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Amylin

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amir mahmodzadeh

The document provides an overview of amylin, also known as islet amyloid polypeptide (IAPP). It discusses amylin's functions as a hormone that regulates glucose homeostasis by slowing gastric emptying, reducing food intake, and inhibiting glucagon secretion. Amylin is co-secreted with insulin from pancreatic beta cells and aggregates to form amyloid deposits associated with type 2 diabetes. Pramlintide is an amylin analog used to treat diabetes by mimicking amylin's actions. The document outlines amylin's synthesis, receptors, effects on glucose regulation and metabolism, role in diabetes, and therapeutic applications including pramlintide.

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Amylin and Glucose Hemostasis 1
Outlines • Introduction • Functions • Metabolism • Amyloid formation • Therapeutic applications 2
Amylin or Islet Amyloid Polypeptide (IAPP)  A neuroendocrine hormone  37-amino acid peptide first reported in 1987  Co-localized and co-secreted with insulin from pancreatic b-cells  actions as a hormone, growth factor, and modifier of behavior  Deficient in diabetes Amylin InsulinHuman amylin 3
• calcitonin family, CGRP, adrenomedullin • A disulfide bridge between amino acid residues 2 and 7 • Amidated COOH terminus 4
Discovery IAPP was discovered through its ability to aggregate into pancreatic islet amyloid deposits, in association with type 2 diabetes in humans and monkeys and cats 5
1.Asahormone 2.Modificationofbehavior increasing satiety,thirst 3.Growthfactor Function 6
An overview of the major actions of amylin activate specific receptors in the brainstem 1. suppression of glucagon release 2.reduction in food intake 3. gastric emptying 7
Synthesis • Expressed by one single-copy gene on the short arm of chromosome 12 • Prepro-IAPP : 89-aa residue containing a 22-aa signal peptide preproprotein and two short flanking peptides, • The signal peptide is cleaved off in the ER • Proislet amyloid polypeptide (proIAPP, proamylin, proislet protein) a 67 amino acid • conversion of proIAPP to IAPP takes place in the secretory vesicles 8
ProIAPP and proinsulin are both processed by he two ndoproteases hormone 1. Protei convertase 2 (PC2) and prohormone convertase 1/3 (PC1/3) 2. carboxypeptidase E (CPE) IAPP and insulin genes contain similar promoter elements, and the transcription factor PDX1 regulates the effects of glucose on both genes 9
FEBS Lett 247: 154–158, 1989 11
Secretion amylin and insulin were co‐localized in, and were co‐secreted factors modulating insulin secretion also appeared to cause an obligatory modulation of amylin secretion glucose , arginine, carbachol, fatty acids somatostatin 12
Amylin And Insulin Were Co‐secreted ranges from 3–5 pM in the fasting state to postprandial concentrations of 15–25 pM 13
14
• Like the C peptide, IAPP is eliminated in the kidney • Insulin-degrading enzyme (IDE,Insulysin and insulinase) • A second protease capable of degrading amyloid is neprolysin Metabolism 15
Amylin degradation is inhibited by excess insulin or amylin. A. B. 16
• approximately 40% amino acid sequence identity with calcitonin • Calcitonin gene related peptide (CGRP)  G protein–coupled receptors (GPCRs) • Receptor Activity-Modifying Proteins (RAMPs) • They are not receptors in themselves, but when they dimerize with the calcitonin receptors, they interact and alter the affinity for ligands Receptors 17 (Hei et al 2015)
Receptor activity-modifying proteins (RAMPs) are a class of protein that interact with and modulate the activities of several Class B G Protein-Coupled Receptors including the receptors for calcitonin(CT), glucagon,and vasoactive intestinal peptide (VIP). There are three distinct types of RAMPs, designated RAMP1, RAMP2, and RAMP3, each encoded by a separate gene 18
