Lecture 17 - Summing up Flashcards
How is insulin secretion regulated?
> glucose-mediated insulin secretion pathway
Pancreatic (ß-cells are equipped with several sensing devices that measure circulating glucose.
Glucose transporter (GLUT1 in humans and GLUT2 in rodents), constitutively expressed in ß-cells, is the first encountered glucose sensor in ß-cells.
Glucose equilibrates in ß-cells via GLUT1-mediated facilitated diffusion.
GLUT1 is the glucose transporter expressed in ß-cells and is should be distinguished from the GLUT4 found on muscle cells, adipocytes.
What is the process of glucose-stimulated insulin secretion (GSIS)?
Imagine an increase blood glucose levels after a heavy meal.
After entering ß-cells via the GLUT1 transporter:
- Glucose is phosphorylated by glucokinase (GK) to glucose-6-phosphate (G6P)
- G6P is converted to pyruvate
- Pyruvate oxidation through the tricarboxylic acid cycle (TCA) by mitochondria
= ATP generated
» Major signaling pathway coupled to ATP-sensitive potassium (KAN) channel-dependent insulin release
- Increase in the ATP:ADP ratio, which reduces K+ permeability by closing the ATP-regulated K+ channels.
- This is followed by accumulation of K+ inside the ß-cells, leading to depolarization of the plasma membrane.
- Voltage-gated calcium channels (VGCC) open and Ca2+ enters the cells.
The subsequent influx of Ca2+ leads to an increase in the concentration of free cytoplasmic Ca2+.
Cytosolic Ca2+ acts in several ways to increase the rate of secretion of insulin from the insulin-storing vesicles or granules.
How does glycerolipid/Free fatty acid amplification of GSIS occur?
After meals or when there is excess nutrients including lipids, there is usually a diversion of free fatty acids (FFAs) away from oxidation and towards esterification with glycerol-3- phosphate to form glycerolipids.
Glycerolipids are used to form triglycerides, which are then hydrolysed to form diacylglycerols (DAG) and further to monoacylglycerols (MAG), including 1-MAG.
1-MAG can bind the insulin granule trafficking protein Munc13-I to enhance insulin granule exocytosis.
FFAs also potentiate GSIS by interaction with the plasma membrane FFA receptor 1 (FFAR1) to phospholipase C (PLC).
PLC activation leads to hydrolysis of the plasma membrane phospholipid phosphatidylinositol bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3).
IP3 liberates Ca2+ from intracellular storage sites (that is, the endoplasmic reticulum (ER)).
DAG and Ca2+ activate PKC.
- PKC (and PKA) stimulate the exocytosis of insulin-containing secretory granules.
- this also stimulates protein kinase D (PKDI) to promote cortical F- actin remodelling and recruitment of secretory granules to the readily releasable pool.
What are GLUT4 transporters?
GLUT4 is a high-affinity glucose transporter that is predominantly expressed in muscle cells and adipocytes.
Glut4 is stored in Glut4 storage vesicles (GSVs) set to fuse with plasma membrane in the presence of insulin
Exclusion Of GLUT4 from the cell surface depends on efficient sorting and sequestration into GSVs that do not readily cycle to the plasma membrane in the absence of stimulation.
What happens when at normal glucose level when insulin is low?
In the absence of insulin, only ~5% of the total GLUT4 transporter pool is found on the cell surface.
What happens when there is an increase in blood glucose levels and insulin level is high?
GSVs translocate to plasma membrane in response to insulin or exercise, which results in a tenfold increase in glucose uptake.
Insulin signalling at target cells
How does insulin binding to the insulin receptor activate the phosphoinositide 3-kinase (P13K)-dependent signalling cascade?
activation of the phosphoinositide 3-kinase (P13K)-dependent signalling cascade by phosphorylating insulin receptor substrate (IRS) proteins, thus producing docking sites for the recruitment and activation of P13K.
- P 13K phosphorylates phosphatidylinositol-4,5-diphosphate (PIP 2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3).
PIP3 serves as a platform for the recruitment of phosphoinositide-dependent kinase 1 (PDK1) and AKT to the plasma membrane.
At the plasma membrane, AKT is phosphorylated by PDKI and mammalian target of rapamycin complex 2 (mTORC2), which results in AKT activation.
