2 - Recombinant Insulin

Structure of insulin?

51 amino acids total

Alpha chain = 21 AA

Beta chain = 30 AA

Connected by disulfide bonds

MW = 5808 Da

heterodimeric protein of 51 amino acids possessing 3 disulfide bonds.

What is the history of insulin development from the 1950s–1982?

1950s → NPH and Lente animal insulins introduced as longer-acting forms

1982 → Human insulin cloning led to replacement of animal insulins

What insulin formulations were developed from 1996 to present, and why?

Short- and long-acting insulins to control glucose in T1D and advanced T2D

What is the history and future of inhaled insulin and newer insulin therapies?

Inhaled insulin approved in 2006, withdrawn, and redeveloped in 2015; future therapies aim to improve glucose control

Overall, insulin as had → nearly 100 years of clinical use

How similar is human insulin to bovine and porcine insulin?

90% amino acid sequence identity

Insulin is an evolutionarily conserved protein → stayed very similar throughout evolution across different animal species

How does C-peptide differ between species?

C-peptide amino acid sequence differs significantly between species

Bovine insulin → 3 amino acid substitutions vs. human insulin

Porcine insulin → 1 amino acid substitution vs. human insulin

What is C-peptide in insulin?

Proinsulin is the inactive precursor of insulin

Proinsulin is then split into insulin + C-peptide

The insulin is made of two chains → A and B

C-peptide is the piece that connects the A and B chains before they are separated

Why were animal insulin preparations sometimes immunogenic?

Allergic reactions were sometimes caused by impurities in animal insulin preparations

Can recombinant human insulin cause allergic reactions?

Recombinant human insulin rarely causes reactions against the insulin protein itself

Preservatives in both animal and human insulin can cause allergic reactions

Where is insulin produced?

In the beta cells of the pancreatic islets of Langerhans

Makes up about 1% of pancreatic cells.

What is the first form of insulin produced in the islets of langerhans?

Preprohormone

What happens after preprohormone is formed?

A signal peptide is removed in the cisternae of the ER and it gets packed into secretory vesicles in the golgi

Becomes proinsulin

What happens after proinsulin is formed and gets packed in the golgi?

It gets folded into its native structure, and is locked by the formation of 2 disulfide bonds

What happens when C-peptide is removed from proinsulin?

It gets removed by prohormone convertases PC1 and PC2

These cleave on the C-terminal side at the dibasic sites

What does carboxypeptidase H (CPH) do after c-peptide is removed?

Removes the two basic residues from the C-terminus of the B-chain

This forms the 51 AA fully function insulin protein

What does the intramolecular disulfide bond in insulin's A-chain connect?

The 6th and 11th amino acids (A6 and A11) within the same A-chain

What is the secondary structure of insulin's A-chain?

Two α-helices (A2–A8 and A13–A19)

Separated by a loop (A9–A12)

What is the secondary structure of insulin's B-chain?

One α-helix (B9–B19)

Followed by a turn (Gly B20)

Then a β-strand (B21–B30)

What holds insulin's A-chain and B-chain together in its tertiary structure?

Two interchain disulfide bonds at →

A7–B7

and

A20–B19

What stays the same across vertebrate insulin, and what differs between species?

The 6 cysteine residues stay in the same positions

But the C-peptide sequence varies between species

What is the average daily human insulin dose?

Approximately 2 mg/day

What is the estimated worldwide annual need for insulin?

Approximately 4,600 kg or 10,000 lbs per year

One pig pancreas yields 150 mg

Approximately 30 million pigs would be needed to meet global demand

What are the advantages of recombinant insulin production?

Produces human insulin

Allows potentially unlimited insulin production

Allows creation of insulin analogues by genetically modifying the human insulin sequence

Why is S. cerevisiae (baker's/brewer's yeast) useful for producing insulin?

Unicellular eukaryotes that can fold, proteolytically process, and glycosylate proteins

Does not produce toxic compounds

Has a well-characterized genome, the entire genome has been sequenced

Used in food production and pharmaceuticals, including insulin and insulin analogues

How is recombinant insulin produced in S. cerevisiae?

Through secretory expression of heterologous proteins into the culture media during continuous fermentation

What is the purpose of the α-factor (leader peptide) in yeast insulin production?

Allows the insulin precursor to be continuously secreted from S. cerevisiae

What enzyme cleaves the α-factor leader peptide in yeast insulin production?

Kex2 (a peptidase)

Allowing single-chain insulin to be secreted into the culture media

What modifications increase the fermentation yield of single-chain proinsulin in yeast insulin production?

Delete Thr (B30)

Replace the C-peptide with Ala-Ala-Lys

How is single-chain insulin converted into insulin in yeast insulin production?

Trypsin and carboxypeptidase B → digest and remove Ala-Ala-Lys

Turning single chain insulin into insulin

Why is Thr (B30) added back during recombinant insulin production?

