5 - Insulin Flashcards
Blood glucose regulation
- High BG stimulates insulin release; low BG stimulates glucagon release
- Glucagon stimulates glycogen breakdown
- Insulin stimulates glycogen formation and stimulates glucose uptake from blood
- Glycogen broken down into glucose (and glucose formed into glycogen) in the liver
Physiological activities of insulin
- Stimulates glucose transport (muscle cells, lipocytes, hepatocytes)
- Stimulates amino acid transport (hepatocytes, muscle cells)
- Increases glycogen synthase activity (hepatocytes)
- Increases protein synthesis and decrease protein degradation (hepatocytes, muscle cells)
- Depresses lipolysis (lipocytes)
Insulin structure
Signal peptide – B-chain – C-peptide – A-chain
- Signal peptide = 24 aa
- Peptide (B chain) = 30 aa
- Propeptide (C peptide) = 31 aa
- Peptide (A chain) = 21
Function of C-peptide of insulin
To measure insulin levels in the blood
Intrinsic property of insulin (charge)
- Net charge of the molecule
- Negatively charged at neutral pH
- Net charge on insulin molecule produced from the ionization potential of 4 glutamate residues (found in A and B chains), 2 histidine residues (found in B chain), lysine residue (found in B chain), arginine residues (found in B chain), and 2 alpha-carboxyl and 2 alpha-amino groups
- Insulin has isoelectric point (pI) of 5.3 in the denatured state
Different association force of insulin molecule
- Hydrophobic interactions at the C-terminus of the B-chain are critical for formation of dimers
- Zinc molecules can be associated w/ 3 insulin monomers at HisB10 residue of each monomer
- Phenolic species (ex: phenol, m-cresol, or methylparaben) bind to specific sites on insulin hexamers, causing a conformational change that increases the chemical stability of insulin in commercial preparations
- Phenolic ligands are bound w/ insulin by H-bonds w/ the carbonyl oxygen of CysA6 and the amide proton of CysA11 as well as numerous van der Waals contacts in a binding pocket between monomers of adjacent dimers
- Binding of these ligands stabilizes a conformation change that occurs at the N-terminus of the B-chain in each insulin monomer
- Shifting the conformational equilibrium of residues B1 to B8 from an extended structure (T-state) to an alpha-helical structure (R-state) strengthens association of insulin molecule
- This conformational change is referred to as the T-R transition
Association of insulin
Monomer dimer higher-order associated states (Zn2+) hexamer (T6) (phenolic preservative) hexamer (R6)
**Monomer is the simplest form that is used in the cells; dimer doesn’t work in the cell
What are insulin analogs?
Modifications of natural insulin, where changes are made in the AA sequence of the insulin molecule that affect the duration of action
Describe the amino acid substitutions at A21, B28 and B29 for humulin and novolin
- A21 = Asn
- B28 = Pro
- B29 = Lys
Describe the amino acid substitutions at A21, B28, B29, and B30 for porcine and bovine insulin
- A21 = Asn
- B28 = Pro
- B29 = Lys
- B30 = Ala (everything else = Thr)
Describe the amino acid substitutions at A21, B28 and B29 for lispro
- A21 = Asn
- B28 = Lys
- B29 = Pro
Describe the amino acid substitutions at A21, B28 and B29 for aspart
- A21 = Asn
- B28 = Asp
- B29 = Lys
Describe the amino acid substitutions at A21, B28, B29, B31, and B32 for glargine
- A21 = Gly
- B28 = Pro
- B29 = Lys
- B31 = Arg
- B32 = Arg
Describe the amino acid substitutions at A21, B28 and B29 for detemir
- A21 = Asn
- B28 = Lys-(N-tetradecanoyl)
- B29 = Pro
Steps of site-directed mutagenesis of insulin
- Determine which site you want to mutate
- Clone gene cDNA into a selectable plasmid
- Amplify gene sequence by PCR
- Remove un-amplified gene sequence
Sites to mutate on regular insulin to make rapid acting lispro
- Proline replaced w/ lysine at B28
- Lysine replaced w/ proline at B29
Design of site-directed mutagenesis primers
- Mutation should be in the middle of the primer
- Primers should be 25-45 nucleotides long and have a GC content of at least 40%
- Melting temp should be 78 C or greater
- 3’-end of the primer is better to end on a C or G
PCR to remove un-amplified DNA sequence
- Methylate plasmid first
- Denature the plasmid and anneal the oligonucleotide primers containing the desired mutation
- Using the non-strand-displacing action of PfuTurbo polymerase, extend and incorporate the mutagenic primers resulting in nicked circular strands
- Digest the methylated, nonmutated parenteral DNA template w/ Dpn 1
- Transform the circular, nicked dsDNA into super-competent cells
Identification of mutated sequence
- Transform into bacteria
- Pick up a colony
- Isolate plasmid DNA from bacteria
- Digest w/ restriction enzymes (BamH1 and Xho1)
- Send out to sequencing facility
- Compare DNA sequence w/ original sequence
Describe the dissociation of regular insulin after SQ injection
Hexamer (R6) hexamer (T6) dimers monomers
- Only monomer can bind to receptor
- Dimer and monomer can cross biological membrane and interact w/ cells
- Phenolic preservative heavily concentrated around hexamer (R6) but gradually released as insulin dissociates into monomers
Lispro (Humalog) – describe the changes made to the structure and what impact they have
- Lysine and proline at the end of the B-chain are reversed => greater steric hindrance and reduced ability to form insulin dimers and hexamers
- Doesn’t alter receptor binding
- Allows larger amounts of active monomeric insulin to be immediately available for postprandial injections
Aspart (NovoRapid) – describe the change made to the structure and what impact it has
- Proline at the end of the B-chain is changed to aspartic acid => greater charge repulsion and steric hindrance due to local conformational change in the B-chain
- To be absorbed quickly into the bloodstream
