Insulin Biosynthesis and Storage

Regulatory Mechanisms of Insulin Secretion

Insulin Receptor Overview
The Molecular Basis of Insulin Resistance
The Metabolic Consequences of Insulin Resistance
Case Study
- cells (60%), - cells (20 30%), - cells
(10%), pancreatic polypeptide -expressing cells
(<5%), and ghrelin - expressing cells (1%)
- cells are highly specialized for the production
and storage of insulin
Insulin comprises approximately 10% of the total
- cell protein
Under normal conditions, >95% of the secreted
product is insulin (and C peptide) and <5% is
released as proinsulin
In patients with T2DM secretion of incompletely
processed insulin precursors is increased
- cells are equipped with mechanisms to detect
changes in:
circulating nutrients
hormone levels
the activity of autonomic nervous system
The major physiologic determinant of insulin
secretion in humans is glucose and other nutrients
The consequent decrease in circulating nutrients is
detected by the - cells, which switch off insulin
secretion to prevent hypoglycemia
- cell response can be modified by potentiators
(hormones and neurotransmitters) which amplify
or occasionally inhibit nutrient - induced responses
The overall insulin output depends on:
relative input from initiators and potentiators on
individual - cells
synchronization of secretory activity between - cells in
individual islets
coordination of secretion between the hundreds of
thousands of islets
Nutrients
The major initiator of insulin secretion is glucose
Insulin secretion occurs in two phases:
transient first phase
prolonged second phase

First phase release
Glucose enters the -cells through GLUT2
Phosphorylated by the glucose sensor Glucokinase
Metabolized to produce ATP (increases ATP:ADP ratio)
Leading to closure of ATP - Sensitive Potassium Channels
(K
ATP
channel ) and membrane depolarization
Calcium ions enter the -cells via Voltage-gated Calcium
ion (Ca
2+
) Channels
Increased intracellular calcium ion concentration causes
the activation of phospholipase C
First phase release = continued
Phospholipase C cleaves PI-4,5-bisphosphate to IP
3
and
DAG
IP
3
leads to the rapid mobilization of intracellular calcium
which amplify the cytosolic calcium concentration
Increased amounts of calcium ions in the cells causes the
release of previously synthesized and stored insulin
Through activation of intracellular calcium sensing
systems within - cells
First phase release = continued
Important among these are the calcium -dependent
protein kinases:
Myosin light chain kinases
Calcium/phospholipid - dependent kinases
Calcium/calmodulin - dependent kinases (CaMKs)
It has been proposed that CaMK II activation initiates
nutrient induced insulin secretion
First phase release = continued
Arachidonic acid liberated by activation of CPLA
2
is
capable of stimulating insulin secretion in a glucose and
calcium -independent manner
But the precise role of it remains unclear
Also elevated intracellular calcium activates calcium -
sensitive adenylate cyclase isoforms, which generate
cyclic AMP from ATP
Although these signaling systems are of undoubted
importance in the regulation of - cells by non -
nutrients, their role in nutrient - induced insulin
secretion is still uncertain
ATP Sensetive Potassium Channel
- cell K
ATP
channel is a hetero - octamer formed from:
4 potassium channel subunits (termed Kir6.2) = (channel) and
4 sulfonylurea receptor subunits (SUR1) = regulatory effect
ATP and sulfonylureas induce channel closure by binding
to Kir6.2 and SUR1 subunits, respectively
ADP activates the channels by binding to a nucleotide -
binding domain on the SUR1 subunit
Activating mutations in the Kir6.2 gene have recently
been shown to be causal for cases of (PNDM)
loss of K
ATP
channel activity has been associated with
(PHHI)
- cells also possess a K
ATP
channel - independent
stimulus secretion coupling pathway termed
amplifying pathway
It is via this pathway glucose stimulates insulin
secretion at concentrations as low as 1 6 mmol/L
It has been suggested that this pathway is impaired
in T2DM
Therapeutic strategies targeting this pathway may
be beneficial in restoring - cell function in
patients with T2DM
Nutrients
Several amino acids stimulate insulin secretion
Most require glucose
Some; (leucine, lysine and arginine) do not require
glucose
Sodium - independent transport system (leucine)
Cationic amino acids specific transport system
Metabolism of leucine decreases K
+
permeability and
activates L-type calcium channels
It is believed that cationic amino acids directly depolarizes
the - cell membrane



