Type 1 Diabetes (2)

Question 1

The pancreas is an organ of the

Answer 1

Endocrine system

Has exocrine (digestive) functions as well but we focus on the endocrine (hormonal) functions in T1D

chemical signalling in the form of hormones that are transported primarily by the bloodstream

Autocrine (affects cell that secreted it)
Paracrine (affects neighboring cells)
Long distance effects (other parts of the body)

Question 2

Pancreas anatomy

Answer 2

Acinus
>cluster of acinar cells, secrete digestive enzymes into the pancreatic duct

Islets of Langerhans
>secrete hormones into the bloodstream
>alpha cells
>beta cells
>exocrine acinus

Question 3

Pancreatic cells: Exocrine

Answer 3

majority of the pancreas consists of acinar and ductal cells

> secrete digestive enzymes into the pancreatic duct that empties into the duodenum of the small intestine
amylase, lipase, trypsinogen, aqueous secretions that contain bicarbonate to neutralise stomach acid

Question 4

Islets of Langerhans

Answer 4

Only 2% of pancreatic cell mass

Normal pancreas insulin staining will stain islets brown

Endocrine cells secrete hormones directly into bloodstream
>beta cells (65-85%): insulin
>alpha cells (15-20%): glucagon
>delta cells (3-10%): somatostatin
>gamma cells (3-5%): pancreatic polypeptide
>epsilon cells (<1%): ghrelin

Question 5

Multiple fate selections allow the development of the pancreatic islet lineages

Answer 5

> Sox17 (endodermal cell)
Sox9 (can split into hepatocytes and liver duct cells, or duodenum epithelial cells and endocrine cells)

> > Pdx1 (pancreatic progenitor cells)
can split into acinar cells or duct cells
Ngn3 (islet progenitor)
beta and alpha cells

Question 6

Insulin

Answer 6

Encoded by INS gene on the short arm of Chr11

> peptide hormone that decreases blood glucose levels
initially translated as this long polypeptide
>beta chain connected to an alpha chain via disulfide bonds, and connecting peptide (c peptide) in the middle
>c peptide is cleaved out on arg/lys then end up with 2 chains connected to each other via disulfide bonds

> insulin is produced and stored as a hexamer coordinated around zinc ions
monomeric form is not stable, thus stored as a hexomer, stable while inside the beta cell waiting for stimulus to be degranulated

Question 7

Glucose is the main trigger for insulin secretion by beta cell

Answer 7

Beta cells - sensitive to glucose concentrations in the blood

Other stimulatory of amplification factors that modulate the secretion of insulin:
>Hormones: glucagon, GIP, GLP-1
>all 3 directly stimulate beta cells to produce insulin
>Increased fatty acid or amino acids in blood also stimulate increase in secretion of insulin

Inhibitory factors:
<

Question 8

The beta cell and insulin:

Describe the Insulin Synthesis Pathway

Answer 8

1) Chr11 INS gene

2) Pre-pro-insulin
>signal sequence (directs PPI to ER) + Beta chain + C-peptide + Alpha chain

3)Endoplasmic reticulum (ER) processing
>cleave signal sequence
>formation of B-A disulphide bonds
>Pro-insulin

4) Shuttle to Golgi Apparatus
>C-peptide cleaved by pro-hormone convertase
>always 1:1 ratio of c-peptide to active insulin

5) Mature biological active insulin stored in insulin granules, secreted by exocytosis

Question 9

The beta cell and insulin:

Describe the Insulin Secretion Pathway

Answer 9

1) Increase in BGL
2) Uptake of glucose into Beta cells via GLUT 1/2/3 Transporters

3) Glycolysis in beta cells > produce ATP and NADH
>Increased ATP:ADP ratio

4) ATP blocks K+/ATP channels
>increased K+ in cell makes cell more positively charged
>depolarisation of cell membrane
>opens up voltage-dependent Ca2+ channels

5) Influx of Ca2+ into cell
>stimulates exocytosis of insulin granules

Question 10

Insulin effects on target cells

Answer 10

Insulin
>anabolic hormone
>promotes conversion of small energy molecules (glucose, fatty acids, amino acids) into large storage molecules

> > binds to cellular receptors (2 alpha 2 beta subunits-RTK) on target cells, and signals upregulation of GLUT4 transporters

Question 11

Insulin promotes:

Answer 11

Insulin promotes *glycogenesis
>Liver
»glucose > glycogen

> > once glycogen storage in liver is full
glycolysis of glucose into pyruvate (+acetyl-coa in adipose tissue)
form fatty acids to store as fat

> Skeletal muscle
>stimulates uptake of glucose and amino acids
promotes protein production and muscle growth

Question 12

Insulin Inhibits:

