder DRVYIHPF DRVYIHPF F
- 12. ACE2 - + - + - - + B0AT1 - - + + - + + 49 6969 98 188 Surface biotinylation Membrane preparation Kowalczuk et al. 2008 Faseb J.
- 13. 0 20 40 60 80 100 120 Leucineuptake(pmol/oocyte*20min) B 0 AT1 - wt R240Q - wt R240Q - wt R240Q ACE2 - - - - - - + + + Collectrin - - - + + + - - - Injected cRNA
- 14. B0AT1 ACE2 Protein Pancreatic proteases Na+ B0AT1 and ACE2 work together
- 17. Overview of presentation • Genes and genetic disorders • Function • Protein restriction • Structure and Drug discovery
- 18. Rats reject isoleucine-deficient diets Basal Imbalanced Devoid Corrected Gietzen and Aja, 2012
- 19. Signaling of protein restriction Na+System A System L System ASC Na+System N H+ RAG mTORC 1 AA Pool 4E- BP1 eIF4E tRNA CAP tRNA GCN2 eIF 2a P P Lysos ome - + eIF2B Lat1 Pat1 SNAT9 tRNA EAAT Na+ K+ H+ AA- AA0 AA+ CAT
- 20. Amino acids Amino acid starvation Gut Adipose tissue FGF21 GLUT1 ? UCP1 Fat Ketone bodies De Sousa-Coelho et al. 2012
- 21. Solon-Biet et al. 2014 Macronutrients and Health
- 22. • Increased neutral amino acids are associated with insulin resistance. – Newgard et al, Cell Metab 2009 – Stancakova et, al Diabetes 2012 – Würtz et al, Diabetes 2012 – Shah et al, Diabetologia 2012 • Neutral amino acids decrease after bariatric surgery much more than after diet with similar weight loss. Branched-chain amino acid levels were correlated with insulin sensitivity. – Laferrère B et al., Sci Transl Med 2011 • Elevated amino acids could be the driver of diabetes development. – Wang TJ, et al. Metabolite profiles and the risk of developing diabetes. Nat Med. 2011 Apr;17(4):448- 53. – Würtz P, et al. Branched-Chain and Aromatic Amino Acids Are Predictors of Insulin Resistance in Young Adults. Diabetes Care. 2012 Nov 5. – McCormack SE et al Circulating branched-chain amino acid concentrations are associated with obesity and future insulin resistance in children and adolescents. Pediatr. Obes 2012 – Cheng et al. Metabolic profiling identifies pathways associated with metabolic risk in humans Circulation 2012 • Mechanism: AA overload activates mTOR, branched-chain fatty acids - Newgard, Cell Metab 2012 • Hypothesis: Reducing protein intake could improve insulin sensitivity
- 23. • Impaired glucose removal (insulin resistance) • Increased hepatic glucose output • Elevated insulin levels • beta cell stress and failure
- 24. Broer et al., 2011 JBC
- 26. Glucose Glucose GTT (2 month old) 0 30 60 90 120 0 5 10 15 20 25 -/-(n=8) +/+(n=8) Time(min) BloodGlucose(mmol/L) GTT (6 month old) 0 30 60 90 120 0 5 10 15 20 25 -/-(n=10) +/+(n=10)*** * * Time(min) BloodGlucose(mmol/L) IPGTT 0 10 20 30 0.0 0.5 1.0 1.5 +/+(n=4) -/-(n=4) * * * time(min) BloodInsulin(ng/ml)
- 27. Body mass +/+(n=11) -/- n=(9) 0 10 20 30 40 *** g Liver mass +/+(n=11) -/- n=(9) 0.00 0.02 0.04 0.06 * %bodymass aWAT +/+(n=11) -/- n=(9) 0.00 0.01 0.02 0.03 0.04 * %bodymass +/+(n=11) -/- (n=9) 0.000 0.005 0.010 0.015 0.020 iWAT %bodymass BAT +/+(n=11) -/- n=(9) 0.000 0.002 0.004 0.006 %bodymass 6 month old mice 0 30 60 90 120 20 25 30 35 40 45 50 55 -/-(n=6) +/+(n=6) +/-(n=6) Days on diet BodyMass/g +/+ -/-
