Insulin

What is diabetes mellitus?

Type 1 (body cannot produce insulin). Type 2 (body produces some insulin - linked to being overweight)

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Describe features of glucose absorption (1)

In the absence of insulin, glucose cannot enter the cell. Insulin signals the cell to insert GLUT4 transporters into the membrane to allow glucose to enter the cell (insulin binds to receptor, signal transduction cascade, exocytosis, glucose enters)

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Describe features of glucose absorption (2)

When blood sugar levels rise (normal after meal), the pancreas secretes insulin. Insulin enters and circulates in the blood, acts on insulin receptors present in muscle/fat/tissues in the body. Binding of insulin to receptors

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Describe features of glucose absorption (3)

Glucose transporters come to the cell surface. Facilitates entry of glucose into cells

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Describe features of type 2 diabetes

Production of insulin is low and there might be insulin resistance. Circulating insulin fails to facilitate absorption of glucose into cells in order to keep blood glucose level at optimum levels. Results in increased blood glucose levels

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Describe features of AGE products (1)

Excess glucose in the blood reacts with proteins in tissues to form advanced glycation end products (proteins modified/unable to function). AGEs create an inflammatory condition in the vasculature (cause heart disease, damage kidneys)

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Describe features of AGE products (2)

Diabetes can eventually cause damage to (heart, arteries, kidneys, nerves, eyes, skin)

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Describe features of AGE products (3)

Reaction of glucose with protein amino groups. Aldehyde reacts with protein amino group, amidori rearrangement (AGE). Non-enzymatic amadori reaction – glucose reacts with amino group, rearrangement, Hb modification (not able to carry O2)

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Outline the change in insulin levels (1)

Insulin secreted by beta cells in the pancreas. Insulin secretion triggered by elevated blood glucose. After meal, blood glucose levels are between 60-80 micro units/L, basal levels of 5-15 micro units/L. Change triggers insulin secretion

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Outline the change in insulin levels (2)

Basal level of insulin (low/constant). Spikes in insulin level. Need to have low insulin before eating. Need spikes in insulin levels

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Describe features of insulin actions

Anabolic hormone. Increases synthesis of glycogen/fat/protein. Increases transport of glucose into cells. Increases activity of enzymes. Gene regulation

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Describe features of insulin structure

51 amino acids, polypeptide, 2 chains linked by inter chain and intra chain disulphides. MW 6000 Da, A chain with 21 amino acids, B chain with 30 amino acids. Protein, 3D structure, alpha helix, beta sheet. pI of 5.3

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What is pI?

Isoelectric point (pH at which the molecule carries no net charge). Insulin is an overall acidic molecule

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What is the difference between bovine insulin and human insulin?

Two amino acids are different (cause of immunogenic reaction)

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State features of disulphide bonds

Disulphide bonds between cystine residues. Number of cystine residues leads to many combinations (table)

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Describe how insulin is produced in the body (1)

Peptide hormone states of pre-proinsulin translocated from mature polyribosome in ER membrane and is spontaneously translocated into ER lumen. In ER lumen, signal sequence is cleaved during translocation forming proinsulin

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Describe how insulin is produced in the body (2)

Folding occurs and disulphides are formed and a hexamer is formed with Zn. Proinsulin travels to Golgi via vesicular transport. Proinsulin moves through Golgi then trans Golgi network and immature granule

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Describe how insulin is produced in the body (3)

It is cleaved by a pro protein convertase yielding insulin with C-peptide. Proinsulin stored in secretory granule. Upon an influx of Ca 2+ the secretory granule fused with basolateral plasma membrane and releases insulin to EC space

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What are the features of pre-proinsulin?

Pre-region (hydrophobic, pass through membrane). B chain. C-peptide. A chain. Disulphide bonds.

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What are the features of proinsulin?

