04 - Biosynthesis and Secretion of Insulin

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TARGET DECK: MED::I::Signaling Pathways in Health and Disease::Cell Signaling::04 - Biosynthesis and Secretion of Insulin

Frederick Sanger and the Chemistry of Insulin

Nobel Lecture, December 11, 1958

“It is great pleasure and privilege for me to give an account of my work on protein structure and I am deeply sensitive of the great honour that has been done to me in recognizing my work in this way. Since the work on insulin has extended over about 12 years it will be necessary to give a somewhat simplified account and to omit most of the work that did not contribute directly to the main problem, the determination of the structure of a protein.”

Mature Insulin Structure

The structure revealed that the longer chain (B chain) forms an α-helix and a β-strand, whereas the A chain consists of two α-helices. These chains are linked by two disulfide bonds. This is the mature form of the insulin molecule, as biosynthesized by the pancreas.

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Insulin: Two Polypeptide Chains Joined by Disulfide Cross-Linkages

• Total: 51 amino acids
• Two polypeptide chains linked by disulfide bonds ($\text{Cysteine}+\text{Cysteine}\to \text{Cystine}$)
• A-chain: 21 amino acids
• B-chain: 30 amino acids

N[C@@H](CS)C(=O) 

(Cysteine residue, donor of the –SH group forming disulfide bonds)

What are the two polypeptide chains of insulin and how many amino acids does each contain?

The A-chain contains 21 amino acids and the B-chain contains 30 amino acids, for a total of 51 amino acids. They are joined by two disulfide bonds.

Anki cloze

Mature insulin consists of {1:two} polypeptide chains linked by {1:two disulfide bonds}, with the A-chain forming {1:two α-helices} and the B-chain forming {1:an α-helix and a β-strand}.

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Quaternary Structure of Insulin: Monomer → Dimer → Hexamer

• The two disulfide-linked polypeptides associate with another insulin molecule to form a homodimer.
• The homodimer aggregates with two additional dimers to form a hexamer.
• Within the pancreatic beta cells, proinsulin is stored in such hexameric, inactive and very stable form before release.

Storage Form

Proinsulin is stored in hexameric, inactive, and very stable form in secretory granules before release.

Anki cloze

Within pancreatic beta cells, proinsulin is stored in a {1:hexameric}, {1:inactive} and very stable form before release.

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Precursor Polypeptides: Chain Lengths

Polypeptide Segment	Length
Signal sequence	23 (+1) amino acids
B-chain	30 amino acids
C-peptide	31 (+4) amino acids
A-chain	21 amino acids
Pre-proinsulin (total)	110 amino acids

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Translation and Translocation of Pre-Proinsulin

Primary Product of the INS Gene

Pre-proinsulin (110 amino acids) is the primary product of the INS gene.

Signal Sequence

• A 23 amino acid segment at the N-terminus
• Directs the passage into secretory vesicles

Step-by-Step Processing

Step 1 – Translation and translocation

• Pre-proinsulin is synthesized by ribosomes and co-translationally translocated into the ER lumen.

Step 2 – Folding, oxidation (S–S), and signal peptide cleavage

• Proteolytic removal of the signal sequence (by Sec11) and formation of 3 disulfide bonds produces proinsulin.
• Proinsulin is stored in hexameric form in secretory granules.

Step 3 – ER export, Golgi transport, vesicle packaging

Step 4 – Protease cleavage liberates C-peptide

• Prohormone convertases PC-1 and PC-2 cleave at dibasic cleavage sites, releasing the C-peptide.

Step 5 – Carboxypeptidase E produces mature insulin (51 amino acids)

Which enzymes cleave proinsulin into mature insulin and C-peptide?

Prohormone convertases PC-1 and PC-2 cleave at dibasic sites; Carboxypeptidase E then trims the ends to yield mature insulin (51 amino acids) and C-peptide (31 amino acids).

Anki cloze

The signal peptide of pre-proinsulin is cleaved by {1:Sec11} in the ER, while proinsulin is later cleaved by {1:PC-1 and PC-2} and {1:Carboxypeptidase E} to yield mature insulin.

