NME2.

32
Insulin and Glucagon

Role in blood glucose

homeostasis
Loranne.Agius@ncl.ac.uk
 Lecture outcomes:
1. Blood profiles of insulin and glucagon

2. Regulation of insulin and glucagon secretion

3. Insulin and Glucagon Action: Where and how?

4. Insulin and Glucagon post-receptor signalling

 Consequences of insulin deficiency in diabetes

 Type 1, Type 2, Monogenic diabetes

 Control of blood glucose levels
Glucose is essential for life, but devastating consequences occur
when the blood glucose concentration deviates from a narrow
range (~5mM).

Hypoglycaemia: A decrease in blood glucose concentration causes

unconsciousness, brain damage and death. ACUTE EVENT

Hyperglycaemia: A sustained increase in blood glucose causes

damage to retina, kidney, nerves, blood vessels (Diabetes mellitus).

• Blood [glucose] ~ 5 mM (90 mg/dl)

• Total amount in blood is < 5 g
• Daily intake of glucose ~ 300 g
• After a meal [glucose] 5-7 mM
• But it potentially could rise to > 20 mM

 Blood glucose homeostasis requires

very effective control of both hormone secretion and action
 Insulin promotes energy storage (glycogen and
triglyceride) and lowers blood [glucose]
Glucagon promotes fuel mobilisation (glycogen and
triglyceride) and raises blood [glucose]

Insulin is secreted when

Blood glucose rises

Glucagon is secreted when

Blood glucose falls

Epinephrine, norepinephrine, cortisol also

promote fuel utilisation and raise blood glucose
Blood profiles of insulin and glucagon after a
high-carbohydrate meal
High carbohydrate
meal Changes in blood glucose (BG), after
a high-carbohydrate meal

Hyperglycaemia (High BG)

Stimulates insulin secretion.
Maximum 30-60 min after a meal.
Insulin Insulin declines in parallel with
decline in blood glucose

Hyperglycaemia (high BG):

Glucagon
Inhibits glucagon secretion.
Lowest level after a high-
carbohydrate meal.
 Pancreatic islets (Islets of Langerhans) are scattered throughout
the pancreas: They make up 1% of pancreatic tissue
They comprise: α,β and δ cells that secrete hormones

Islet Hormone %
Cells

Beta Insulin 60

Alpha Glucagon 30

Delta Somatostatin 8-10

2 Insulin production: Transcription:
Sequence of events synthesis of mRNA

Translation: binding of
Transcription mRNA to ribosomes
Translation
Pre-proinsulin

Enters ER

Cleavage to Proinsulin

Enters Golgi

Cleavage to insulin + C-peptide

Insulin accumulates in granules

Secretion- Exocytosis: fusion

of granules with membrane
2
Proinsulin  Insulin conversion

C-Peptide

NH2
S S A Chain
COOH

S
S
S
B Chain S

Intramolecular disulphide bonds form

 2
Proinsulin  Insulin conversion

C-peptide
Inactive, stable
Diagnostic value
A21

NH2
B30 S S A Chain
COOH

S
S
S
B Chain S Insulin
A-chain (21 AAs)
B-chain (30 AAs)
Active, short
half-life
2
Formation of Insulin from Pre-proinsulin

Insulin is synthesized as a pre-prohormone which is converted in the

RER to proinsulin.
Proinsulin folds into the correct conformation and intramolecular
disulphide bonds form between cysteine residues.
It is transported in microvesicles to the Golgi complex
A protease cleaves proinsulin into insulin and C-peptide.
The vesicles also contain Zinc ions.
Insulin is less soluble than proinsulin and it precipitates with Zinc in
the granules during cleavage.
During vesicle fusion both insulin and C-peptide are secreted.
C-peptide has no physiological function but it has a longer half-life than
insulin and can be a useful measure of endogenous insulin secretion.
 2
Regulators of insulin release
• Major
– GLUCOSE +++
• Other
– Amino acids +
– Cholinergic nerves +
– Gut peptides (incretins) +
ex: GLP1, GIP
– Adrenaline -
– Somatostatin -

Glucose is the major determinant. But parasympathetic innervation

& peptides secreted by the gut can markedly enhance secretion.
Trauma and stress through secretion of adrenaline can inhibit secretion
& over-ride the effect of glucose.
 Glucose 2
How does glucose stimulate
Insulin Secretion?

Glucose metabolism Glucose

1
by glucokinase
Glucokinase
Glycolysis
Glucose-6-P
Pyruvate oxidation 2
Pyruvate Pyruvate
ATP formation Insulin 8
3 granules
Closure of K-ATP
(Potassium) TCA 7
4
Channels
ATP Ca2+
K+
Membrane 6 ↓
Depolarisation 5

Ca-entry through
voltage channel
Sulphonylureas
Insulin exocytosis
 2
What is the Potassium channel?

