Block C Lecture 2 - Regulated Exocytosis

Question 1

Other than vesicle fusion with the membrane, what process does exocytosis mediate?

Answer 1

The delivery of membrane proteins to the plasma membrane

(Slide 4)

Question 2

What are the 2 types of exocytosis?

Answer 2

Constitutive and Regulated

(Slide 5)

Question 3

What is constitutive exocytosis and what is it important for?

Answer 3

It is when vesicles bud from the Golgi and fuse directly with the plasma membrane. This is important for the transport of newly synthesised proteins and lipids to the plasma membrane and for secretion of various molecules

(Slide 5)

Question 4

What is regulated exocytosis?

Answer 4

When vesicles are retained in the cytoplasm until a signal arrives (which is usually a rise in cytoplasmic Ca2+ concentration). This signal then promotes the fusion of vesicles with the plasma membrane

(Slide 5)

Question 5

What are 3 examples of regulated exocytosis?

Answer 5

Neurotransmitter Secretion

Insulin Secretion

GLUT4 translocation

(Slide 6)

Question 6

What does phosphorylation of glucose to glucose-6-phosphate result in?

Answer 6

It traps glucose in the cell and commits it to further metabolism

(Slide 10)

Question 7

What 2 enzymes catalyse the phosphorylation of glucose to glucose-6-phosphate?

Answer 7

Hexokinase and Glucokinase

(Slide 10)

Question 8

Hexokinase is ubiquitously expressed. What does this mean?

Answer 8

It’s widespread throughout the body and isn’t limited to a specific cell or tissue

(Slide 10)

Question 9

Why does hexokinase work at full capacity even with low glucose concentrations and what does this ensure?

Answer 9

As it has a high affinity for glucose, with this ensuring cells have sufficient glucose metabolism even when blood glucose is low

(Slide 10)

Question 10

How does glucokinase activity vary with glucose concentration and why is this the case?

Answer 10

It varies within the physiological range of blood glucose concentration as it has a lower affinity for glucose than hexokinase does

(Slide 10)

Question 11

How does glucose-6-phosphate lead to an increase in ATP?

Answer 11

As once glucose is phosphorylated to glucose-6-phosphate it enters the glycolysis pathway

(Slide 10)

Question 12

How is insulin secretion biphasic?

Answer 12

As there is first a rapid increase of secreted insulin followed by a slowly increases second phase

(Slide 11)

Question 13

What occurs in the 2 phases of insulin secretion?

Answer 13

First phase: Fusion of granules that are docked at the plasma membrane (called “readily-releasable granules)

Second phase: Requires recruitment of cytosolic pool of granules (known as the “reserve pool”)

(Slide 12)

Question 14

Which proteins mediate the exocytosis of insulin in response to a Ca2+ influx and how do they do this?

Answer 14

SNARE proteins mediate this by vesicle SNAREs (aka vSNAREs) forming a complex with tSNARES in the membrane with the formation of this complex driving the fusion (exocytosis) of the vesicle with the membrane

(Slide 13)

Question 15

Other than glucose and SNARE proteins, what is needed for insulin to be secreted by beta cells?

Answer 15

Ca2+-secretion coupling

(Slide 14)

Question 16

What is synaptotagmin?

Answer 16

A Ca2+-binding protein which is present on insulin granules

(Slide 15)

Question 17

How does synaptotagmin link increased calcium levels induced by elevated blood glucose levels to the secretion of insulin?

Answer 17

As the Ca2+-bound form of synaptotagmin interacts with SNARE proteins and membrane phospholipids to enhance the probability of membrane fusion taking place

(Slide 15)

Question 18

What are the 3 major targets of insulin?

Answer 18

The liver

Adipocytes (fat cells)

Skeletal muscle

(Slide 16)

Question 19

What regulated exocytosis pathway does insulin activate via intracellular insulin signalling pathway in adipocytes and skeletal muscle cells?

Answer 19

The exocytosis of vesicles containing GLUT4

(Slide 16)

Question 20

What is GLUT4?

Answer 20

A glucose transporter protein

(Slide 16)

Question 21

What are the steps of GLUT4 exocytosis triggered by insulin?

Answer 21

1. Binding of insulin to the insulin receptor (IR) leads to receptor auto-phosphorylation
2. Phosphorylated residues on the IR acts as binding sites for insulin receptor substrate (IRS) proteins
3. IR phosphorylates 4 tyrosine residues in IRS proteins
4. The lipid kinase, phosphoinositide 3-kinase binds to phosphorylated residues on IRS proteins, and then converts membrane PIP2 to PIP3
5. Binding to PIP3 activates PDK1, which then phosphorylates and activates kinases such as PKB (aka Akt)
6. Activated PKB (phosphorylated on Serine-473 and threonine-308) phosphorylates proteins on GLUT4 vesicles to promote exocytosis

(Slide 17)

Question 22

What interactions does the exocytosis of GLUT4 require?

Answer 22

Interactions between SNARE proteins; VAMP located on the vesicle interaction with syntaxin and SNAP23 at the plasma membrane, with these leading to vesicle fusion

(Slide 18)

Question 23

What does vesicles containing GLUT4 binding to the plasma membrane lead to?

Answer 23

The insertion of GLUT4 receptors into the plasma membrane and an increased flux of glucose into the cells through GLUT4 binding to said receptors

(Slide 20)

Question 24

What is the function of AS160 and how does protein kinase B (PKB) influence this?

Answer 24

AS160 acts to retain vesicles containing GLUT4 inside the cell.

PKB phosphorylates AS160, inactivating it and allowing GLUT4 vesicles to fuse to the plasma membrane leading to increased levels of the glucose transporter at the cell surface

(Slide 22)

Question 25

What is type 1 diabetes and how is it caused?

Answer 25

It is a chronic autoimmune disease where the destruction of beta cells results in insulin deficiency and hyperglycaemia.

It occurs in genetically susceptible individuals, likely as a result of an environmental trigger.

Question 26

In what 2 ways does type 1 diabetes contribute to hyperglycaemia?

Answer 26

1. Loss of insulin-stimulated glucose uptake into target cells
2. Loss of insulin-mediated repression of gluconeogenesis and glycogen breakdown in the liver

(Slide 24)

Question 27

What is type 2 diabetes?

Answer 27

High blood glucose as a result of insulin resistance of target tissues leading to insufficient production / secretion of insulin (beta cell dysfunction)

It’s usually diagnosed later in life

(Slide 25)

Question 28

Which type of diabetes is most common?

Answer 28

Type 2

(Slide 25)

Question 29

What are 4 reasons type 2 diabetes is becoming more popular?

Answer 29

Increased obesity

Lack of exercise

Poor diet

An ageing population

(Slide 25)

Question 30

What allows type 2 symptoms to be controlled and what may be prescribed?

Answer 30

Eating a healthy diet with regular exercise may allow control of symptoms.

Oral medications may be required as the disease progresses and insulin can also be prescribed if glucose control is not adequate

(Slide 27)