- 29. How can this be explained? Reduced nutrient uptake in the intestine (only the weight development not the IPGTT) Waste of energy Fast removal by insulin-dependent tissues Constant removal by insulin-independent mechanisms
- 30. Amino acids Amino acids Short chain fatty acids Proximal intestine Distal intestine Faecal dry mass +/+(n=5) -/- n=(4) 0 50 100 150 200 250 mg/d Faecal energy +/+(n=5) -/- n=(4) 0 5 10 15 20 J/mg Energy output +/+(n=5) -/- n=(4) 0 1 2 3 4 KJ/d propanoic acidbutyric acid valeric acid isovaleric acidhexanoic acid 0 1 2 3 4 5 +/+(n=5) -/-(n=5) * * Short Chain Fatty Acids * RelativeSignal
- 31. 14 16 18 20 22 24 90 100 110 +/+ -/- VO2 (ml/h) Lean body mass (g) 0 1 2 3 4 5 6 7 VCO2 Gasexchange[ml/(h*g)] VO2 RER 0.0 0.2 0.4 0.6 0.8 1.0 1.2 +/+ -/- RER Glucose uptake in BAT +/+(n=4) -/-(n=4) 0 50 100 150 200 250 mol/100g/min Glucose uptake (iWAT) +/+(n=4) -/-(n=4) 0 5 10 15 20 mol/100g/min *
- 32. Amino acids Proximal intestine Portal vein 1 Adipose tissue Amino acid starvation responseFGF21FGFR βKlotho Fatty acids Ketone bodies Fuel for other organs
- 33. Oral foodOral food Amino acids Amino acids Proximal intestine Distal intestine GIP/GLP-1 GIP 0m in 60m in 0 100 200 300 400 500 +/+(n=5) -/-(n=5) * * GIPpg/ml GLP-1 0 m in 60 m in 0 50 100 150 200 +/+(n=4) -/-(n=4) * pM
- 34. Tissue glucose uptake LiverK idneyIntestineM uscle W A T B rain H eart B A T Lung 0 50 100 150 200 250 slc6a19 +/+(n=4) slc6a19 -/-(n=4) umol/100g/min Liquid phase gastric emptying 30 60 90 120 0 20 40 60 -/-(n=4) +/+(n=4) time(min) mg/L
- 35. ? ? ?
- 36. Overview of presentation • Genes and genetic disorders • Function • Protein restriction • Structure and Drug discovery
- 37. Slc6a19 transporter is related to LeuT
- 40. Na+ Amino acids CHO slc6a19/collectrin Flip-in #1 cell control A rg Phe Ser G ly 0 20 40 60 +Na (net) -Na Competitor (10mM) Isoleucineuptake(nmol/mg*6min) CHO F1 #392 parental cell control A rg Phe Ser G ly 0 20 40 60 80 -Na +Na (net) Competitor (10mM) Isoleucineuptake(nmol/mg*6min) B0AT1 collectrin Na/K-ATPase -- ++ 0 30 60 90 120 150 80 100 120 140 160 - Ile +Ile Time(sec) %ofBaseline
- 41. 1E-3 0.01 0.1 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -Na +Na (net) NormalizedIsoleucineUptake [NEC 63912] (µM) 1E-3 0.01 0.1 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -Na +Na (net) NormalizedIsoleucineUptake [NSC39706] (µM) Benztropine 1-(4,4-diphenylbut-3-enyl)piperidine IC50 (+Na): 109.06±16.33µM
- 42. Urine samples Treatment of mice with Benztropine Faecal samples
- 43. IC50: 1±0.5µM
- 44. Summary • Protein restriction is a powerful signal to modulate cellular metabolism. • Nutrient signalling by the intestine is an important mechanism to modulate metabolism. • Slc6a19(-/-)mice are protein restricted and at the same time have enhanced signalling from the intestine. • Slc6a19 inhibitors can modulate glycaemic control and potentially protect beta cells.
