Diabetes Mellitus Type 1 Diabetes and Its Current Treatments

1. Diabetes MellitusType 1 Diabetes and Its Current Treatments Michelle Adams CHEM 5389 April 3, 2007

2. Presentation Outline Diabetes • What is Diabetes? • How do people get diabetes? • What are the signs and symptoms? • Importance of Control – Complications of Diabetes Control • Being In Control • Methods for Control Insulin • History of Insulin • Insulin Structure • Insulin Synthesis • Insulin Secretion • Insulin Receptor • Insulin Synthetically Produced • Insulin Analogues Developments • Recent developments in the field

3. What is Diabetes? • Diabetes Mellitus – the presence of elevated glucose levels in the blood due to absolute or relative insufficiencies of insulin • 171 million people suffer from diabetes Type 1 • Autoimmune destruction of the b cells in the pancreas which are used to product insulin 1. Insulin administration Type 2 • Combination of defective insulin secretion and increased insulin resistance • Generally seen in overweight patients (85%) • Thought that obesity contributes to insulin resistance due to secretion of hormones called adipokines that impairs glucose tolerance • Used to be seen in older patients, now disease is becoming more prevalent in children as obesity grows among children 1. Life style change 2. Oral medications i. Improve insulin production ii. Regulate abnormal release of glucose by the liver iii. Decrease insulin resistance 3. Insulin administration Gestational diabetes • Insulin resistance caused by hormones during pregnancy • Usually improves or disappears after giving birth • Seen in 5% of pregnant women 1. Diet control

4. How do people get diabetes? Type 1 • Genetic element/mutation, susceptibility to triggers: • Viral infections • Stress • Environmental exposure - exposure to certain chemicals or drugs • White blood cells, T lymphocytes, produce immune factors called cytokines which attack and destroy b cells of pancreas • Can take 7yrs. or longer to develop to absolute, by the time know something is wrong 80% - 90% of b cells are destroyed • 10% chance of inheriting if first degree relative has diabetes • Most likely to inherit from father • Increase incidences would take at least 400 years if genetic factors were the only cause Viruses • Infection introduces a viral protein that resembles a b cell protein • T-cells and antibodies tricked by this resemblance into attacking b protein and virus • Cases rising in certain areas of U.S. – particularly Northeastern region • Cow’s milk – certain protein which may trigger attack on b cells • Breast milk – hormones which protect body from attack on b cells Type 2 • Inheritance pattern, first degree relatives with type 2 have much higher risk for developing • Perhaps inheriting a tendency towards obesity since 85% obese Gestational • Genetically predisposed, have greater chance for developing type 2 later in life

5. What are the Signs and Symptoms? Polyuria (frequent urination) • Glucose concentration in blood is high • Reabsorbtion of glucose in the proximal renal tubuli is incomplete, glucose remains in urine • Osmotic pressure of urine increases • Inhibits reabsorbtion of water by kidney, resulting in increase urine production Dehydration • Lost watervolume in kidney replaced from water held in body, increased thirst and increased fluid intake - polydipsia Polyphagia • Increased appetite, no glucose delivered to muscles, tissues, body sends signal to brain to eat something to renourish Weight loss and weakness • glucose cannot participate in crib cycle to be used as energy, use of fat as alternative energy source Vision changes • changes shapes of lens in eye

6. Importance of Control – Complications of Diabetes Diabetic Ketoacidosis • Fat break down accelerates and increase the production of fatty acids • Fatty acids converted into ketone bodies • Ketones are toxic at high levels • Symptoms - rapid, deep breathing, polyuria, nausea, vomiting, abdominal pain, altered states of mind such as hostility, mania, confusion, lethargy, and hypotension, coma, death Hypoglycemia • Low blood sugar, too much insulin or not enough glucose to cover insulin treatment • Symptoms - sympathetic activation of the autonomic nervous system: immobilized panic, dread, agitated, sweaty, seizures Amputations • Heal slowly • Fail to heal • Infection Vascular diseases • Damage to blood vessels • Diabetic retinopathy – growth of poor quality new blood vessels in retina, retinal damage, blindness • Diabetic nephropathy – damage to kidney, chronic renal failure – dialysis • Damage to arteries • Coronary artery disease, stroke, peripheral vascular, diabetic myonecrosis (‘muscle wasting’) • Diabetic foot – neuropathy and arterial disease