Mnemonic – Processing Steps

“Translate, Trim, Transport, Cleave, Carboxypeptidase” = T-T-T-C-C

1. Translate & translocate (ribosome → ER)
2. Trim signal peptide + form S–S bonds → proinsulin
3. Transport (ER → Golgi → secretory vesicle)
4. Cleave with PC-1/PC-2 (liberates C-peptide)
5. Carboxypeptidase E → mature insulin

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Release: Mature Insulin and C-Peptide

Secretion

When blood glucose is sufficiently elevated, proinsulin is converted to mature (active) insulin by proteases (PC-1, PC-2) and is released into the blood by exocytosis, mixed in equimolar amounts with the C-peptide (31 amino acids).

In what ratio are insulin and C-peptide secreted?

They are secreted in equimolar amounts (1:1 ratio) by exocytosis from pancreatic beta cells.

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Three-Dimensional Structure of Insulin

• Tertiary structure: A-chain (green), B-chain (cyan), disulfide bonds (yellow)
• Quaternary structure: monomer → dimer → hexamer (in secretory vesicles)

Anki cloze

Insulin quaternary structure progresses from monomer → {1:dimer} → {1:hexamer}, the latter being the storage form in {1:secretory vesicles}.

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Nobel Prizes Related to Insulin

Year	Laureate(s)	Contribution
1923	Frederick G. Banting & John Macleod	Discovery of insulin
1958	Frederick Sanger	Chemical structure of insulin
1964	Dorothy C. Hodgkin	3D structure of insulin (X-ray crystallography)
2013	James E. Rothman, Randy W. Schekman, Thomas C. Südhof	Machinery regulating vesicle traffic

Still an Active Field

More than fifty years after the structure of insulin was first determined, and after four Nobel prizes, there are still new structures of insulin being deposited in the PDB, and amazingly, the structure of insulin bound to its receptor was only published in early 2013.

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Glucose-Stimulated Insulin Secretion in Pancreatic β-Cells

Overview of the Pathway

1. Glucose enters the β-cell via GLUT2 (glucose transporter)
2. Glucokinase (Hexokinase IV) phosphorylates glucose → Glucose-6-phosphate
3. Glycolysis → Citric acid cycle → ↑ ATP
4. ↑ ATP/ADP ratio → closure of ATP-gated ${\text{K}}^{+}$ channels ($K_{ATP}$)
5. Membrane depolarization
6. Opening of voltage-gated ${\text{Ca}}^{2+}$ channels
7. ↑ intracellular ${\text{Ca}}^{2+}$ → exocytosis of insulin from secretory granules

Key Molecular Players (Summary)

• GLUT transporters
• Glycolysis
• ATP
• Ligand-gated channels
• Voltage-gated channels
• ${\text{Ca}}^{2+}$

What triggers closure of KATP channels in pancreatic β-cells?

An increase in the ATP/ADP ratio resulting from glucose metabolism (glycolysis + citric acid cycle) inhibits the $K_{ATP}$ channel, causing membrane depolarization.

Anki cloze

In β-cells, glucose uptake via {1:GLUT2} leads to increased {1:ATP/ADP ratio}, which closes {1:KATP channels}, causing membrane {1:depolarization}, opening of {1:voltage-gated Ca²⁺ channels}, and triggering {1:insulin exocytosis}.

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Molecular Structure of the β-Cell ATP-Sensitive Potassium Channel ($K_{ATP}$)

Subunit Composition

The functional $K_{ATP}$ exists as an octamer comprising:

• 4 Kir6.2 subunits (pore-forming)
• 4 SUR1 subunits (regulatory; ATP-binding cassette superfamily)

Kir6.2 Subunit

• Binding of ATP (in red) to Kir6.2 inhibits channel activity by stabilizing the closed state of the pore.
• Each Kir6.2 subunit in the tetramer is capable of binding a molecule of ATP.
• Binding to one subunit is sufficient to cause channel closure → membrane depolarization.

Paradox of KATP Activity

$K_{ATP}$ channels in the pancreatic β-cell are predicted to be largely closed, even at resting glucose concentrations, since $K_{ATP,1/2}$ in isolated membrane patches is ~10 μM, yet cellular ATP concentrations in the energized cell reside in the low mM range. However, measured $K_{ATP}$ activity “on-cell” is significantly higher, likely reflecting the net stimulatory input from MgADP that reduces ATP-inhibition in the cellular milieu.

