BIO 4127 - 5 Insulin and Glucagon

Why is it important to tightly control glucose levels in the body?

- glucose is key energy source for cellular function

- brain cannot synthesize or store glucose (too much or too little can be rapidly lethal)

How does the pancreas develop?

it develops as a glandular outgrowth of the intestine

2 parts of the pancreas

endocrine and exocrine

Endocrine pancreas

produce peptide hormones which are secreted into the blood

Cells of the endocrine pancreas (4)

Alpha cells

Beta cells

Delta cells

PP cells

Alpha cells secrete..?

glucagon

Beta cells secrete..?

insulin

Delta cells secrete...?

somatostatin

PP cells secrete...?

pancreatic polypeptide

Exocrine pancreas

digestive enzymes secreted into pancreatic duct leading to small intestine

Enzymes secreted by the exocrine pancreas

amylase (glycogen breakdown)

lipase (triglyceride breakdown)

trypsin/chymotrypsin and carboxypeptidase (protein breakdown)

The endocrine pancreas is made up of many?

islets of Langerhans

What is an Islet of Langerhans

the functional unit of the endocrine pancreas. Contains alpha, beta, delta, and PP cells.

Acinar cells

enzyme-secreting cells of the exocrine pancreas

Islet of Langerhans organization

Insulin secreting beta cells are in the middle of the islet and the other types of cells are on the periphery

Appearance of endocrine pancreas cells along the vertebrate evolutionary tree

over 400 million years ago; these cells are ancient

Pancreatic hormones (4)

- Insulin

- Glucagon

- Somatostatin (SST, SRIF)

- Pancreatic polypeptide

Insulin

- rapidly secreted from beta-cells when plasma glucose increases

- acts on many cells (e.g., liver, muscle, adipose)

Insulin function

Increases glucose uptake and conversion of glucose to glycogen

Glucagon

29 amino acids, single chain peptide that stimulates hepatic glucose synthesis and release

Glucagon function

- Stimulates hepatic glucose synthesis and release

- stimulates conversion of amino acids to glucose, and stimulates lipolysis

Somatostatin (SST, SRIF)

prohormone (28 a.a.) but active peptide is 14 a.a. (1 cysteine bond)

- paracrine mode of action

Somatostatin function

- inhibits secretion of insulin and glucagon from pancreas

- inhibits gastric function (inhibits acid secretion, H2CO3 -, and pancreatic enzyme secretion)

Pancreatic polypeptide

- 37 a.a. in human, 36 a.a. in rat

- single peptide

- Not well studied

Pancreatic polypeptide function

decreases liver glycogen and pancreatic secretion

Oral glucose tolerance test (OGTT)

Given a certain amount of glucose to ingest, then blood samples are taken periodically to assess your clearance of glucose (activity of your beta-cells)

Normal blood sugar level before a meal (fasting)

4-7mmol/L

As blood glucose levels increase, what happens to glucagon?

glucagon secretion is shut off

As blood glucose levels increase, what happens to insulin?

insulin secretion increases

Human insulin gene codes for...?

Pre-proinsulin with signal peptides that tells the cell where the polypeptide should go

Insulin biosynthesis is controlled by which molecule?

glucose, so that insulin is only made when needed

Pre-proinsulin to insulin conversion

Proteases remove the signal peptide and fold the remaining polypeptide

Proinsulin to insulin conversion

cleavage of the c-chain

insulin C-chain

Portion of the proinsulin peptide that links the A and B-chain. It is cleaved to form insulin

C-chain cleavage site type

Dibasic cleavage site

How are the A and B insulin chains linked?

by two cysteine bridges

What class of proteases cleave proinsulin to form insulin?

Prohormone convertases

Insulin synthesis steps

- Preproinsulin is secreted into the endoplasmic reticulum

- Post-translational processing cleaves the N-terminal signal sequence and forms the disulfide bridges

- Proinsulin passes through the Golgi and is packaged into vesicles

- PC enzymes within the acidic vesicle cleave proinsulin at two positions

- Secretory vesicle now contains active insulin and free C-chain

Insulin secretion is dependent on which ion?