It is temporarily deleted to improve yeast production yield

Then added back using trypsin-mediated transpeptidation to restore the correct human insulin sequence

Where is single-chain insulin secreted during yeast fermentation?

Into the culture media → where it is subsequently processed using trypsin and carboxypeptidase B

How does porcine insulin differ from human insulin?

Porcine insulin has Ala instead of Thr at B30

Originally → Trypsin-mediated transpeptidation replaces Ala with Thr → converting it to human insulin

How are recombinant human insulin analogues produced in E. coli?

By cloning the insulin gene into a plasmid

How did early recombinant insulin production in E. coli work?

The A-chain and B-chain were produced separately

Then combined to form the correct disulfide bonds

How does the improved method produce recombinant human insulin in E. coli?

Clone a synthetic human insulin gene with a shorter C-peptide into an expression vector

Allow the protein to fold properly

Specific proteases cleave the protein to produce insulin

ow are rapid-acting and long-acting insulin analogues created with E. Coli?

Site-directed mutagenesis changes specific codons, substituting amino acids in the insulin sequence

What is the purpose of regulating insulin secretion from pancreatic β-cells?

Maintain stable blood glucose during fasting and feeding

What factors coordinate the regulation of insulin secretion?

Nutrients

GI hormones

Pancreatic hormones

Autonomic neurotransmitters

Which nutrients promote insulin secretion?

Glucose

Amino acids

Fatty acids

Ketone bodies

What is the primary insulin secretagogue?

Glucose

Insulin secretion closely follows extracellular glucose concentration

What is the incretin effect?

The same amount of glucose causes greater insulin secretion when given orally versus intravenously

Which GI hormones are responsible for the incretin effect?

GIP and GLP

Which autonomic nerves innervate pancreatic islets?

Adrenergic and cholinergic nerves

How do α2 and β2 adrenergic receptors affect insulin secretion?

α2 stimulation → inhibits insulin secretion

β2 agonists → enhance insulin release

How does vagal nerve stimulation affect insulin secretion?

Enhances insulin release

How does sympathetic activation affect insulin secretion, and why?

Suppresses insulin secretion by stimulating α2 adrenergic receptors

What situations can activate the sympathetic nervous system and suppress insulin secretion?

Hypoxia

Hypoglycemia

Exercise

Hypothermia

Surgery

Severe burns

What is the resting state of pancreatic β-cells during fasting in insulin release?

The beta-cell is hyperpolarized

How does glucose stimulated insulin begin in insulin release?

The transport of glucose into the beta cell via GLUT2

What happens to glucose after it enters the beta cell via GLUT2 in insulin release?

It gets phosphorylated by glucokinase to generate G6P

This is the rate-limiting step in glucose metabolism

What happens after G6P is generated in insulin release?

It enters the glycolytic pathway causing changes in the NADPH and the ratio of ADP/ATP → increasing ATP

What happens after G6P increases ATP in insulin release?

The elevated ATP inhibits an ATP-sensitive K+ channel → closing the KATP channel

This leads to cell membrane depolarization

What does the KATP channel consist of?

Inward rectifying K+ channel → Kir6.2

A close associated protein → SUR1

What happens following membrane depolarization from increased ATP in insulin release?

It leads to opening of a voltage dependent Ca2+ channel and increase intracellular Ca2+

This results in exocytotic release of insulin from the storage vesicles

How is each insulin receptor monomer structurally organized?

Each monomer contains 8 distinct domains.

How are the insulin receptor α- and β-chains connected, and how are the two monomers held together?

Within each monomer → One disulfide bond connects the α- and β-chains

Between monomers → Two disulfide bonds extend from each α-chain to maintain the dimer

What is the overall 3D structure of the insulin receptor ectodomain?

An inverted “V” shape with four ligand-binding sites

What happens when insulin binds to the α-subunits of the insulin receptor in muscle / adipose tissue?

It triggers transphosphorylation of the β-subunits → activating the insulin receptor

What happens after the insulin receptor is activated in muscle / adipose tissue?

It initiates signaling → by phosphorylating a set of intracellular proteins / IRS receptor

What happens after the IRS receptor is phosphorylated in muscle / adipose tissue?

The phosphorylation of the IRS receptor regulates downstream kinases Akt

Akt2 coordinate the translocation of glucose transporter 4 (GLUT 4) → to the plasma membrane

Where is GLUT4 in muscle / adipose tissue?

In basal state → it resides in the intracellular space

After being activated by Akt2 → it gets rapidly moved to the plasma membrane

What happens after GLUT4 is activated and moved to the plasma membrane in muscle / adipose tissue?

It allows glucose to enter the cell

What happens after glucose enters the cell in muscle / adipose tissue?

Hexokinase II converts the glucose into → G6P

What is hexokinase II regulated by?

It is regulated transcriptionally by insulin

What happens after G6P is produced in the muscle / adipose tissue?