lung, stomach fundus, spleen, and brain 19
A.WhatIsAmyloid? • a specific protein aggregation state in which molecules in β - sheet conformation are bound to each other predominantly by hydrogen bonds but also by other bonds • This state of aggregation creates thin (10 nm), stable fibrils in which the β-strands are oriented perpendicular to the fibril axis. ISLET AMYLOID POLYPEPTIDE AND AMYLOID Human IAPP is one of the most amyloidogenic peptides known. 20
AmyloidFormation 21 ( Karen et al 2013)
22
Amylin ReducesTheAd PathologyAnd Improves Cognitive Impairment In TheAnimal Models ForAD amylin type peptides enhance the removal of neurotoxic Aβ out of the brain. 23 (Qui et al, 2014)
Amylin increases expression of the synaptic marker synapsin I and the kinase cyclin-dependent kinase-5 in the hippocampus, as well as decreased oxidative stress and inflammatory markers in the hippocampus 24
Glucose Homeostasis • Amylin is released during the feeding/fed state in response to nutrient entry into the gastrointestinal tract • appetite • glucagon • Gastric empting 25
InsulinDeficiencyandGlucagonHypersecretionin Diabetes 26
PostprandialGlucagon NotCorrectedby ExogenousInsulin 27
Deregulationofamylinin diabetesmellitus There is an absence of secretion of b-cell hormones in type 1 diabetes, whereas β-cell deregulation in type 2 diabetes (depending on its severity) can range from mild to severe. 28
Gastric Emptying and Diabetes. Gastric emptying as a therapeutic target 29
Pramlintide • An analog of amylin that overcomes the tendency of human amylin to: • Aggregate, form insoluble particles • Adhere to surfaces • Pharmacokinetic and pharmacodynamic properties similar to human amylin Human amylin Pramlintide (analog of amylin) Amide S S A Y T N S G V N T T T T N A A A L I K S S C C Q R L N N NF G F L V H Amide P P P Y T N S G V N T T T T N A A A L I K S S C C Q R L N N NF G F L V H 30 (Fineman et al 2002)
PramlintideMimickedThree Important Actions of AmylinThat ImpactGlucose Appearance       Amylin* Pramlintide Slows gastric emptying Promotes satiety and reduces caloric intake Inhibits inappropriately high postprandial glucagon secretion 31
Pramlintide Reduces Postprandial Glucagon T1DM Time (h) Placebo Pramlintide Placebo or 25 µg/h pramlintide infusion -20 0 10 20 30 -10 Insulin 0 2 3 4 51 T2DM, Late Stage Time (h) PlasmaGlucagon(pg/mL) Insulin 60 40 30 50 Placebo or 100 µg/h pramlintide infusion 0 1 2 3 4 5 PlasmaGlucagon(pg/mL) 32 feedingfeeding
%Emptiedperhr afterbreakfast Placebo 30 ug Pramlintide 60 ug Pramlintide PramlintideSlowedGastric Emptying-T1DM Insulin + Placebo Insulin + Pramlintide Gastric Emptying Is Accelerated in T1DM 33
PramlintideReducedCaloric Intake in Type2 Diabetes 0 250 500 750 1000 1250 Protein CHO Fat CHO Fat Protein -202 kcal (-23%) P <0.01 Ad-Libitum Caloric Intake (kcal) Placebo Pramlintide 34
Pramlintide Improved Postprandial Glucose 100 150 200 250 300 0 60 120 180 240 Time Relative to Meal and Pramlintide (min) Mean (SE) Plasma Glucose (mg/dL) 100 150 200 250 300 0 60 120 180 240 Mean (SE) Plasma Glucose (mg/dL) Lispro Insulin Pramlintide 60 ug + Lispro Insulin Regular Insulin Pramlintide 60 ug + Regular Insulin TYPE 1 DIABETES 35
Pramlintide Clinical Effects -0.8 -0.6 -0.4 -0.2 0 -4 -2 0 2 4 6 8 -2 -1 0 1 *** *** *** ** * *** *** *** Week 4 Week 13 Week 26Week 4 Week 13 Week 26Week 4 Week 13 Week 26 Insulin Use (%)A1C (%) Weight (kg) Placebo + Insulin 30 or 60 ug Pramlintide TID or QID + Insulin TYPE 1 DIABETES COMBINED PIVOTALS 36
PramlintideReducedFastingand PostprandialGlucose 120 140 160 180 pre-bf post-bf pre-lu post-lu pre-di post-di bedtime Glucose(mg/dL) Baseline 6 Months * * TYPE 1 DIABETES 37