Channel Conductance vs. Glucose

Glucose Concentration	$K_{ATP}$ Conductance (% of maximal)
5 mM	7%
10 mM	3%

This tiny change in conductance can cause a marked change in membrane potential.

SUR1 Subunit

• Member of the ABC superfamily
• Confers sensitivity of $K_{ATP}$ to stimulatory ${\text{Mg}}^{2+}$-nucleotides (MgADP and MgATP)
• Conformational changes associated with SUR1 MgATP binding → hydrolysis at the NBDs (nucleotide-binding domains) are transduced to the activation gates of Kir6.2, stabilizing the opening state of the channel pore.

What are the two subunit types of the KATP channel and what are their roles?

Kir6.2 (×4): pore-forming subunit; ATP binding closes the channel. SUR1 (×4): regulatory subunit (ABC superfamily); MgADP/MgATP binding opens the channel.

Anki cloze

The KATP channel is an {1:octamer} of {1:4 Kir6.2} and {1:4 SUR1} subunits; ATP binding to {1:Kir6.2} closes the pore, while MgADP binding to {1:SUR1} promotes {1:opening}.

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Clinical Correlates: KATP Channel Mutations

Mutation Type	Subunit	Effect	Clinical Consequence
Loss-of-function (LOF)	SUR1	Closed $K_{ATP}$ pore	Congenital Hyperinsulinism (HI)
Gain-of-function (GOF)	SUR1	Open $K_{ATP}$ pore	Permanent (PNDM) or Transient Neonatal Diabetes Mellitus (TNDM)

Selected SUR1 Loss-of-Function Mutations (orange) – Congenital Hyperinsulinism

E208K, D209E, L225P, D212I/N, R370G, L451P, G1382S, AF1388, R1394H, I1425V, G1478V, R1353H, R1380L, K1374R, S1386P, R1539Q, V1524M

Selected SUR1 Gain-of-Function Mutations (purple/green) – PNDM/TNDM

(See figure for individual variants)

What clinical syndrome results from a loss-of-function mutation in the KATP channel?

Congenital Hyperinsulinism (HI): the channel remains closed even at low glucose, causing constitutive insulin secretion.

What clinical syndrome results from a gain-of-function mutation in the KATP channel?

Permanent (PNDM) or Transient Neonatal Diabetes Mellitus (TNDM): the channel remains open even at high glucose, preventing insulin secretion.

Anki cloze

Loss-of-function mutations in SUR1 cause the KATP channel to remain {1:closed}, leading to {1:congenital hyperinsulinism}, while gain-of-function mutations cause it to remain {1:open}, leading to {1:neonatal diabetes mellitus}.

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Pharmacology: Sulfonylureas and KATP

Therapeutic Relevance

Sulfonylurea drugs can bind and close the $K_{ATP}$ channel, mimicking the effect of high glucose. This is used therapeutically to stimulate insulin release in Type 2 Diabetes, and also in GOF $K_{ATP}$ neonatal diabetes (where oral sulfonylureas can replace insulin injections).

What is the mechanism of action of sulfonylureas in stimulating insulin secretion?

Sulfonylureas bind to the SUR1 subunit of the KATP channel and close it, causing membrane depolarization → Ca²⁺ influx → insulin exocytosis, independent of glucose metabolism.

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SNARE-Mediated Insulin Exocytosis

SNAREs: Core Fusion Machinery

SNARE = Soluble N-ethylmaleimide-sensitive factor Attachment Protein REceptor

• t-SNARE: target membrane SNARE (e.g., Syntaxin)
• v-SNARE: vesicle SNARE (on secretory granule)
• SNARE complex formation drives vesicle fusion with the plasma membrane

What does SNARE stand for?

Soluble N-ethylmaleimide-sensitive factor Attachment Protein REceptor

Anki cloze

Vesicle fusion during insulin exocytosis is mediated by the {1:SNARE} complex, which includes {1:t-SNAREs} on the target membrane and {1:v-SNAREs} on the vesicle.

