Pancreatic β-Cell Nucleus Envelop Nucleol Chromatin Lysosome Mitochondria Glucose Glucose Glucokinase Glucose TtTransporter Nonmaturated Vesicles Glucokinase (sensor of glucose level) Maturated Vesicles + Closing the K ATP Channel Endoplasmic Reticulum Depolarization of Golgi Apparatus the Membrane Exocytosis Opening the Ca+ Channel secretory vesicle secretory vesicle ProInsulin Insulin Normal 102 ~ < 102 Pro-diabetes ProAmylin Amylin Amylin Deposition is a Hallmark of Type-2 Diabetes in Humans Diabetes 56:1324-1332 (2007) HUMAN PANCREAS 20 µm Amylin Transmissible Spongiform Dementia, Parkinson’s Type-2 Encephalopathies Alzheimer’s Disease Disease Diabetes Hyperamylinemia, Amylin Oligomerization, & the Risk of Cardio-Cerebrovascular Diseases β‐Cell Dysfunction & Apoptosis Pancreatic β‐Cell AMYLIN HYPERGLYCEMIA OLIGOMERIZATION LOOD (INSULINOGENIC DRUGS) BB HYPERAMYLINEMIA HYPERINSULINEMIA Department of Pharmacology Florin Despa University of California, Davis Relevance to Our Mission This research project is relevant to understanding, preventing, and, possibly, treating diabetes complications in two ways: 1. It uncovers an early pathogenic mechanism linking age-related metabolic disorders with diabetic brain injury, dementia, and CVD; 2. It identifies hyperamylinemia as a feasible therapeutic target to reduce accumulation of proteinaceous debris in the CV and CN systems, and thus to limit/ delay diabetes complications. Outline of Research in My Laboratory 1. Mechanisms of amylin oligomer formation and accumulation: a. understanding the etiology of hyperamylinemia; b. testing the circulating amylin oligomer hypothesis. 2. Amylin oligomer-induced cardiac dysfunction: a. primary structural defect induced by oligomers in myocytes; b. hypertrophy, remodeling; c. oxidative and inflammatory stress. 3. Role of oligomeric amylin in diabetic brain damage and dementia: a. interaction & co-localization of amylin with Aβ; b. amylin oligomer-mediated inflammatory and oxidative damage. 4. Curbing amylin deposition to delay/reduce the CVD risk: a. pro-fibrinolytic molecules limit amylin attachment to sarcolemma and red u ce ROS produ ction in cardiac moctesmyocytes. Collaborators • KlKaleena Jac kson • Heike Wulff • Kathy Guglielmino • Elva Diaz • Brian Koch • Gustavo Barisone • Sanda Despa • Dave Speca • Bruce Hammock • Peter J Havel • Anne Knowlton AD Center • Donald Bers • Charles DeCarli • Heinrich Taegtmeyer (UTHS) • Lee-way Jin • Keneth B. Margulies (U Penn) • Donald Steiner (U Chicago) • Simon Xie (Stanford) Funds: AHA, NSF, UCD AD Center, Vision Grant - UC Davis Cardiotoxicity of Hyperamylinemia (RATIONALE) Lean Non-Failing Hearts Amylin Oligomers Lean, Non-diabetes Failing Hearts Obese/Overweight Non-Failing Hearts distinct amylin oligomer size distributions Obese/Overweight Amylin Failing Hearts Oligomers Type-2 Diabetes Failing Hearts Amylin Oligomers Accumulate in Heart Failing vs. Non-failing AmylinT Trimers Anti-Amylin Antibody 500 ** ontrol) 400 L-NF OW/OB-HF CC ** 50 300 ** octamer 200 -NF -HF tetr am er (% rimers BB BB 15 FF 100 10 trimer kDa 0 L-NF L-HF OW/O OW/O DM-H Amylin T Larger Amylin Oligomers 500 16-MER ** FAILING ontrol) 400 CC * OCTAMER 300 TETRAMER 200 W/OB B-HF -NF Level (% F OO nn TRIMER NON-FAILING 100 OO OW/ DM-H 0 