PramlintideSafety and Tolerability in Type1 Diabetes • Nausea: • Mostly mild-to-moderate nausea. Occurred more frequently during initiation and then decreased with time but can increase risk of hypoglycemia. • Nausea reduced by dose titration • Could increase risk of insulin-induced severe hypoglycemia due to reduced food intake • Insulin-Induced Severe Hypoglycemia: – More common in type 1 diabetes; risk reduced by appropriate patient selection, careful patient instruction and insulin dose adjustments as stated in the Boxed Warning 38
AmylinHelps Regulate PostprandialGycemiaBy MultipleMechanisms • Enhances feeling of fullness at meals • Slows inappropriately accelerated gastric emptying • Decreases hepatic glucose output via suppression of postprandial pancreatic glucagon secretion 39 Young A. Adv Pharmacol. 2005;52:67-77. 39
40
1. Hay, D.L., et al., Amylin: Pharmacology, Physiology, and Clinical Potential. Pharmacol Rev, 2015. 67(3): p. 564-600. 2. Westermark, P., A. Andersson, and G.T. Westermark, Islet amyloid polypeptide, islet amyloid, and diabetes mellitus. Physiological reviews, 2011. 91(3): p. 795-826. 3. Westermark, P., et al., A novel peptide in the calcitonin gene related peptide family as an amyloid fibril protein in the endocrine pancreas. Biochemical and biophysical research communications, 1986. 140(3): p. 827-831. 4. Woods, S.C., et al., Pancreatic signals controlling food intake; insulin, glucagon and amylin. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 2006. 361(1471): p. 1219-1235. 5. Marzban, L., et al., Erratum. Small Interfering RNA-Mediated Suppression of Proislet Amyloid Polypeptide Expression Inhibits Islet Amyloid Formation and Enhances Survival of Human Islets in Culture. Diabetes 2008;57:3045-3055. Diabetes, 2016. 65(3): p. 818. 6. Lee, S.M., D.L. Hay, and A.A. Pioszak, Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: Insights into Peptide-binding Modes and Allosteric Modulation of the Calcitonin Receptor by Receptor Activity-modifying Proteins. J Biol Chem, 2016. References 41
• 7. Ilitchev, A.I., et al., Human Islet Amyloid Polypeptide N-Terminus Fragment Self-Assembly: Effect of Conserved Disulfide Bond on Aggregation Propensity. J Am Soc Mass Spectrom, 2016. • 8. Bower, R.L., et al., Mapping the calcitonin receptor in human brainstem. Am J Physiol Regul Integr Comp Physiol, 2016: p. ajpregu.00539.2015. • 9. Van Hulst, K., et al., Islet amyloid polypeptide/amylin messenger RNA and protein expression in human insulinomas in relation to amyloid formation. European journal of endocrinology, 1999. 140(1): p. 69-78. • 10. Middleton, C.T., et al., Two-dimensional infrared spectroscopy reveals the complex behaviour of an amyloid fibril inhibitor. Nature chemistry, 2012. 4(5): p. 355- 360. • 11. Westermark, P., A. Andersson, and G.T. Westermark, Is aggregated IAPP a cause of beta-cell failure in transplanted human pancreatic islets? Current diabetes reports, 2005. 5(3): p. 184-188. • 12. Goldsbury, C., et al., Amyloid fibril formation from full-length and fragments of amylin. Journal of structural biology, 2000. 130(2): p. 352-362. 42
• 13. Drucker, D.J., The role of gut hormones in glucose homeostasis. The Journal of clinical investigation, 2007. 117(1): p. 24-32. • 14. Lutz, T.A., The role of amylin in the control of energy homeostasis. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2010. 298(6): p. R1475-R1484. • 15. Higham, C.E., et al., Processing of synthetic pro‐islet amyloid polypeptide (proIAPP)‘amylin’by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro. European Journal of Biochemistry, 2000. 267(16): p. 4998-5004. • 16. Hay, D., et al., Amylin receptors: molecular composition and pharmacology. Biochemical Society Transactions, 2004. 32(5): p. 865-867. • 17. Qi, D., et al., Fatty acids induce amylin expression and secretion by pancreatic β-cells. American Journal of Physiology-Endocrinology and Metabolism, 2010. 298(1): p. E99-E107. • 18. Wookey, P.J., et al., Amylin in the periphery. The Scientific World Journal, 2003. 3: p. 163-175. 43