Calcium

Insulin across evolution

is conserved from protochordates like amphioxus to fish to mammals

Insulin A and B chain conserved residues

A-chain - cysteines at pos. 6, 7, 11, and 20 (considerable variation at 8, 9, 10)

B-chain - cysteines conserved at 7, 19 (variation around position 30)

Fish Pancreas

endocrine and exocrine parts are separate unlike mammals

Endocrine fish pancreas is called the...?

Brockmann body

- location where amino acids will signal insulin secretion and synthesis

Insulin receptor type

tyrosine kinase

- Tetramer

- Two identical α subunits linked via a disulfide bond

- Two beta subunits

Tyrosine kinase domain

Enzyme-like domain that is activated upon insulin binding to receptor. -

What happens to IR when insulin binds IR

IR autophosphorylation: adds a phosphate to tyrosine molecule

What IR subunit does insulin bind?

IR alpha subunits

Insulin receptor activation cascade

phosphorylation cascade

Phosphorylation

the addition of a phosphate group (PO4) to a molecule

Insulin action (insulin triggering IR) steps

1. Mature IR

2. Insulin binds to the alpha subunit of the receptor

3. Autophosphorylation of beta subunits leads to a conformational change in the IR

4. Kinase activity of the IR increases

5. Phosphorylation of intracellular proteins initiates the signal transduction cascade for insulin action

How are the two types of insulin receptors derived?

alternative splicing of mRNA from the IR gene

Types of Insulin receptors

Isoform A and B

IRa binds...?

insulin and insulin-like growth factor 2

IRb binds..?

IRS (insulin receptor substrate) preferentially and insulin

Insulin receptor isoforms affinity

Affinity of IRa for insulin is 2-fold higher (in vivo)

- Preferentially IRa is the target for insulin

- IRb couples more efficiently to signalling substrates IRS-1 (in vivo)

How do cells express insulin receptor isoform?

A cell may express both or either isoforms of insulin

Most insulin signals are produced or modulated through...?

tyrosine phosphorylations

Insulin Receptor Substrate Proteins

IRS1 and IRS2

- crucial cytoplasmic adaptor proteins that transduce signals from insulin and IGF-1 receptors to downstream effectors, regulating growth, metabolism, survival, and differentiation

- They act as docking stations, linking receptor tyrosine kinases to pathways like PI3K, essential for glucose homeostasis

IRS1 function

controls body growth and peripheral insulin action

IRS2 function

regulates brain growth, body weight control, glucose homeostasis, involved in female fertility

What downstream kinase does insulin activate?

PI3 kinase

After PI3 kinase is activated, what other kinases are recruited and where?

- 3-phosphoinositide-dependent protein kinase-1 (PDK1)

- Protein Kinase B (also known as AKT, several forms)

To the inner membrane

PDK1 is activated by steps of phosphorylations, and then PKB is activated

PKB phosphorylates serines in multiple other proteins

What proteins do PKB phosphorylate downstream? (3)

- BAD (BCL2-associated agonist of cell death)

- FOXO-1 (forkhead box O1)

- Glycogen synthase kinase-3 beta

What amino acid does PKB phosphorylate?

serine

Insulin phosphorylation cascade steps

IR tyrosine kinase → PI3 kinase →PDK1 → PKB → BAD + FOXO-1 + Glycogen synthase-3 beta

BAD

Cell survival protein; positively regulates apoptosis

- Member of the BCL-2 family known to be regulators of programmed cell death.

FOXO-1

Transcription factor that promotes β-cell function

- decrease in FOXO-1 inhibits gluconeogenesis and adipocyte differentiation

Glycogen synthase kinase-3 beta

Regulates growth and glycogen synthesis

Excess glucose in blood is stored as...?

glycogen

2 enzymes needed for glucose-glycogen interconversion

- Glycogen synthase

- Glycogen phosphatase

Glycogen synthase

Synthesizes glycogen from glucose

- inactive when phosphorylated

- active when dephosphorylated

Insulin action on glycogen synthase

Insulin de-phosphorylates (activates) glycogen synthase because insulin promotes glucose conversion