Comprised of 2 subunits:

Illustration
– Kir6.2 - Channel-forming
subunit
– SUR1 - Transmembrane
protein

 Resting state:
– Channel is open
– Efflux of K+ maintains membrane potential at -60mv.
 Glucose stimulus:
– Channel is closed
– Suppresses efflux of K+ depolarises plasma membrane

** Mutations in the K+ channel cause diabetes or hypoglycaemia depending

depending on whether they block opening or closing of the channel.
** Sulphonylureas used to treat Type 2 diabetes close the channel
 2
Regulation of insulin secretion by glucose
• Glucose is transported across the membrane by the GLUT2 transporter.

• Glucose is phosphorylated by glucokinase (similar to liver).

• Glucose 6-P is converted to pyruvate by glycolysis.

• Pyruvate enters mitochondria  acetyl-CoA  Krebs cycle  NADH.

• NADH is used to generate ATP by oxidative phosphorylation.

• Increase in ATP/ADP ratio closes the K-ATP (Potassium) channels.

• Closure of K-channels causes depolarisation of the plasma membrane.

• Depolarisation allows calcium entry and stimulation of insulin secretion.

• Mutations in glucokinase of K-channels cause either diabetes (MODY2)

or hypoglycaemia.
2
Glucagon

Pre-proglucagon (160 amino acids)  Proglucagon

Glucagon (29 aminoacids)

Glucagon is produced as preproglucagon on the RER and converted to

Proglucagon as it enters the RER.

Proglucagon is cleaved to glucagon (29 amino acids) and glucagon-like

peptides (GLP-1 and GLP-2).

Gluagon is rapidly degraded by the liver and kidney with a plasma half-life of
3-5 min.

The major target organ for glucagon is the liver.

2
Regulators of glucagon release

• Major
– GLUCOSE -
– Insulin -
– Somatostatin -
– Amino acids +
• Minor
– Neural input (stress) +
– Gut hormones +
– Epinephrine +
– Cortisol +
 Liver, Adipose tissue & Muscle are insulin-sensitive tissues.
Glucose (& lipid) metabolism in L, M, AT is insulin-dependent.
The brain is glucose-dependent but insulin-independent.

Liver function Adipose tissue

Glucose homeostasis Stores and degrades Triglycerides
Fatty acid  KB
Amino acid metabolism Regulated by:
Insulin & counter-regulatory
Regulated by: hormones
Insulin & glucagon

The brain is critically

Muscle fuels for energy: dependent on Glucose
Glucose (insulin-dependent) But insulin-independent
Fatty acids or
It use Ketone bodies
Ketone Bodies
But NOT Fatty acids
Adipose: fed state (High insulin) Adipose: fasted state (Low insulin)
High glucose uptake Low glucose uptake
High lipoprotein lipase (LPL) Low lipoprotein lipase (LPL)
Low hormone sensitive lipase (HSL) HiGH hormone sensitive lipase (HSL)

Fasted
Fed

HSL

LPL

High Insulin Low insulin

Triglyceride storage Triglyceride breakdown
Muscle:
Muscle: fed state (High insulin)
High glucose transport
Glucose used as fuel
Protein degradation balanced by
synthesis

Muscle: fasted state (Low insulin)

Glucose not used as fuel
Fatty acids are the main fuel
Ketone Bodies can be used
Protein degradation not balanced
by synthesis
Amino acids are released and used
for gluconeogenesis in liver
Liver Metabolism

Fed state (high insulin)

Glucose conversion 
glycogen & Triglyceride
(secreted in VLDL)

Major site of protein

synthesis & degradation

Fasted state (low insulin):

Glucose produced by
Glycogenolysis &
Gluconeogenesis
FA oxidation  Ketone
Bodies

Type 1 diabetes (v. Low

insulin)
Excessive
The Brain always requires Glucose: but it can also uses
ketone bodies during starvation

Fed state Fasted state

 33. Insulin action in: Liver, muscle & adipose tissue

Liver
+ Glycogen synthesis
+ Fatty acid / TG synthesis
+ Protein synthesis
- Glycogen degradation
- Gluconeogenesis
Ketone
bodies Muscle
Fatty acids + Glucose transport
+ Glucose oxidation
Glycogen + Glycogen synthesis
+ Protein synthesis

Adipose tissue
+ Glucose transport
+ Triacylglycerol synthesis
- Triacylglycerol breakdown
3 3 Glucagon stimulates glucose production by the liver

Glycogen

Amino acids
1
2
Glucose

Glucose

Glucagon:
1. Increases Glycogen degradation (Rapid)
2. Increases Gluconeogenesis (Slower)
 3 3. Insulin and glucagon exert opposite effects on enzyme
activation / inactivation and gene transcription

1. Rapid changes in enzyme activity Phosphorylation causes (-) or (+)

Glucagon causes phosphorylation Glycogen synthase (-)
Insulin causes dephosphorylation Glycogen phosphorylase (+)

2. Regulation of gene transcription  Changes in enzyme concentration.

Insulin ++ ↑ Glucokinase (Gene transcription)

↑ Phosphofructokinase (↑ F2,6-P2)
Glucagon -- ↑ Enzymes of lipogenesis (gene
transcription).