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Amylin

  • 2. Outlines • Introduction • Functions • Metabolism • Amyloid formation • Therapeutic applications 2
  • 3. Amylin or Islet Amyloid Polypeptide (IAPP)  A neuroendocrine hormone  37-amino acid peptide first reported in 1987  Co-localized and co-secreted with insulin from pancreatic b-cells  actions as a hormone, growth factor, and modifier of behavior  Deficient in diabetes Amylin InsulinHuman amylin 3
  • 4. • calcitonin family, CGRP, adrenomedullin • A disulfide bridge between amino acid residues 2 and 7 • Amidated COOH terminus 4
  • 5. Discovery IAPP was discovered through its ability to aggregate into pancreatic islet amyloid deposits, in association with type 2 diabetes in humans and monkeys and cats 5
  • 7. An overview of the major actions of amylin activate specific receptors in the brainstem 1. suppression of glucagon release 2.reduction in food intake 3. gastric emptying 7
  • 8. Synthesis • Expressed by one single-copy gene on the short arm of chromosome 12 • Prepro-IAPP : 89-aa residue containing a 22-aa signal peptide preproprotein and two short flanking peptides, • The signal peptide is cleaved off in the ER • Proislet amyloid polypeptide (proIAPP, proamylin, proislet protein) a 67 amino acid • conversion of proIAPP to IAPP takes place in the secretory vesicles 8
  • 9. ProIAPP and proinsulin are both processed by he two ndoproteases hormone 1. Protei convertase 2 (PC2) and prohormone convertase 1/3 (PC1/3) 2. carboxypeptidase E (CPE) IAPP and insulin genes contain similar promoter elements, and the transcription factor PDX1 regulates the effects of glucose on both genes 9
  • 10. FEBS Lett 247: 154–158, 1989 11
  • 11. Secretion amylin and insulin were co‐localized in, and were co‐secreted factors modulating insulin secretion also appeared to cause an obligatory modulation of amylin secretion glucose , arginine, carbachol, fatty acids somatostatin 12
  • 12. Amylin And Insulin Were Co‐secreted ranges from 3–5 pM in the fasting state to postprandial concentrations of 15–25 pM 13
  • 13. 14
  • 14. • Like the C peptide, IAPP is eliminated in the kidney • Insulin-degrading enzyme (IDE,Insulysin and insulinase) • A second protease capable of degrading amyloid is neprolysin Metabolism 15
  • 15. Amylin degradation is inhibited by excess insulin or amylin. A. B. 16
  • 16. • approximately 40% amino acid sequence identity with calcitonin • Calcitonin gene related peptide (CGRP)  G protein–coupled receptors (GPCRs) • Receptor Activity-Modifying Proteins (RAMPs) • They are not receptors in themselves, but when they dimerize with the calcitonin receptors, they interact and alter the affinity for ligands Receptors 17 (Hei et al 2015)
  • 17. Receptor activity-modifying proteins (RAMPs) are a class of protein that interact with and modulate the activities of several Class B G Protein-Coupled Receptors including the receptors for calcitonin(CT), glucagon,and vasoactive intestinal peptide (VIP). There are three distinct types of RAMPs, designated RAMP1, RAMP2, and RAMP3, each encoded by a separate gene 18
  • 18. lung, stomach fundus, spleen, and brain 19