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Synaptotagmin-1: The ${\text{Ca}}^{2+}$ Sensor for Vesicle Fusion

• Synaptotagmin-1 is a synaptic vesicle ${\text{Ca}}^{2+}$-sensor
• Essential for ${\text{Ca}}^{2+}$-triggered vesicle fusion
• Located on the synaptic vesicle membrane at the active zone
• Works together with: RIM, Complexin, Syntaxin, ${\text{Ca}}^{2+}$-channels

What is the role of Synaptotagmin-1 in vesicle exocytosis?

Synaptotagmin-1 acts as the Ca²⁺ sensor on synaptic vesicles; upon Ca²⁺ binding, it triggers SNARE-mediated vesicle fusion and neurotransmitter/hormone release.

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Nobel Prize in Physiology or Medicine 2013

Nobel Committee

he Nobel Prize in Physiology or Medicine 2013 was awarded jointly to James E. Rothman, Randy W. Schekman, and Thomas C. Südhof “for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells”.

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Beyond Glucose: The Cephalic Phase and Incretins

Primary Signal for Insulin Secretion

It is worth noting that in vivo the primary signal for insulin secretion is not usually glucose, but:

1. Neurotransmitters released in response to the sight or smell of food (the cephalic phase of release)
2. Incretins (peptides released from the gut due to the presence of food in the gut lumen)

Together, these mechanisms prepare the body for the subsequent increase in plasma glucose and prevent blood glucose levels from rising too high after a meal.

This also explains why insulin secretion is greater in response to an oral glucose challenge than an intravenous one.

Neural Pathway (Cephalic Phase)

1. Food-related sensory inputs (e.g., taste bud cells) relayed by cranial nerves
2. → Sensory processing areas in the brainstem
3. → Efferent signals carried through cholinergic fibers in the vagus nerve
4. → In rodents, some fibers make direct contacts with beta cells in pancreatic islets
5. → Stimulation causes release of acetylcholine (ACh)
6. → ACh binds to muscarinic receptors on the surface of beta cells
7. → In humans, the neural mechanisms that stimulate beta cells are less clear because pancreatic islets appear to have sparse innervation

Mechanism of ACh Action

The binding of ACh to muscarinic receptors on beta cells is thought to activate a $K_{ATP}$-independent depolarizing current.

Why is insulin secretion greater after oral glucose than after intravenous glucose?

Because oral glucose triggers incretin release from the gut (e.g., GLP-1) and activates the cephalic phase (vagal/cholinergic stimulation), both of which amplify insulin secretion independently of blood glucose.

Anki cloze

The cephalic phase of insulin secretion is mediated by {1:cholinergic fibers} of the {1:vagus nerve}, releasing {1:acetylcholine} that binds to {1:muscarinic receptors} on beta cells, activating a {1:KATP-independent depolarizing current}.

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Pancreatic α-Cells and Glucagon

Glucagon Structure

The mature form of glucagon is a single polypeptide chain of 29 amino acids.

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Proglucagon: A Precursor to Multiple Hormones

Proglucagon-Derived Peptides

Embedded in the proglucagon sequence, there are at least two additional peptides, which have important roles in physiology and which can have major impact in human disease when manipulated pharmacologically.

Proglucagon Processing Map

Residues	Peptide
1–30	GRPP (glicentin-related polypeptide)
33–61	Glucagon
64–69	IP-1 (intervening peptide 1)
72–107/8	GLP-1 (Glucagon-like peptide 1)
123–126	IP-2 (intervening peptide 2)
126–158/160	GLP-2 (Glucagon-like peptide 2)

Enzymatic Processing

Proglucagon is cleaved by prohormone convertases (similar to furin). Three hormones are produced: glucagon (PDB: 1gcn), GLP-1 (PDB: 3iol), and GLP-2 (PDB: 2l63).

Name the three hormones derived from proglucagon.

Glucagon, GLP-1 (Glucagon-like peptide 1), and GLP-2 (Glucagon-like peptide 2), all cleaved from proglucagon by prohormone convertases.

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GLP-1 and GLP-2: Physiological Effects and Therapeutics

Source

GLP-1 and GLP-2 are produced in intestinal entero-endocrine cells (L-cells) as a result of post-translational processing of proglucagon.