- 45. Molecular Nutrition group at ANU Greg Amino acid homeostasis Qi Pharmacology HTS Kiran Biomarker development Nishank Docking Protein expression Angelika Cancer metabolism Stephen Protein-Protein interactions Weidong Flippases ANU Collaborators Brian Billups Anselm Enders Sydney Collaborators Jeff Holst Industry Collaborators Sanofi Merck GmbH
- 46. pyruvate challenge 0 15 30 45 60 75 90 0 5 10 15 slc6a19 -/-(n=6) slc6a19 +/+(n=5) time/min BloodGlucose(mmol/L)
- 47. Insulin Glucose IPITT(2 month old) 0 15 30 45 60 4 6 8 10 12 -/- (n=5) +/+(n=7) ** ** * * Time(min) BloodGlucose(mmol/L) IPITT(6 month old) 0 15 30 45 60 4 6 8 10 -/- (n=8) +/+(n=7) * * Time(min) BloodGlucose(mmol/L)
- 48. Kazmier et al., . Nature Structural & Molecular Biology 2014 Apr 20;21(5):472-9.
- 49. Amino acid transport in kidney and intestine are similar Hartnup disorder Transporter defect Renal secretion of Neutral amino acids
- 52. 1E-3 0.01 0.1 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -Na +Na (net) NormalizedIsoleucineUptake [NEC 63912] (µM) 1E-3 0.01 0.1 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -Na +Na (net) NormalizedIsoleucineUptake [NSC39706] (µM) Compound 11 Compound 51
- 53. Protein restriction and ER stress Transporter transcription MIN6 cells Krokowski et al., 2013
- 55. GTT (IP) high-fat diet
- 56. ITT (IP) high-fat diet
- 57. Wilcken et al., 1977 Wilcken et al., 1980 Total screening 800,000 newborns
- 58. Hartnup individual Male/ Female Clinical phenotype Aminoaciduria 1 M Not known Yes 2 M None Yes 3 F None Yes 4 F Unrelated health issues Yes 5 M None Yes 6 M Unknown Yes 7 F None Yes 8 F Skin rash Yes 9 M Skin rash Yes 10 M Not known Yes
- 60. Research School of Biology
- 61. ACE2 LTA (S1) overlay ACE2 B0AT1 overlay ACE2 B0AT1 overlay Kowalczuk et al. 2008 Faseb J.
- 62. Research School of Biology Gl c Glucose Glucose Glycogen Gut Pancreas Portal vein Insulin Adipose tissue Glycogen Acetyl-CoA VLDL Glc Glc GLUT4 GLUT4
- 63. FGF21 in mouse and man 0 2 0 0 0 4 0 0 0 6 0 0 0 F G F 2 1 FGF21(pg/ml) F e d F a stin g Mouse data
- 64. Collectrin binding site in B0AT1
- 65. • Protein restriction as a signal • Transporters as a tool to regulate protein restriction? • The role of amino acids in insulin sensitivity • Assay development and compound screening Overview
- 66. • Protein restriction as a signal • Transporters as a tool to regulate protein restriction? • The role of amino acids in insulin sensitivity • Assay development and compound screening Overview
- 67. Research School of Biology
- 68. Overview • Protein restriction as a signal • Transporters as a tool to regulate protein restriction? • The role of amino acids in insulin sensitivity • Assay development and compound screening Research School of Biology
- 71. Neutral AA Loss of neutral AA Plasma AA pool mTOR activation GIP Glp1 Insulin fgf21 mTOR activation Little effect Ketone body production Glucose consumption Ketone body production GIP Glp1 Glucose output Preservation of beta cells Fatty acids Ucp1
- 73. • Protein restriction as a signal • Transporters as a tool to regulate protein restriction? • The role of amino acids in insulin sensitivity • Assay development and compound screening Overview
- 74. Heterodimeric design of transporters is not unusual
- 75. LTA (Proximal tubule marker) B0AT1