It can either →

Be used to produce energy

Become isomerized to G1P and eventually stored as glycogen

This is how insulin increase glycogen synthase activity!!

What is insulin resistance?

Cells in muscle and fat tissue do not respond effectively to insulin

Reducing glucose uptake and causing blood glucose to rise

How does insulin resistance affect glucose production in the liver?

The liver continues producing and releasing glucose through gluconeogenesis → increasing blood glucose

How does insulin resistance affect fat cells and circulating lipids?

Reduces uptake of circulating lipids

Increases production and release of triglycerides and fatty acids into the blood

How does insulin resistance contribute to weight gain?

Stimulates formation of new fat tissue and accelerates weight gain

How do pancreatic β-cells initially respond to insulin resistance?

Increase insulin production to compensate for elevated blood glucose

Causing hyperinsulinemia → high blood insulin

What causes the transition from insulin resistance to type 2 diabetes?

Pancreatic β-cells can no longer produce enough insulin to compensate for insulin resistance

Resulting in persistent hyperglycemia

What conditions are commonly associated with insulin resistance?

Visceral adiposity (abdominal fat)

Hypertension

Hyperglycemia

Dyslipidemia

What lipid abnormalities are associated with insulin resistance?

Elevated triglycerides

Increased small, dense LDL particles (sdLDL)

Decreased HDL cholesterol

What form does natural human insulin exist in at concentrations below 0.1 mM?

Monomer (single insulin molecule)

What happens to insulin at concentrations above 0.1 mM?

Insulin oligomerizes into dimers and hexamers

What coordinates and stabilizes the insulin hexamer?

Two zinc atoms

Where can natural human insulin form zinc-containing hexamers?

Storage vesicles in pancreatic β-cells

Blood

How does insulin's oligomeric state affect its pharmacology?

Influences its pharmacokinetic profile and receptor binding

Which form of insulin binds to the insulin receptor?

Only the monomer form

What was Lente (zinc) insulin used for?

One of the earliest dosage forms of porcine insulin → designed for slow release

Stimulates the basal insulin produced by beta-cells representing 50% of the daily insulin requirement

• Prandial insulin (mealtime) makes up for the remaining 50%

Why were short-acting, non-hexameric human insulins developed?

To improve mealtime (prandial) insulin by allowing faster absorption after injection

Which amino acids are essential for binding to the insulin receptor?

A1Gly

A4 Glu

A5Gln

A19Tyr

A21Asn

B12Val

B16Tyr

B23Gly

B24Phe,

B25Phe

TyrB26

What amino acid is not essential for binding to the insulin receptor?

B27-30

This has been the preferred site for structural modifications for modifying the pk profile of insulin

What conditions cause insulin fibril formation?

Elevated temperatures

Contact with hydrophilic surfaces

Shear stress (repeated up-and-down plunger movement)

Can also form in → infusion pumps

What are the characteristics of insulin fibrils?

Inactive

Do not resuspend once formed

What additives help prevent insulin fibril formation?

Phosphate buffer and other additives

What happens to insulin at low concentrations (less than 5 IU/mL) in tubing?

Insulin can adsorb / stick to the tubing → reducing the amount delivered

Adding albumin blocks insulin from adsorbing to tubing!

What happens to insulin during long-term storage at low pH (≤4)?

Deamidation occurs at Asn A21 → potentially causing crosslinking of the A and B chains

At 4°C → 1–2% per month

At 25°C → 90% after 6 months

Why was insulin glargine (Lantus) modified at position A21?

Glycine replaces asparagine (Asn) at A21 → to prevent low-pH deamidation at that site

What happens to insulin at neutral pH?

Deamidation occurs at Asn B3 → forming isoaspartate-containing insulin

That can trigger an immune response

Which insulins appear cloudy?

NPH and lispro protamine

All pre-mixed/combo insulins

What is Humulin R (U-100), and how is it made?

Regular insulin

Short-acting human insulin produced using recombinant DNA technology in E. coli

It is a clear colorless solution

When should regular insulin (Humulin R) be administered relative to meals?

Approximately 30 minutes before a meal

The patient should eat within 30 minutes of administration

What are the onset, peak, and duration of regular insulin?

Onset → 30–60 minutes

Peak → 2–3 hours

Duration → Approximately 6 hours

What is the typical daily dose range of regular insulin?

0.5–1 unit/kg/day

Individualized based on patient needs

What insulins can regular insulin be used with?

Intermediate-acting insulin

Long-acting insulin

Oral antihyperglycemic agents

How should unopened Humulin R be stored?

Refrigerate at 2–8°C (36–46°F)

Do not freeze

What is the major risk of regular insulin overdose?

Hypoglycemia

How is severe insulin-induced hypoglycemia treated?

IM/SC glucagon

Concentrated IV glucose

Why is continued carbohydrate intake and observation important after severe hypoglycemia?

Hypoglycemia can recur even after apparent clinical recovery