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

100 UCD-T HIP 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 expressi on  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 Cardiac Accumulation of Oligomeric Amylin Induces Contractile Dysfunction in Pre-diabetic HIP Rats

120 8000 *

Maximum Rate of Hg/s) 6000 (mmHg)

80 mm PP PRiPressure Rise

(m 4000

40 max 2000 Systolic P P P P P/dt II II II II dd d R H 0 R H 0 En Left-Ventricular End Systolic Pressure Left-Ventricular End Diastolic Pressure Hg) s)

// 8

6000 mm * 6 4000 (mmHg Maximum Rate of

in 4 olic P P (m olic tt mm 2000 Pressure Fall 2 -dP/dt RIP HIP 0 0 RIP HIP collab. with A. Knowlton (UC Davis) End Dias 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.