Glargine (Lantus) – describe the changes made to the structure and what impact they have
- Synthesized from non-disease producing strain of E. coli
- Modification of 3 amino acids:
- 2 positively charged arginine molecules added to the c-terminus of the B-chain -> shifts pI from 5.4 to 6.4
- Asparagine at position 21 in the A chain is replaced by glycine -> prevents deamination (loss of amino group) and dimerization (production of polymers) of the arginine residue
- The shift in isoelectric point from 5.4 to 6.7 makes insulin less soluble at physiological pH (7.4) and more soluble at acidic pH
- Glargine formulated at pH 4
- At injection site (pH 7.4), the increase in pH causes acidic insulin solution to precipitate
- Precipitate slowly dissolves, causing gradual release of monomers into blood
- Release of insulin further delayed by addition of Zn2+ to the formulation
Detemir (Levemir) – describe the changes made to the structure and what impact they have
- Synthesized from bakers’ yeast
- Modifications:
- 14 carbon fatty acid chain (myristic fatty acid or tetradecanoil) is attached to the lysine residue on position 29 of insulin B-chain -> promotes self-association of insulin molecules and binding to albumin at injection site
- Removal of threonine from position 30 of the insulin B-chain
- Fatty acid chain:
- Allows insulin to be formulated as a neutral solution which doesn’t precipitate after injection
- Contributes to hexamer formation and delays hexamer dissociation allowing the solubilized insulin to remain in depot
- Allows insulin to be 99% albumin-bound, which buffers against sudden changes in insulin concentration and absorption thus reducing risk of hypoglycemia
- Albumin binding also acts to slow diffusion of insulin into interstitial compartment
Summary of insulin structure
- 2 zinc molecules help insulin to form hexamer
- Extra zinc molecules on outside of insulin hexamer increase association of insulin molecule
- Phenolic species help to induce T -> R transition
- pH shift from neutral to acidic helps insulin to precipitate at physiological pH
- Addition of fatty acid allows insulin binding to albumin
- Addition of a protein (ex: protamine) helps form an insulin-protamine complex
- Formation of crystal delays release of insulin
Intermediate acting insulin – examples and what general changes are made
- NPH (neutral protamine Hagedorn) – insulin co-crystallized w/ protamine; antigenic properties
- Lente – insulin formulated w/ zinc; intermediate action due to crystallization and zinc hexamer formation which increases duration of action and slows onset time
NPH insulin – describe the changes made to the structure and what impact they have
- Neutral crystalline suspension prepared by co-crystallization of zinc hexamers of insulin w/ protamine
- Protamine = small proteins composed greatly of arginine; when mixed w/ insulin, slows down onset and extends duration of action
- Very minimal levels of soluble insulin or protamine in solution
- Crystalline formulation of insulin allows an extended time to action b/c of required dissolution time
Lente insulin – describe the changes made to the structure and what impact they have
- Preparation of 2 insoluble insulins w/ zinc (70% rhombohedral zinc insulin crystals, 30% amorphous insulin particles)
- Forms hexamers (6 insulin molecules, 2 zinc atoms, 6 water molecules)
- Excess zinc binds to outside of hexamers, increasing dissolution time
- Only monomers are physiologically active, dissociation is required => extended time-action effect
Ultralente insulin – describe the changes made to the structure and what impact they have
- Like NPH, is a crystalline insulin formulation
- Larger rhombohedral crystals
- No protamine
- Crystallized at pH 5.5 w/ zinc, NaCl and acetate buffer
- Adjusted to pH 7.4 and addition of excess zinc and methylparaben as a preservative
Premixed insulin
- Several different mixes available, the following are available in Canada:
1. Humalog mix 25 = 25% lispro (rapid acting) and 75% lispro protamine (intermediate acting)
2. Humulin (20/80, 30/70) = 20% regular insulin and 80% NPH insulin (intermediate acting)
3. Novolin ge (30/70, 40/60, 50/50) = 30% Toronto (regular insulin) and 70% NPH insulin (intermediate acting)
Chemical stability of insulin formulations
- Hydrolytic transformation of amide to acid groups
- Asparagine (AsnA21) is the primary degradation mechanism of insulin formulation at acidic pH (asparagine -> aspartic acid)
- Deamidation of AsnB3 is the primary degradation mechanism insulin formulation at neutral pH (asparagine -> aspartic acid or iso-aspartic acid)
- Formation of covalent dimers and higher order polymers
- High molecular weight protein (HMWP) can be formed at both storage and room temp; higher temps = higher order insulin oligomers
- Rate of HMWP formation can be affected by strength of insulin formulation and addition of glycerol as an isotonicity agent
- Biopotency of HMWP is significantly less (1/10 to 1/5 of insulin) than monomeric species
Physical stability of insulin formulations
- Mediated by non-covalent aggregation of insulin
- Hydrophobic forces typically drive the aggregation although electrostatics plays a subtle but important role
- Aggregation leads to a loss in potency of the formulation
- Physical changes in soluble formulations = colour or clarity change, formation of precipitate
Amino acid modifications of insulin
- Disulfide bond 31 -> 96 interchain
- Disulfide bond 43 -> 109 interchain
- Disulfide bond 95 -> 100
How do you identify success of site mutagenesis of insulin?
- Transform into bacteria
- Antibiotic selection of positive clone
- Restriction enzyme digestion and sequencing gene
- Compare gene sequence w/ normal gene sequence
Which codons encode lysine?
AAA and AAG
Which codons encode proline?
CCU, CCC, CCA, and CCG