Insulin -- ↑ Phosphoenolpyruvate carboxykinase

↑ Fructose bisphosphatase
Glucagon ++ ↑ Glucose 6-phosphatase
 44. Insulin Signalling
Insulin receptor (IR)
Tetramer: ααββ
β=Tyrosine kinase 
Activated by insulin
 
Autophosphorylates > β(P)
GT
IR substrate (IRS) P P 
Binds to phosphorylated IR
Becomes phosphorylated by IRβ
IRS P
Kinase cascade GT
Activation of PI3kinase
Activation of PDK1 PI3K P
“ of Protein kinase B (PKB)
“ Signalling enzymes PI-3,4,5-P3 Kinase
“ of Transcription factors
Protein Synthesis Cascade
GLUT4 translocation in PDK1
muscle & adipose tissue

Activation of glycogen sythase Glycogen synthesis

P
Inactivation of “ phosphorylase PKB/Akt P
Fat synthesis
Gene transcription:
Induction of Glucokinase P nucleus
Repression: Gluconeogenic TF
enzymes (G6Pase, FDPase
TF GK
PEPCK).

TF X PEPCK
44. Insulin signalling: a cascade of protein phosphorylation

1. Binding of insulin to the insulin receptor (IR) alpha-units causes a

conformational change in the beta-subunits and activation of the receptor
tyrosine kinase activity.

2. The IR becomes autophosphorylated on tyrosine residues (Y).

3. The Insulin receptor substrate (IRS), a large protein, binds to the

phosphorylated IR and becomes phosphorylated on tyrosine residues by the
IR kinase.

4. Phosphatidylinositol 3-kinase (PI 3-kinase) binds to the phosphorylated IRS

and becomes activated and generates PI-3,4,5-P3.

5. PDK1 is activated by PI-3,4,5-P3 and causes phosphorylation (activation) of

Protein kinase B (Akt) on serine and threonine residues.

6. Akt causes phosphorylation of various substrates on serine and threonine

residues leading to stimulation of glucose transport, glycogen synthesis and
protein synthesis, and control of gene transcription.
 4 Glucagon Signalling
4.
1 Glucagon receptor binding 1 Adenylate
cyclase
2 Interaction with G-protein-GDP
3
3. GTP exchanges with GDP
GDP 2 GTP
4. GTP-Gs-α binds and activates
adenylcyclase GTP GDP
ATP cAMP
5  Adenylate cylcase  cAMP

4  cAMP-dep-PK
4
5 Protein phosphorylation
Phosphorylase Kinase (P) 5 cAMP-dependent
Glycogen Protein kinase
Breakdown Glycogen phosphorylase (P) activation

Gene transcription 5
CREB (P)
(Gluconeogenic enzymes)
 5 Consequences of insulin deficiency
1. Decreased uptake of glucose by muscle and adipose tissue.

2. Increased production of glucose by the liver (glycogen breakdown

& gluconeogenesis).

3. Decreased conversion of glucose to glycogen and fat in the liver.

4. Increased breakdown of triglyceride by adipose tissue and release

of fatty acids.

5. Conversion of fatty acids to ketone bodies in liver (low malonyl-

CoA)

6. Protein breakdown and loss of amino acids by muscle

↑ Blood glucose ↑ Fatty acids ↑ Ketone Bodies

 Blood [glucose], [fatty acids] & [ketone body] (mM) during 8 day fasting

Diabetes
Days Fed 1 2 3 4 8
Substrate

Glucose 5.5 4.7 4.1 3.8 3.6 3.5

36% ?
Fatty acids 0.30 0.42 0.82 1.04 1.15 1.88
600 ?
%
Ketone 0.01 0.03 0.55 2.15 2.89 5.34
bodies
500X ?
 Blood [glucose], [fatty acids] & [ketone body] (mM) during 8 day fasting

Diabetes
Days Fed 1 2 3 4 8
Substrate

Glucose 5.5 4.7 4.1 3.8 3.6 3.5

>20
36%
Fatty acids 0.30 0.42 0.82 1.04 1.15 1.88
600
%
Ketone 0.01 0.03 0.55 2.15 2.89 5.34
bodies
500X
 Types of Diabetes mellitus

Polygenic forms (involvement of multiple predisposing genes):

Type 1 DM: (~ 10%) Autoimmune destruction of islet -cells (IDDM)

Type 2 DM: (~90%) Insulin resistance and relative insulin insufficiency (NIDDM).

Monogenic diabetes (Maturity Onset diabetes of the Young, MODY): (1-2%)

Single gene defects of : Glucokinase, transcription factors,
potassium channels

Glucokinase (MODY2)
Transcription factors: HNF4, HNF1, PDX-1