Answer 12

Insulin inhibits *gluconeogenesis
>Liver
»prevent formation of glucose from lactic acid and non-carbohydrate molecules

Insulin inhibits *lipolysis
>adipose tissue
»prevent breakdown of fatty acids

Question 13

Glucagon (counter-regulator to insulin)

Answer 13

Encoded by GCG gene on long arm of Chr2
>Peptide hormone that increases BGL

> stored in granules in the alpha cell where it waits to be released (monomer form)

> Preproglucagon
(leader signal peptide moves it into ER and golgi)
proglucagon
glucagon

Question 14

Glucose is the main regulator of glucagon secretion by alpha cells

Answer 14

Alpha cells - sensitive to low glucose concentrations in the blood

Stimulatory factors:
>Adrenaline
»sympathetic NS
»activated by stress

> Cholecystokinin
>intestinal cells
>helps with digestion: stimulates digestion and absorption

Inhibitory factors:

Question 15

Glucagon effect on target cells

Answer 15

Glucagon
>catabolic hormone
>promotes breakdown of large storage molecules

> binds to receptors on target cells (7TM receptor)
signals cell to breakdown glycogen and fat

Glucagon promotes
>glycogenolysis (breakdown of glycogen into glucose)
>gluconeogenesis (synth of glucose from non-carbohydrate carbon sources)
>*lipolysis
»>causes blood glucose levels to rise
»>breakdown of fatty acids converted into ketone bodies in liver (might lead to diabetic ketoacidosis)

Question 16

Interplay between insulin and glucagon to maintain blood glucose levels

Answer 16

> Increase in blood glucose concentration (e.g. after a meal)
Increase insulin secretion by pancreatic beta cells
»glucose uptake by liver (adipose tissue, muscle)
»increase in glycogenesis (also lipogenesis)
«Decrease in blood glucose concentration (e.g. fasting, sleeping, exercise)
Increase in glucagon secretion by pancreatic alpha cells
»Breakdown of glycogen by liver
»increase in glycogenolysis, glyconeogenesis (also lipolysis in adipose tissue)
«

Question 17

Pancreas interplay with brain, liver, gut, adipose and muscle tissues

Answer 17

Pancreatic beta and alpha cells receive constant feedback from various organs in the body
>brain-islet axis
>liver-islet axis
>gut-islet axis
>adipose/muscle tissue

Highly sophisticated network using various hormones, neurotransmitters, and cytokines

Question 18

How to measure glucose?

Answer 18

> > > Urine sample (not as accurate as blood)
Glucose oxidase enzyme (and peroxidase) located on detection strip

hydrogen peroxide H2O2 from glucose oxidase reaction is coupled to peroxidase enzyme to form h20, causes potassium iodide chromogen dye to get oxidised
>colour change proportional to glucose concentration in the sample

> > > Blood sample (finger prick)
Alternatively
Glucose meter use an electrode instead of 02 to take up electrons needed to oxidise glucose
Electrons produce an electronic current in proportion to glucose concentration

Question 19

How to monitor glucose?

Answer 19

Urine glucose monitoring
- Kidneys typically remove glucose from the bloodstream when blood glucose concentrations >10mmol/L
-not possible to determine exact blood glucose concentration from a urine test
(its the overflow of blood glucose into the kidney and you dont know the exact amount that has caused this overflow)

Blood glucose monitoring

• finger prick (capillary blood sample - small volume)
• some variation is normal, depends on when measured (e.g. empty stomach or after a meal)
• measurement of blood glucose for diagnosing diabetes done by a certified laboratory (POCT devices are not clinically diagnostic)

Hyperglycaemia = BGL > 7.8mmol/L
Hypoglycaemia = BGL < 3.3mmol/L

Question 20

Measure glucose for diagnostic purposes

Answer 20

Random venous plasma glucose measurement
1. A blood sample is taken randomly (intravenous collection done in the clinic)
2. Plasma glucose concentrations measured by a certified laboratory
»»» >11.1mmol/L = Diabetes Mellitus
(Basically use the same glucose oxidase enzymatic reaction but a little more controlled with certain types of standards)

Question 21

Why can’t a diagnosis of T1D be made based on this value of random venous blood glucose >11.1mmol/L alone?

Answer 21

T1D diagnosis also requires detection of autoantibodies against beta cell antigens in serum

Question 22

How to measure insulin and C-peptide

Answer 22

Radioimmunoassay

1. Add known amount of radiolabelled insulin (or c-peptide) to an insulin (or c-peptide) specific antibody
   >let it bind
2. Add unlabelled patient serum: contains the unknown insulin and c-peptide amount to be measured
3. Insulin (or C-peptide) in unlabelled patient serum displaces radiolabelled insulin (or c-peptide)
4. Separate antibody-bound insulin (c-peptide) from unbound insulin (c-peptide)
   >using a secondary antibody to pull down by the Fc portion
   >immunoprecipitate this antibody
   >measure the radiation in both fractions and compare to standard curves to measure PTs serum
5. Measure radioactivity in bound and unbound fractions
6. Compare to standard curve to determine insulin (or c-peptide) amount in patient serum

Question 23

What can be determined from a radioimmunoassay?