7. Being In Control Non-diabetic Generally between 80mg/dL-120mg/dL • Fasting glucose level: <110mg/dL • 2 hours after a 75g carb meal: <140mg/dL • 110mg/dL-125mg/dL: impaired fasting glucose • By definition 2 fasting glucose above 126 mg/dL – positive for diabetes Diabetic Goals • 90mg/dL-130mg/dL before meals • 110mg/dL-150mg/dL bedtime HbA1c (glycosylated hemoglobin) – measures the level of glucose irreversibly bound to hemoglobin, 90 day measure of average blood sugar – can be misleading • <6.0% for non diabetics = 114mg/dL • <7.0% for diabetics = 147mg/dL • Control best obtained with pre-meal testing, 2 hour post meal testing, and bed time = 7x per day • Lows more frequent in controlled diabetics, can’t feel them as well • Long term diabetics, may not feel lows as well • Lows can occur more in less educated diabetics • Exercise – increases insulin sensitivity

8. Methods for Control • Daily Injections • Bolus insulin • Basal insulin • Two to five shots a day • Insulin Pump Therapy • Constant insulin delivery • One insulin type become the bolus insulin and the basal insulin • Catheter changed every three days

9. History of Insulin • 1920 – Insulin is first discovered by Fredrick Banting and Charles Best • November 1922 - Eli Lilly and Company is first to produce large amounts of insulin • 1923 - MacLeod and Banting receive Nobel Prize in Physiology and Medicine for insulin discovery • 1964 - Dorothy Crowfoot Hodgkin receives Nobel Prize in Chemistry for determination of spatial conformation of molecule • 1980 - Frederick Sanger, British molecular biologist, receives Nobel Prize in Chemistry for amino acid structure determination

10. What is Insulin?

11. 3D Structure of Insulin

12. Insulin Synthesis • Produced within the pancreas by b cells, islets of langerhans – identified by Paul Langerhans in 1869 Islets of Langerhans – 1 million islets a cells – secrete glucagons b cells – produce insulin, most abundant d cells – secrete somatostatin • It is very difficult for small proteins to fold into stable structures so larger precursors are synthesized first Stored as b granule • When b cell is appropriately stimulated, insulin is secreted from the cell by exocytosis and diffuses into islet capillary blood

13. Insulin Secretion • Glucose transported into b cell by a glucose transporter • Results in membrane depolarization and an influx of extracellular calcium • Fusion of insulin storage vesicle in plasma occurs • Hexamer released from cell as crystal and dissolves to monomer Reasons for monomer transformation: • Change in pH • Loss of ligands due to dilution, dissociation of allosteric ligands • Endogenous chelator removes the His B10 Zn2+ ions

14. Insulin Receptor • 2 a subunits linked by disulfide bonds to 2 b subunits • Insulin binds to a subunits • Causes autophosphorylation of b subunits within plasma • Activates catalytic activity of receptor • Phosphorylates a number of intracellular proteins • Website: http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/pancreas/insulin_phys.html

15. How is Insulin Synthetically Produced by Recombinant DNA? • Isolate Gene (pig) • Prepare target DNA (cut plasmid ring open with special proteins) • Insert DNA into plasmid (circular piece of DNA) • Insert Plasmid back into cell • Plasmid multiplies • Target cells gathered and purified • Cells used are e-coli and yeast

16. Normal Insulin Secretion and Insulin Analogues

17. Insulin Stability Insulin decomposition • Hydrolysis and intermolecular transformation leading to covalent insulin dimers Hexamer Structure • Hexamer – helps with storage in pancreas • Zn2+ and Ca2+ binding likely becomes significant only at a tetrameric stage of assembly • Insulin hexamer much more stable to chemical and/or physical degradation than the insulin monomer

18. Regular Insulin • No alteration in structure of human insulin • Insulin present in hexameric and monomeric form • Fast absorption of the insulin is due to the monomeric form already present • Does not peak until 1 to 2 hours later because hexameric form must be converted to monomeric form • Lasts about 4 to 6 hours in body