Physiological Effects of GLP-1

Target Organ	Effect
β-cell	↑ Insulin secretion; ↑ Insulin biosynthesis
α-cell	↓ Glucagon secretion
Brain	↓ Food intake; ↑ Neuroprotection; ↑ Aversive response
Cardiovascular	↑ Heart rate; ↑ Cardioprotection; ↓ Stroke

Physiological Effects of GLP-2

Target	Effect
Gut	↓ Gut growth (↑ barrier function; ↓ nutrient absorption)

Triggers for GLP-1/GLP-2 Release

• Nutrients
• Endotoxin
• Bacterial metabolites

Licensed Drug Classes from Proglucagon Research

Drug Class	Target Condition
GLP-1 receptor agonists (GLP-1 RA)	Type 2 diabetes; Obesity
DPP-4 inhibitors (DPP4i)	Type 2 diabetes
GLP-2 receptor agonists (GLP-2 RA)	Short bowel syndrome

What are the three drug classes derived from proglucagon research and their indications?

1. GLP-1 RA – Type 2 diabetes and obesity; 2. DPP-4 inhibitors – Type 2 diabetes; 3. GLP-2 RA – Short bowel syndrome.

Anki cloze

GLP-1 and GLP-2 are produced in {1:intestinal entero-endocrine (L) cells} by post-translational processing of {1:proglucagon}, and GLP-1 receptor agonists are used to treat {1:type 2 diabetes} and {1:obesity}.

Key Reference

O’Rahilly, The islet’s bridesmaid becomes the bride: Proglucagon-derived peptides deliver transformative therapies, Cell (2021). https://doi.org/10.1016/j.cell.2021.03.019

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TLDR

Lecture A.04 – Complete Summary

Insulin Biosynthesis:

• Pre-proinsulin (110 aa) is the primary product of the INS gene; signal sequence (23 aa) directs ER entry.
• Processing: signal peptide cleavage (Sec11) + 3 disulfide bonds → proinsulin → PC-1/PC-2 cleavage + Carboxypeptidase E → mature insulin (51 aa, A-chain 21 aa + B-chain 30 aa) + C-peptide (31 aa) released equimolarly.
• Storage: hexameric, inactive form in secretory granules.

Quaternary Structure: monomer → dimer → hexamer; A-chain has 2 α-helices; B-chain has α-helix + β-strand; joined by 2 disulfide bonds.

Nobel Prizes: 1923 (Banting/Macleod – discovery), 1958 (Sanger – sequence), 1964 (Hodgkin – 3D structure), 2013 (Rothman/Schekman/Südhof – vesicle trafficking).

Glucose-Stimulated Insulin Secretion:

• GLUT2 → glucokinase → glycolysis → ↑ATP/ADP → KATP closure → depolarization → voltage-gated Ca²⁺ channel opening → Ca²⁺ influx → exocytosis.

KATP Channel:

• Octamer: 4× Kir6.2 (pore, ATP inhibits) + 4× SUR1 (ABC superfamily, MgADP activates).
• LOF mutations → Congenital Hyperinsulinism; GOF mutations → PNDM/TNDM.
• Sulfonylureas close KATP → therapeutic insulin release.

Exocytosis Machinery:

• SNARE proteins (v-SNARE + t-SNARE) drive membrane fusion.
• Synaptotagmin-1 is the Ca²⁺ sensor essential for triggered fusion.

Cephalic Phase & Incretins:

• Primary in vivo signal for insulin secretion is NOT glucose but neurotransmitters/incretins.
• Vagus nerve → ACh → muscarinic receptors on β-cells → KATP-independent depolarization.
• Explains oral > IV glucose effect on insulin secretion.

Glucagon and Proglucagon:

• Glucagon: 29 aa single polypeptide from α-cells.
• Proglucagon encodes: GRPP, Glucagon, IP-1, GLP-1, IP-2, GLP-2; cleaved by prohormone convertases (furin-like).
• GLP-1: ↑ insulin, ↓ glucagon, ↓ food intake, cardioprotective.
• GLP-2: gut growth and barrier function.
• Drug classes: GLP-1 RA (T2D/obesity), DPP-4i (T2D), GLP-2 RA (short bowel syndrome).