  • 19. A.WhatIsAmyloid? • a specific protein aggregation state in which molecules in β - sheet conformation are bound to each other predominantly by hydrogen bonds but also by other bonds • This state of aggregation creates thin (10 nm), stable fibrils in which the β-strands are oriented perpendicular to the fibril axis. ISLET AMYLOID POLYPEPTIDE AND AMYLOID Human IAPP is one of the most amyloidogenic peptides known. 20
  • 21. 22
  • 22. Amylin ReducesTheAd PathologyAnd Improves Cognitive Impairment In TheAnimal Models ForAD amylin type peptides enhance the removal of neurotoxic Aβ out of the brain. 23 (Qui et al, 2014)
  • 23. Amylin increases expression of the synaptic marker synapsin I and the kinase cyclin-dependent kinase-5 in the hippocampus, as well as decreased oxidative stress and inflammatory markers in the hippocampus 24
  • 24. Glucose Homeostasis • Amylin is released during the feeding/fed state in response to nutrient entry into the gastrointestinal tract • appetite • glucagon • Gastric empting 25
  • 27. Deregulationofamylinin diabetesmellitus There is an absence of secretion of b-cell hormones in type 1 diabetes, whereas β-cell deregulation in type 2 diabetes (depending on its severity) can range from mild to severe. 28
  • 28. Gastric Emptying and Diabetes. Gastric emptying as a therapeutic target 29
  • 29. Pramlintide • An analog of amylin that overcomes the tendency of human amylin to: • Aggregate, form insoluble particles • Adhere to surfaces • Pharmacokinetic and pharmacodynamic properties similar to human amylin Human amylin Pramlintide (analog of amylin) Amide S S A Y T N S G V N T T T T N A A A L I K S S C C Q R L N N NF G F L V H Amide P P P Y T N S G V N T T T T N A A A L I K S S C C Q R L N N NF G F L V H 30 (Fineman et al 2002)
  • 30. PramlintideMimickedThree Important Actions of AmylinThat ImpactGlucose Appearance       Amylin* Pramlintide Slows gastric emptying Promotes satiety and reduces caloric intake Inhibits inappropriately high postprandial glucagon secretion 31
  • 31. Pramlintide Reduces Postprandial Glucagon T1DM Time (h) Placebo Pramlintide Placebo or 25 µg/h pramlintide infusion -20 0 10 20 30 -10 Insulin 0 2 3 4 51 T2DM, Late Stage Time (h) PlasmaGlucagon(pg/mL) Insulin 60 40 30 50 Placebo or 100 µg/h pramlintide infusion 0 1 2 3 4 5 PlasmaGlucagon(pg/mL) 32 feedingfeeding
  • 32. %Emptiedperhr afterbreakfast Placebo 30 ug Pramlintide 60 ug Pramlintide PramlintideSlowedGastric Emptying-T1DM Insulin + Placebo Insulin + Pramlintide Gastric Emptying Is Accelerated in T1DM 33
  • 33. PramlintideReducedCaloric Intake in Type2 Diabetes 0 250 500 750 1000 1250 Protein CHO Fat CHO Fat Protein -202 kcal (-23%) P <0.01 Ad-Libitum Caloric Intake (kcal) Placebo Pramlintide 34
  • 34. Pramlintide Improved Postprandial Glucose 100 150 200 250 300 0 60 120 180 240 Time Relative to Meal and Pramlintide (min) Mean (SE) Plasma Glucose (mg/dL) 100 150 200 250 300 0 60 120 180 240 Mean (SE) Plasma Glucose (mg/dL) Lispro Insulin Pramlintide 60 ug + Lispro Insulin Regular Insulin Pramlintide 60 ug + Regular Insulin TYPE 1 DIABETES 35
  • 35. Pramlintide Clinical Effects -0.8 -0.6 -0.4 -0.2 0 -4 -2 0 2 4 6 8 -2 -1 0 1 *** *** *** ** * *** *** *** Week 4 Week 13 Week 26Week 4 Week 13 Week 26Week 4 Week 13 Week 26 Insulin Use (%)A1C (%) Weight (kg) Placebo + Insulin 30 or 60 ug Pramlintide TID or QID + Insulin TYPE 1 DIABETES COMBINED PIVOTALS 36
  • 36. PramlintideReducedFastingand PostprandialGlucose 120 140 160 180 pre-bf post-bf pre-lu post-lu pre-di post-di bedtime Glucose(mg/dL) Baseline 6 Months * * TYPE 1 DIABETES 37
  • 37. PramlintideSafety and Tolerability in Type1 Diabetes • Nausea: • Mostly mild-to-moderate nausea. Occurred more frequently during initiation and then decreased with time but can increase risk of hypoglycemia. • Nausea reduced by dose titration • Could increase risk of insulin-induced severe hypoglycemia due to reduced food intake • Insulin-Induced Severe Hypoglycemia: – More common in type 1 diabetes; risk reduced by appropriate patient selection, careful patient instruction and insulin dose adjustments as stated in the Boxed Warning 38