B chain. A chain. C-peptide. Pre-region dropped off, binds to Zn, disulphide bones, hexamer formed. Enzyme to cleave off signal peptide to form pro-insulin. 86 amino acids, 6 of amino acids form disulphide bones in specific way

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Describe features of insulin secretion (1)

Insulin synthesised on ribosome as pro-insulin (86 amino acids). Folds into tertiary structure with 3 disulphides. Pro-insulin forms hexamer by complexing with Zn ions binding to His-10. Octahedral complex. Process takes 30 min

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Describe features of insulin secretion (2)

Pro-insulin hexamers stored in Golgi. Cleaved by trypsin like enzyme selective for double charges residues e.g. Lys-Lys or Arg-Arg. Insulin hexamers slowly formed over 1-2 hr. Conversion of pro-insulin hexamer to insulin hexamer

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Describe features of insulin secretion (3)

Removes 6 x C-peptide, contains 24 glutamate and 6 aspartate residues. Insulin hexamer is less soluble and precipitates. Storage vesicle contains (insulin)6Zn2+ precipitates. Survives for hrs/days until next meal

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Describe features of insulin secretion (4)

pH of vesicle is 5.5. and [Zn 2+] = 10^-3 M. Insulin released into blood. pH is 7.4 and [Zn 2+] < 10^-6 M. Zn 2+ dissociates. Hexamer unstable at low concentration. 10^-3 M vesicle. 10^-6 M blood. Insulin dissociates into monomer - active species

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Describe features of insulin secretion (5)

C-peptide removed from proinsulin to form insulin (A chain, B chain, disulphide bonds). Insulin hexamer is effectively a delivery system. Release of monomer is gradual. Very difficult to mimic in therapeutic doses of insulin when given by injection

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What are the main functions of chymotrypsin, trypsin and elastase?

Chymotrypsin (recognises aromatic groups and breaks the bond) . Trypsin (recognises basic residues and cleaves). Elastase (cleaves amino acids with small side chains)

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What are the differences in amino acids for human/bovine/porcine insulin?

Human (Thr, Ile, Thr). Bovine (Ala, Val, Ala)

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Describe the semi-synthesis of human 'actrapid' Novo (1)

Porcine insulin treated with enzyme, remove alanine amino acids. Trypsin residue recognises basic residue. Use Thr(tBu)COOtBu. SEC, anion exchange chromatography. Trifluoroacetic acid. RP-HPLC

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Describe the semi-synthesis of human 'actrapid' Novo (2)

Trypsin attacks amide in porcine insulin, removal of alanine, amino acid group attacks to remove enzyme group. Use of trifluoroacetic acid. Size exclusion, reverse phase purification. Human insulin formed

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What is the strategy for synthesising insulin?

Synthesise insulin with three different groups protecting thiols - selective disulphide formation, protecting groups not available - synthesis of A chain and B chain separately. Refold insulin - difficult task

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Outline how recombinant insulin is synthesised (1)

Gene encoding polypeptide chain and gene encoding beta-galactosidase incorporated into plasmid. Inserted into E.coli. Protein production. Separation and purification. A and B chains formed separately. A and B chains combined to form insulin

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Outline how recombinant insulin is synthesised (2)

Collect bacteria, break open cells, purify insulin protein expressed from recombinant human insulin gene

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Outline the rearrangement of disulphide bonds

Reduced substrate protein and oxidised PDI (protein disulphide isomerase). Reduced PDI, oxidised substrate protein. Protein with incorrect disulphide bonds to protein with correct disulphide bonds. Disulphide bonds formed in lumen of RER

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State features of protein expression systems

Properties of insulin depends on the type of cells the protein grows in e.g. some folding in E.coli, folding in insect cells

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Give examples of insulin preparations

Regular (Zn and cresol - antioxidant). NPH insulin (Zn, cresol, phenol, protamine - protein with lots of amino acids with + charge, highly basic, acidic insulin binds to protamine and is released slowly, lasts longer). Lente Zn. Ultaralente

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What do insulin properties depend on? (1)

pH, temperature, ionic strength, concentration and ions. Monomeric insulin (pH 2, low concentration). Dimerises as pH increases. Complex formation above pH 4 in presence of Zn. pH 4-7 complexes precipitates as amorphous aggregates

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What do insulin properties depend on? (2)

Amorphous precipitate crystallises at pH 5.6-6.0. Insulin - monomer, dimer, insulin aggregates, insulin hexamer T6, insulin hexamer R6 (tense/relax, conformation). 10^-3 M to 10^-5 M to 10^-8 M. Insulin monomer enters capillary

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How is an insulin dimer formed?