Answer 23

Insulin: detect insulinomas (i.e. insulin-secreting tumour)

Insulin: limited utility for diabetes - does not reliably identify patients who require insulin therapy (can still detect residual insulin in patients with T1D)
>it will depend on if they’ve had a meal or what stage of T1D they’re at

C-peptide: typically used in research studies top determine beta cell function
>usually research context, not a clinical context
>identify individuals at risk of developing T1D
(in combination with other things such as autoantibodies)
>Determine residual beta cell mass in long-standing T1D patients
>Measure beta cell function in patients after pancreas/islet transplantation

Question 24

The practical side of regulating blood glucose

Answer 24

> Healthy adult ~75kg
blood volume 5L
glucose 5g
glucose concentration = 5.5mmol/L

> > glucose homeostasis healthy range is 4-6mmol/L
BGL stays level overnight because of glucagon - kicks in to maintain a certain threshold level

Insulin (along with glucagon)
>always a basal level of each hormone, glucagon making sugar and putting it in the bloodstream, stimulating a bit of insulin production, and vice versa
>homeostatic effect
»»>tightly regulates blood glucose concentration 4-6mM
»»>Basal (fasting) insulin <25uU/mL
»»> Postprandial insulin can rise >10-fold

Question 25

Physiological glucose levels in a healthy individual vs T1D

Answer 25

4-6mM

Insulin spikes post-prandial (after meal) and glucagon dips

Above normal physiological glucose level = hyperglycaemia
Below normal physiological glucose level = hypoglycaemia

T1D
>glucose level will be above normal range
>difficulty in keeping glucose levels within a normal range
> no insulin being produced because beta cells are being destroyed by immune cells
>some glucose will still be absorbed by the cells in the body and be secreted out in the urine so there is still a decrease on occasion down to the normal range, but storage and uptake of glucose by liver, adipose and muscle is severely impaired which is enough to keep glucose levels high in the bloodstream

Question 26

Goal of treatment in T1D

Answer 26

Mimicking physiological insulin with exogenous insulin injections

> rapid acting insulin injections
taken just before meals
(monomer form which isnt coordinated with zinc)
(during this time, alpha cells are still working and not destroyed by immune cells, glucagon is still coming on and stimulating release of insulin, so you need to counteract that increase in BGL)

> long-acting insulin injections
taken twice daily, one morning and one night
(hexomer coordinated with zinc so it slowly dissipates and releases)
(in this way, trying to regulate BGL in opposition with glucagon)

Question 27

How well does exogenous insulin treatment work in T1D?

Answer 27

Child with T1D and treated with exogenous insulin
>can see that it is a struggle to keep it within the normal range
>hyperglycaemia is still occurring but there were no severe hypoglycaemic events
>an adult with T1D will typically be better and more disciplined with injections and managing their BGL in a normal range

Question 28

Diabetic Ketoacidosis

Answer 28

Lack of insulin
>increased glucagon:insulin ratio
>release of FFA from adipose tissue (lipolysis)
>converted by liver into ketone bodies (beta oxidation)
>ketone bodies have low pKa
(can be used by cells for energy, mainly in brain)
»>decreased blood pH
»>Acetone (fruity breath)

Stress of infection (even with insulin therapy)
>adrenaline (epinephrine) released
>stimulates glucagon secretion by alpha cells
»>increased glucagon:insulin ratio
»>feed back into above cycle
>also increased glucose by liver (gluconeogenesis)
>increased glucose removed by kidney along with water and solutes (osmotic diuresis)
»>polyuria
»>dehydration
»>polydipsia

*if not treated with insulin and fluids, can cause nausea, vomiting, confusion, and loss of consciousness

Question 29

Type 1 vs Type 2 Diabetes

Answer 29

T1D:
>loss of insulin production = hypoerglycaemia at diagnosis
(significantly reduced or no beta cell mass)
>exogenous insulin treatment

T2D:
>insulin resistance
(loss of insulin responsiveness in target tissues)
>pancreatic beta cells compensate = hyperinsulinemia
(produce more insulin, excessive blood insulin levels)
>pancreatic beta cell failure = hyperglycaemia at diagnosis
(failure = cannot secrete enough insulin to overcome resistance to insulin in tissues)
>exogenous insulin treatment ONLY AT LATE STAGES OF DISEASE
>is preventable by lifestyle modifications (e.g. diet and exercise)