Glycogen phosphatase

Removes phosphate groups from glycogen

Insulin effects (phosphorylation)

- stimulate phosphatase

- inhibit phosphorylation

- stimulate glucose transporter (on alpha and beta cells)

Insulin/Glucagon effects (ATP to ADP)

- Insulin inhibits cAMP dependent kinase stopping ATP conversion to ADP

- Glucagon activates adenylate cyclase and stimulates cAMP dependent kinase (promotes ADP → ATP)

Glucagon effects (molecular)

- adenylate cyclase/ cAMP increases

- activation of a cAMP-dependent kinase

- phosphorylation = inactivation of glycogen synthase

What does metabolism of glucose do in beta-cells?

produce various metabolites that will specifically augment insulin secretion

- increase ATP/ADP ratio

- increase intracellular pH

- phospholipid metabolism

Metabolic signals effect on insulin synthesis

- increase insulin transcription,

- Decrease mRNA insulin degradation,

- Increase mRNA translation

Insulin Secretion steps**

- Rise in blood glucose levels

- Uptake of glucose by the GLUT2 transporter of beta-cell

- Glycolytic phosphorylation of glucose causes a rise in the ATP:ADP ratio

- This rise inactivates the potassium channel that depolarizes the membrane (charge in beta cell becomes increasingly positive)

- The calcium channel to open up allowing calcium ions to flow inward

- The rise in levels of calcium leads to the exocytotic release of insulin from their storage granule

IDDM (type 1 Diabetes)

- lack of insulin (beta cells destroyed)

- treat with tissue grafts, add insulin, or stem cell therapies

Why is stem cell therapy for T1D difficult to implement?

The islet of Langerhans is the functional unit (beta cells function best with the other types of cells around) so another therapy is islet of langerhans transplantation (Edmonton protocol)

NIDDM (type 2 Diabetes)

Beta cell insensitivity - many reasons

- insulin autoantibodies, mutant insulin, insulin receptor malfunction, lack of glucose transporters, mutant glucokinase or other enzymes

- treat with drugs to increase insulin sensitivity exercise, diet

T2D risk can increase due to...?

Poor in utero nutrition, obesity, lack of exercise

How can enlarged adipocytes contribute to obesity and diabetes?

In some obese people, enlarged fat cells have reduced IR, so the cells are less likely to respond to insulin

How does the body compensate for diabetes?

Increased insulin secretion, however normal/responsive cells respond to high levels of insulin with receptor down regulation

- Result: hyperglycemia

Nutritional factor controlling an endocrine cell example

Blood glucose levels regulating insulin secretion

Signalling regulation of islets of langerhans

autocrine, paracrine, endocrine, and nutritional signals

Another action insulin has on IR

IR internalization

What is IR internalization?

When insulin binds IR it also promotes the downregulation of IR and some leave the beta-cell surface.

- With too much insulin secretion, IR internalization occurs at such a high level that the cell cannot respond to insulin and glucose is not cleared from the blood.

Primary regulator of insulin secretion

glucose

Other secondary potentiators and inhibitors of insulin secretion origins

- From alpha cell

- From delta cell

- parasympathetic/sympathetic innervation of pancreatic inlet

Pancreastatin effect on insulin release

inhibits insulin release; autocrine (from beta-cell)

Glucagon effect on insulin release

promotes insulin release; paracrine (from alpha cell)

Peptide YY effect on insulin release

inhibits insulin release; paracine (from alpha cell)

Galanin and NPY effect on insulin release

inhibits insulin release; neurocrine (from sympathetic nerves)

ACh, VIP, gastrin releasing peptide effect on insulin release

promotes insulin release; neurocrine (from parasympathetic nerves)

CCK effect on insulin release

promotes insulin release; neuroncrine

Epinephrine effect on insulin release

Can inhibit and promote insulin release; endocrine (from blood)

Gamma aminobutyric acid (GABA) and insulin secretion

GABA inhibits insulin secretion by autocrine feedback

- GABA may have some protective effects on β-cells

What enzyme converts glutamic to GABA?

glutamic acid decarboxylase (GAD)