Form dimer (COOH, NH2, electrostatic interaction between NH3+ and COO-), hydrophobic interaction (chains in opposite directions)

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How would you stop an insulin dimer being formed?

Stop electrostatic interactions, insulin will form a monomer (fast acting)

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Describe features of Lispro (1)

Humolog. First fast acting insulin (can have 10-15 mins before eating) – don’t have electrostatic interaction, dimer not formed, only form monomer, absorption at millimolar concentration (proline/lysine swapped round). 10^-3 M, insulin in capillary

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Describe features of Lispro (2)

Lysine and proline exchanged at positions B28 and B29 and the equilibrium shifts away from hexamer formation thus enhances the rate at which it dissociates into dimers and then into monomers and the rate of absorption is increased

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Describe features of Aspart (1)

Novolog. New aspartic acid instead of a proline, add a negative charge, two negative charges (repel). Aspartate is substituted for proline at B28 that shifts the equilibrium away from hexamer formation that reduces the tendency to form hexamers

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Describe features of Aspart (2)

Increases rate of absorption (faster onset of action)

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Describe features of Glulisine

Apidra. Glutamic acid present, faster absorption, also has a lysine chain. Eliminates ability to form dimer of Zn hexamer. Faster absorption rate. Insulin of choice in insulin pumps - no clogging

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Describe features of Glargine (1)

Lantus. Two arginines, pI changes, micro-precipitate, binds to albumin, slowly released, long lasting insulin. pH 4 solution, low solubility at neutral pH. Upon SC injection solution is neutralised, micro-precipitate. Binds to albumin

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Describe features of Glargine (2)

Slowly released over 24hr. Can be used as basal insulin injection OD

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Describe features of Determir

Levimer. B-chain Thr(30) deleted. B chain Lys (29), amino acid group myristoylated (C14). Lipid moiety slows absorption. Once in circulation bound to albumin. Slows absorption across endothelium. Less weight gain in type 2 diabetes

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Describe features of Degludec (1)

Most recent insulin on the market. B-chain Thr(30) deleted. B chain Lys (29), amino acid group myristoylated (C14). Lipid moiety slows absorption. Once in circulation bound to albumin. Slows absorption across endothelium. Less weight gain in T2DM

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Describe features of Degludec (2)

Multimeric chains follow SC injection. PK/PD half life of 25h. In presence of phenol and Zn (pharmaceutical formulation). Forms stable soluble dihexamer. After injection, phenol diffuses away, reorganisation to form multi-hexamer chains

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Describe features of Degludec (3)

That reside a long time as injection depot. With gradual diffusion, Zn chains disassemble to release insulin monomers. Terminal COOH of fatty acid chain is essential. Co-ordination of terminal COOH residue of fatty acid to Zn atom of other hexamer

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Describe features of Degludec (4)

Injected formation (T3R3 state). Depot formulation (T6 multi hexamer state). Dimer to monomer - absorption

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What are the fast acting insulin preparations?

Lispro, Aspart and Glulisine (table)

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What are the long acting insulin preparations?

Glargine and Detemir (table)

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Describe features of the different insulin preparations (1)

Basal insulin, constant peak, less kinetic profile that mimics normal basal insulin secretion. Poorly soluble insulin protamine - NPH insulin. High Zn formulations (Lente insulin). pH dependent precipitation - Glargine

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Describe features of the different insulin preparations (2)

Local albumin binding - Determir. Glargine/Detemir - improved profiles. Truly stable 24h basal blood glucose lowering achieve steady state kinetics. Absorption rate correlates with mwt. Multimeric insulin could provide a new approach - link hexamers

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State the structure complex of insulin in the formulations (1)

Degludec (dihexamer to multihexamer). Glargine (U300 or U100 hexamer, aggregates). Detemir (dihexamer, bind to albumin). NPH (hexamer-protamine). Regular insulin (hexamers, hexamers/dimers/monomers)

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State the structure complex of insulin in the formulations (2)

Lispro/aspart/glulisine (hexamer/multimers, hexamer/dimers/monomers). Fasting acting aspart (hexamers, hexamers/dimers/monomers). Rank slow-fast, rank long acting - short acting order (graphs)

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Insulin

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