L-NF L-HF DIMER Amyli Despa S, …, Despa F. Circ Res. 2012;110:598 Cardiac Amylin Deposition in Humans with Type-2 Diabetes PANCREAS HEART Control HEART HEART 20 µm 20 µm 20 µm 20 µm HEART HEART ntibody Staining AA ngo Red -Amylin ii oo C Ant 20 µm 20 µm Despa S, …, Despa F. Circ Res. 2012;110:598 Selection of Human Brain Samples Type-2 Diabetes + CD and/or AD Amylin Accumulation Late-onset AD no diabetes Amylin Accumulation ? Age-matched, lean (?), non-diabetics, without AD Amylin Deposition in the Brain of Patients with Dementia A 20 µm B 20 µm C 20 µm 20 X 20 X 20 X D 20 µm E 20 µm F 20 µm G 20 µm 20 X 20 X 20 X PANCREAS 20 X Amylin co-localizes with Aβ T2D-AD Group ACB 10 µm 10 µm 10 µm D E F 10 µm 10 µm 10 µm 40 X Not All Amylin Species are Amyloidogenic ! Human Amylin (amyloidogenic) KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY Rat Amylin (non-amyloidogenic) KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY Despa et al., Biol. Phys. (2008) Pancreas Heart PancreasHeart 25 15 10 kDa HIP rats UCD-T2DM rats Animal Models Human Amylin pancreas HIP rat Rat Amylin pancreas 200 Pre-Diabetes 180 UCD-T2DM rat 160 g/dl) DM Glucose -fasted 22 mm 140 ( non 120 Blood UCD-T HIP 100 Summary (1) Circulating Amylin Oligomers mitochondrial attachment to sarcolemma attachment to endothelial dynamics ? cells ors Ca transients ROS RAGE CaMKII/HDAC Calcineurin/NFAT ? Other fact activa tion activa tion NF-κB Hypertrophic signaling eostasis; mm SERCA express ion TNF-α; IL-6; IL-10 ? Diastolic [Ca]i / lipid ho Slower Ca transient inflammation ee relaxation d glucos e e Diastolic Ca transients dysfunction Oligomeric amylin accelerates Alter diabetic HF ! Systolic dysfunction End SystolicPressure Left-Ventricular -dP/dtmin (mmHg/s) End Systolic P (mmHg) 2000 4000 6000 Cardiac Accumulation of Oligomeric Amylin Induces Oligomeric Amylin Cardiac Accumulationof 120 40 80 Contractile Dysfunction in Pre-diabetic HIP Rats Contractile DysfunctioninPre-diabetic HIP 0 0 RIP RIP * HIP * HIP Pressure Fall Maximum Rateof collab. with A. Knowlton (UC Davis) collab. with Fall PRi P Maximum Rateof ressure Ri se End Diastolic Pressure Left-Ventricular dP/dtmax (mmHg/s) End Diastolic P (mmHg) 2000 4000 6000 8000 6 8 0 2 4 0 RIP RIP HIP HIP Cerebral Accumulation of Oligomeric Amylin Induces Behavioral Changes in HIP Rats collab. with S. Xie (Stanford) Therapeutics: Reversingggyg/Preventing Amylin Oligomer-Induced Injjyury Patent US20070031955 “Compositions and Methods for Refolding of Denaturated Proteins” (sold to Maroon Biotech). Circulating Amylin Oligomers ↑ EETs ↓ sEH APAU attachment to sarcolemma s s mitochondrial dynamics Ca transients ROS her factor tt Ca MKII/HDAC CliCalcineur i/NFATin/NFAT activation activation stasis; O o o Hypertrophic signaling SERCA expression pid home Diastolic [[]Ca] ii i Slower Ca transient relaxation glucose/ l Ca transients Diastolic dysfunction Altered Systolic dysfunction Summary Hyperamylinemia and consequent amylin oligomerization are an early pathogenic mechanism linking age-related metabolic disorders with diabetic brain injury, dementia, and CVD. Hyperamylinemia is a feasible therapeutic target to reduce accumulation of proteinaceous debris in the CV and CN systems, and thus to limit/ delay diabetes complications..