Insulin Preparations

Question 1

Why is Hemoglobin A1c (HbA1C) known as glycated hemoglobin instead of glycosylated hemoglobin?

Answer 1

Glycosylated refers to an enzyme PTM reaction.

Glycation of hemoglobin takes place via non-enzymatic condensation rxn b/w glucose and amino end of the beta chain in hemoglobin.

Question 2

What are 2 causes of high HbA1c?

Answer 2

1. High blood glucose levels
2. Longer period of high blood glucose levels

Question 3

Why is HbA1c used for long-term monitoring of diabetic condition?

Answer 3

Glycated Hb remains throughout RBC lifespan ~3months

Question 4

Describe the different between the structure of preproinsulin and proinsulin.

What is the final insulin structure?

Answer 4

Preproinsulin has an additional 23 AA terminal signal peptide sequence attached to the B chain

Proinsulin - 2 disulfide bonds form between the A chain and B chain, C chain from proinsulin is cleaved and released as a C peptide to form insulin

Insulin - A chain (21 AAs), B chain (30 AAs), joined by 2 disulfide bond

Question 5

Bovine insulin has more variations than Porcine insulin, when compared to human insulin

TRUE or FALSE

Answer 5

TRUE

Bovine insulin - 3 variations at A8, A10, B30

Porcine insulin - B30

Question 6

Using animal insulin or proinsulin may lead to immune response. What are these immune response?

Answer 6

1. Lipoatrophy
2. Local allergic reaction (itchiness, redness)

Question 7

What does DNA encode for in recombinant human insulin?

Answer 7

DNA encodes for a single polypeptide chain that consist of the A, B, and C chain of proinsulin.

Question 8

(Recombinant human insulin)
Methionine (ATG) start codon is chemically synthesized and attached to ____ of proinsulin cDNA

Answer 8

5’ end

Question 9

(Recombinant human insulin)
What is the role of cyanogen bromide (CnBr)?

Answer 9

Cyanogen bromide removes the Met residue at N terminus (of B chain) via chemical cleavage

Question 10

(Recombinant human insulin)
Name the 2 enzymes involved in cleavage of the C chain, to release C peptide from the active insulin.

Answer 10

1. Trypsin (cleaves peptide bond b/w carboxyl group of arg/lys and adjacent AA)
2. Carboxypeptidase (cleaves AA at C terminal)

Question 11

Explain why natural insulin is short acting but does not have rapid onset.

Answer 11

No rapid onset because insulin has intrinsic ability to form dimers and higher order associated states due to favorable hydrophobic interactions.

Question 12

What does insulin form in the presence of zinc?
Describe how this dissociates at the site of injection.

Answer 12

Zinc-containing hexamer.

Hexamer dissociates into monomers at site of injection when injected concentrations fall to physiological levels.

Monomers can bind to insulin receptors.

Question 13

Name 3 rapid acting insulin.
Where are the modifications made for each, and explain their purpose.

Answer 13

All 3 have modifications on the B chain only. These modifications are meant to decrease association between insulin monomers such that they do not dimerize or form hexamers.

1. Insulin Lispro - B28 and B29 reversed to resemble insulin-like growth factor 1 that poorly self associates [ProB28, LysB29 ==> LysB28, ProB29] *Lispro
2. Insulin Aspart - [ProB28 ==> AspB28] reduction in association, non-hexameric
3. Insulin Glulisine - [LysB29 ==> GluB29, AsnB3 ==> LysB3], reduction in association, non-hexameric

Question 14

Name 2 long-acting insulin.

Answer 14

1. Insulin Glargine
2. Insulin Determir

Question 15

Explain the structural modifications of insulin glargine.
How does it contribute to longer duration of action (slower release)?

Answer 15

A chain: AsnA21 ==> GlyA21
B chain: 2 Arg residues added to C terminus (at B30)

Change of glargine’s pI to 6.7 (vs 5.4)
Glargine is more soluble at acidic pH, it is less soluble at neutral pH. Additionally, protein solubility is also lowest at pI.
Hence, at physiological pH 7.4, Glargine is less soluble, allowing slow release.

Question 16

Even though Glargine pI is shifted to make it less soluble in physiological pH, it appears as a clear solution when formulated. Why? (also explain why it is formulated this way)

Answer 16

It is formulated at acidic pH 4, where it is soluble.

It is formulated at pH 4 because pH4 stabilizes insulin hexamer, resulting in prolonged and predictable absorption form subcutaneous tissues.

Question 17

Explain the structural modifications of insulin detemir.
How does it contribute to longer duration of action (slower release)?

Answer 17

ThrB30 is deleted, replaced with C14 fatty acid (myristic acid) covalently attached to LysB29

This results in increase hydrophobicity, allowing determir binding to serum albumin (at site of injection + upon gaining entry intro circulation), hence causing slow dissociation into blood and prolonged release

Question 18

Explain how detemir binds to serum albumin at the site of injection.

Answer 18

Apart from circulation, albumin is found in interstitial fluid as well.
This is due to leaky capillaries + higher hydrostatic pressure at arterial end, that moves albumin out into interstitial fluids at site of injection.

Question 19

Describe the components of intermediate acting Isophane insulin (NPH insulin).

Answer 19

Regular insulin + Protamine + Zinc

Protamine: highly basic 30AA peptide, +ve charge

insulinandprotamineCo-crystalized with zinc in neutral pH (phosphate buffer)
=> Neutral crystalline insulin suspension, appears cloudy
*Intermediate acting as need to dissolve and dissociate into monomer

Mixed with regular insulin - latter (70/30, 50/50), ensure coverage of post meal high blood glucose with short acting insulin

Question 20

Which insulin analogue is the only one with A chain modification?

Answer 20

Insulin Glargine (long acting)
A chain: Asn21 ==> Gly21
+
Additional 2 Arg residues on C terminal of B chain