19. Fast-acting Insulin • Lispro (Humalog): B28 and B29 amino acids reversed • Aspart (Novolog): B28 replaced with Asp • Glulisine (Apidra): B3 replaced with Lys, B29 replaced by Glu • Inhibiting the molecule's natural tendency to form hexamers by self-association, means better, faster absorption, more rapid onset and peak, and shorter duration

20. NPH • Neutral Protamine Hagedorn • Protamine and Zinc added to the insulin structure • These additions resulted in more of the hexameric form present in the mixture than the monomeric form • Thus, more hexamers had to be transformed to monomers for insulin absorption • The duration, onset, and peak of the insulin prolonged

21. Glucagon • Linear peptide, 29 amino acids • Synthesized as proglucagon • Brain can only use glucose, not alternative energy sources like fatty acids • Insulin stimulates liver to store glucose in the form of glycogen In liver: 1. Stimulates break down of glycogen stored in liver 2. Activates hepatic gluconeogenisis: non-hexose substrate (amino acids) are converted to glucose Injection of this hormone is given to patients when seizures from low blood sugars occur Seizures most likely to occur during the middle of the night, most specifically when using Regular insulin and NPH insulin

22. Insulin Glargine - Lantus • Addition of positively charged amino acids • Isoelectric point of human insulin is at pH = 5.4 • Isoelectric point of insulin glargine is at pH = 6.8 • Soluble in acidic environment, formulated to a pH of 4 • Forms microprecipitate after injection because of neutral pH environment • Crystals slowly dissolve – slow release of insulin dimers and monomer to tissue and blood stream • Very little peak

23. Insulin Detemir - Levemir • Addition of fatty acid moiety on lysine in position B29 and removal of threonine from position B30 • Fatty acid is a 14-carbon fatty acid side chain, Myristic acid • Binds reversibly to albumin, contributing to long duration action (20hrs) • Very little peak

24. Recent Developments • Constant glucose monitoring – sensor records blood glucose every 10 seconds and sends an average of the glucose measurements every 5 minutes to the pump for 3 days • Calibration by glucometer still required • Symlin – analog of human amylin, a hormone that contributes to glucose control during postprandial periods • Slows gastric emptying • Supresses glucagon secretion • Implanted Insulin Pump (not yet fully developed) • Transplants • Pancreas and kidney dual transplants have been successful, must take immunosuppressant for rest of life • Islet cell injections – several injections must take place, not viable for the whole population because costly and not enough supply, most take immunosuppressant for rest of life

25. Conclusions • Diabetes is a rapidly increasing disease • Controlling diabetes is extremely important for good health • Control for type 1 diabetes must be obtained by insulin administration • The hexamer form of the insulin molecule is a very important aspect in designing insulin analogues • Several insulin analogues can be used in conjuction for treatment • Recent developments offer better control but much research is still needed to help control diabetes and possibly find a cure for it

26. References B. Cheatham, C. Kahn, Endocrine Reviews, 1995, 16, 117-142 W. Duckworth, R. Bennet, F. Hamel, Endocrine Reviews, 1998, 19, 608-624 S. Taylor, A. Cama, D. Accili, F. Barbetti, M. Quon, M. De La Luz Sierra, Y. Suzuki, E. Koller, R. Levy-Toledano, E. Wertheimer, V. Moncada, H. Kadowaki, T. Kadowaki, Endocrine Reviews, 1992, 13, 566-595 Z. Vajo, J. Fawcett, W. Duckworth, Endocrine Reviews, 2001, 22, 706-717 J. Brange, Diabetologia, 1997, 40, S48-S53 U. Derewenda, Z. Derewenda, E. Dodson, G. Dodson, X. Bing, J. Markussen, Journal of Molecular Biology, 1991, 220, 425-433 M. Dunn, BioMetals 2005, 18, 295-303 J. Goldman-Levine, K. Lee, The Annals of Pharmacotherapy, 2005, 39, 502-507 A. Barnett, Diabetic Medicine, 2003, 20, 873-885 T. Levien, D. Baker, J. White Jr., R. Campbell, The Annals of Pharmacotheraphy, 2002, 36, 1019-1027 http://en.wikipedia.org/wiki/Diabetes http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/index.html