AK T promotes GSV exocytosis by:
phosphorylating and inactivating two GTPase-activating proteins (GAPs) which regulate small GTPases that are involved in GSV retention and targeting, respectively.
- AS160 (Akt substrate of 160 kDa) and
- the RAL—GAP complex (RGC, consisting of a regulatory subunit (RGCI) and a catalytic subunit (RGC2))
What happens to the GLUT4 transporters when insulin levels drop?
Endocytosis of Glut4 such as via Clathrin coated vesicles
Internalization of GLUT4 through clathrin-mediated endocytosis requires the adaptor protein AP2.
AP 2 coordinates packaging of this glucose transporter into endocytic vesicles by recruiting clathrin to the plasma membrane and by binding to an amino-terminal F5QQ1 sequence in GLUT4.
The clathrin coat is another coat protein that functions to bend the membrane to aid it in forming the vesicle budding inwards at the plasma membrane.
Vesicle scission from the plasma membrane requires the GTPase dynamin, which assembles at the neck of the invaginating vesicle and, following GTP hydrolysis, constricts and breaks the membrane.
Note that this dynamin is also involved at the TGN when cells are delivering enzymes to the lysosomes.
COPII coats do-not require dynamin.
Cytoskeletal structures and motor proteins are involved
How to measure levels of Glut4 at the plasma membrane and interior of the cell?
Total tagged HA-GIut4-GFP can be
detected using fluorescence microscopy.
- total = plasma membrane (surface) and
internal
Measurement of the GFP signals can be
done using imaging software.
Plasma membrane (surface) HA-GIut4-GFP can be detected using anti-HA anti-bodies and the appropriate secondary anti-bodies. These anti-bodies do not detect the internal HA-GIut4-GFP as long as the cells are NOT permeabilised.
This is because the anti-HA anti-bodies cannot pass through the intact plasma membrane.
Measurement of the HA signals can be done using imaging software.
So one can measure the total GFP signals of a number of cells and take the mean and do the same for the surface HA signals.
Then take a ratio of the surface to total GFP to obtain a more quantitative data for different types of studies.
What is a low-density lipoprotein (LDL)?
Cholesterol (from food ingested or synthesis in the liver) is insoluble in say the bloodstream and must be transported by a water-soluble carrier.
Low-density lipoprotein (LDL) is one of several complexes that carry cholesterol through the bloodstream.
An LDL particle is a sphere with an outer monolayer membrane of phospholipids and cholesterol, in which one molecule of a very large protein, called apo-B, is embedded.
Endocytosis
How does uptake of Low-density lipoproteins occur?
An LDL particle binds to an LDL receptor on the plasma membrane via the ApoB.
The receptor-ligand complex is internalized in a clathrin-coated pit that pinches off to become a coated vesicle with the help of dynamin.
The clathrin coat then depolymerizes to triskelions, resulting in an early endosome.
This endosome fuses with a sorting vesicle, known as a late endosome, where the low pH (~5) causes the LDL particles to dissociate from the LDL receptors.
A receptor-rich region buds off to form a separate vesicle that recycles the LDL receptors back to the plasma membrane.
A vesicle containing an LDL particle ultimately fuses with a lysosome to form a larger lysosome.
Lysosomal hydrolases degrade the apo-B protein of the LDL particle to amino acids.
Cholesterol esters in the LDL particles are cleaved into cholesterol and fatty acids.
The cholesterol is incorporated directly into cell membranes or is re-esterified and stored as lipid droplets in the cell for later use; the fatty acids are used to make new phospholipids or tri-glycerides.
Cholesterol also is converted to steroid hormones in adrenal cortical cells and to bile acids in hepatocytes.
What are the consequences of defects in the uptake of low-density lipoproteins?
Hyper-cholesterolemia - high levels of blood cholesterol
Familial hyper-cholesterolemia: high cholesterol in the blood leading to a buildup of cholesterol deposits that ultimately block the arteries.
+ genetic disease
+ heart attacks, death at a young age
Due to different genetic defects:
E.g. mutations in components of the endocytic process
+ LDL receptor mutations leading to inability to recognise ApoB
+ reduced expression levels of the LDL receptor