  • 38. AmylinHelps Regulate PostprandialGycemiaBy MultipleMechanisms • Enhances feeling of fullness at meals • Slows inappropriately accelerated gastric emptying • Decreases hepatic glucose output via suppression of postprandial pancreatic glucagon secretion 39 Young A. Adv Pharmacol. 2005;52:67-77. 39
  • 39. 40
  • 40. 1. Hay, D.L., et al., Amylin: Pharmacology, Physiology, and Clinical Potential. Pharmacol Rev, 2015. 67(3): p. 564-600. 2. Westermark, P., A. Andersson, and G.T. Westermark, Islet amyloid polypeptide, islet amyloid, and diabetes mellitus. Physiological reviews, 2011. 91(3): p. 795-826. 3. Westermark, P., et al., A novel peptide in the calcitonin gene related peptide family as an amyloid fibril protein in the endocrine pancreas. Biochemical and biophysical research communications, 1986. 140(3): p. 827-831. 4. Woods, S.C., et al., Pancreatic signals controlling food intake; insulin, glucagon and amylin. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 2006. 361(1471): p. 1219-1235. 5. Marzban, L., et al., Erratum. Small Interfering RNA-Mediated Suppression of Proislet Amyloid Polypeptide Expression Inhibits Islet Amyloid Formation and Enhances Survival of Human Islets in Culture. Diabetes 2008;57:3045-3055. Diabetes, 2016. 65(3): p. 818. 6. Lee, S.M., D.L. Hay, and A.A. Pioszak, Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: Insights into Peptide-binding Modes and Allosteric Modulation of the Calcitonin Receptor by Receptor Activity-modifying Proteins. J Biol Chem, 2016. References 41
  • 41. • 7. Ilitchev, A.I., et al., Human Islet Amyloid Polypeptide N-Terminus Fragment Self-Assembly: Effect of Conserved Disulfide Bond on Aggregation Propensity. J Am Soc Mass Spectrom, 2016. • 8. Bower, R.L., et al., Mapping the calcitonin receptor in human brainstem. Am J Physiol Regul Integr Comp Physiol, 2016: p. ajpregu.00539.2015. • 9. Van Hulst, K., et al., Islet amyloid polypeptide/amylin messenger RNA and protein expression in human insulinomas in relation to amyloid formation. European journal of endocrinology, 1999. 140(1): p. 69-78. • 10. Middleton, C.T., et al., Two-dimensional infrared spectroscopy reveals the complex behaviour of an amyloid fibril inhibitor. Nature chemistry, 2012. 4(5): p. 355- 360. • 11. Westermark, P., A. Andersson, and G.T. Westermark, Is aggregated IAPP a cause of beta-cell failure in transplanted human pancreatic islets? Current diabetes reports, 2005. 5(3): p. 184-188. • 12. Goldsbury, C., et al., Amyloid fibril formation from full-length and fragments of amylin. Journal of structural biology, 2000. 130(2): p. 352-362. 42
  • 42. • 13. Drucker, D.J., The role of gut hormones in glucose homeostasis. The Journal of clinical investigation, 2007. 117(1): p. 24-32. • 14. Lutz, T.A., The role of amylin in the control of energy homeostasis. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2010. 298(6): p. R1475-R1484. • 15. Higham, C.E., et al., Processing of synthetic pro‐islet amyloid polypeptide (proIAPP)‘amylin’by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro. European Journal of Biochemistry, 2000. 267(16): p. 4998-5004. • 16. Hay, D., et al., Amylin receptors: molecular composition and pharmacology. Biochemical Society Transactions, 2004. 32(5): p. 865-867. • 17. Qi, D., et al., Fatty acids induce amylin expression and secretion by pancreatic β-cells. American Journal of Physiology-Endocrinology and Metabolism, 2010. 298(1): p. E99-E107. • 18. Wookey, P.J., et al., Amylin in the periphery. The Scientific World Journal, 2003. 3: p. 163-175. 43