5 - Insulin Flashcards
1
Q
Blood glucose regulation
A
- 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
2
Q
Physiological activities of insulin
A
- 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)
3
Q
Insulin structure
A
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
4
Q
Function of C-peptide of insulin
A
To measure insulin levels in the blood
5
Q
Intrinsic property of insulin (charge)
A
- 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
6
Q
Different association force of insulin molecule
A
- 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
7
Q
Association of insulin
A
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
8
Q
What are insulin analogs?
A
Modifications of natural insulin, where changes are made in the AA sequence of the insulin molecule that affect the duration of action
9
Q
Describe the amino acid substitutions at A21, B28 and B29 for humulin and novolin
A
- A21 = Asn
- B28 = Pro
- B29 = Lys
10
Q
Describe the amino acid substitutions at A21, B28, B29, and B30 for porcine and bovine insulin
A
- A21 = Asn
- B28 = Pro
- B29 = Lys
- B30 = Ala (everything else = Thr)
11
Q
Describe the amino acid substitutions at A21, B28 and B29 for lispro
A
- A21 = Asn
- B28 = Lys
- B29 = Pro
12
Q
Describe the amino acid substitutions at A21, B28 and B29 for aspart
A
- A21 = Asn
- B28 = Asp
- B29 = Lys
13
Q
Describe the amino acid substitutions at A21, B28, B29, B31, and B32 for glargine
A
- A21 = Gly
- B28 = Pro
- B29 = Lys
- B31 = Arg
- B32 = Arg
14
Q
Describe the amino acid substitutions at A21, B28 and B29 for detemir
A
- A21 = Asn
- B28 = Lys-(N-tetradecanoyl)
- B29 = Pro
15
Q
Steps of site-directed mutagenesis of insulin
A
- Determine which site you want to mutate
- Clone gene cDNA into a selectable plasmid
- Amplify gene sequence by PCR
- Remove un-amplified gene sequence
16
Q
Sites to mutate on regular insulin to make rapid acting lispro
A
- Proline replaced w/ lysine at B28
- Lysine replaced w/ proline at B29
17
Q
Design of site-directed mutagenesis primers
A
- 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
18
Q
PCR to remove un-amplified DNA sequence
A
- 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
19
Q
Identification of mutated sequence
A
- 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
20
Q
Describe the dissociation of regular insulin after SQ injection
A
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
21
Q
Lispro (Humalog) – describe the changes made to the structure and what impact they have
A
- 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
22
Q
Aspart (NovoRapid) – describe the change made to the structure and what impact it has
A
- 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
23
Q
Glargine (Lantus) – describe the changes made to the structure and what impact they have
A
- 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
24
Q
Detemir (Levemir) – describe the changes made to the structure and what impact they have
A
- 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
25
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
26
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
27
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
28
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
29
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
30
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)
31
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
32
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
33
Amino acid modifications of insulin
- - Disulfide bond 31 -> 96 interchain
- - Disulfide bond 43 -> 109 interchain
- - Disulfide bond 95 -> 100
34
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
35
Which codons encode lysine?
AAA and AAG
36
Which codons encode proline?
CCU, CCC, CCA, and CCG