Non-nutrients
Most, if not all influence the - cell by binding to and
activating specific extracellular surface receptors
- cell expresses receptors for a wide range of
biologically active peptides, glycoproteins and
neurotransmitters with limited number of intracellular
effectors
Islet Hormones
Insulin
Glucagon
Somatostatin
Pancreatic polypeptide
Non-nutrients
Most, if not all influence the - cell by binding to and
activating specific extracellular surface receptors
- cell expresses receptors for a wide range of
biologically active peptides, glycoproteins and
neurotransmitters with limited number of intracellular
effectors
Islet Hormones
Insulin
Glucagon
Somatostatin
Pancreatic polypeptide
Ghrelin
Non-nutrients
Neural peptides and neurotransmitters
Islets are innervated by cholinergic, adrenergic and peptidergic
autonomic nerves
In - cells, acetylcholine acts predominantly via M3 receptors to
activate PLC and also PLA
2
, beside that it augments membrane
depolarization by affecting sodium conductivity

On other hands norepinephrine has dual effect (stimulatory
2
)
and (inhibitory
2
)
Parasympathetic neuropeptides, including VIP, PACAP and GRP all
stimulate the release of insulin and glucagon
Sympathetic NPY and galanin act through specific G
i
-coupled
receptors
Non-nutrients
Incretins
Gastrointestinal - derived incretin hormones (GLP-1, GIP and
CCK)
They are secreted in response to nutrient absorption
They interact with specific receptors on the - cell surface to
stimulate insulin secretion
Glucagon - like peptide 1 (GLP-1)
GLP - 1 analogue, liraglutide
Selective DPP - 4 inhibitor sitagliptin
GLP - 1, exenatide and liraglutide act at specific G
s
-protein coupled
receptors
Non-nutrients
Incretins
Glucose - dependent insulinotropic peptide (GIP)
Stimulates insulin secretion in a glucose - dependent manner
It binds to G
s
-protein coupled receptors
because GIP stimulates glucagon secretion and inhibits GLP - 1
release, it is unlikely that that GIP - related peptides will be
developed
Cholecystokinin (CCK)
CCK - 8 acts at specific G
q
- coupled receptors on - cells to
activate PLC
High concentrations are required for its effects on insulin secretion
Insulin has 2 main effects in the body


Metabolic effects
regulation of whole body fuel homeostasis
Mitogenic effects
diminishes protein catabolism and increases translation, and enhances
cell growth, differentiation, and survival
Insulin Receptor
Tyrosine
kinase
Insulin-mediated glucose transport signaling pathway
Insulin
P
IRS
PI3K
Akt
P
IR
a
a
b
b
Glut4
Insulin-mediated glucose transport signaling pathway
Cell membrane
Xiao Chen, 2006
Insulin
P
IRS
PI3K
Akt
P
IR
a
a
b
b
glucose
Insulin-mediated glucose transport signaling pathway
Cell membrane
Insulin-mediated glucose transport signaling pathway
Xiao Chen, 2006
Membrane bound
Glycoproteins
Heteroteramers
2 and 2 subunits
The subunits insulin binding
The subunits udergoes
autophosphorylation at tyrosine residues and
has tyrosine kinase activiy
Product of single gene with 22 exons and 21
introns
Exsons 1- 11 express subunit
Exons 12 -22 express subunit
2 isoforms due to alternative splicing of exon 11
isoform A and isoform B
Isoform A lack exon 11 and has more affinity to
IGF2
Isoform B has exon 11 and more affinity to
insulin
Intracellular proteins
Phosporylated at tyrosine residues by ligand
bound iunsulin receptor s

IRS ( 1-6 )
Grb2 associated binder
Cbl
Src homology containing proteins

Insulin receptor substrates propagate signal
transduction
They act as adaptor molecules
They act by docking , apposition , interaction
and activation
Insulin Signaling Pathways by C. Hooper http://www.abcam.com/index.html?pageconfig=resource&rid=10602&pid=7
2 subunits p85 regulatory and p110 catalytic
Convert PIP2 amembrane phospholipid to
PIP3
PIP3 mediate the effect through activation of
PDK1 ( phosphoinositoid dependent protein
kinase 1 )
PDK 1 activate 2 different pathways :
Akt \ PKB and atypical PKC isoforms

Mediate various insulin induced cell response
such as :
1. Glucose translocation
2. Decrease activity of GSK3
3. Increase SREBP mediated lipogensis and
cholesterol synthesis
4. Decrease apoptosis
5. Inhibition of FOXO mediated gluconeogesis

3 isoforms
Akt 1 , akt 2 and akt 3
Akt 2 is most important

Akt \ PKB
Not activated by Ca nor DAG
Includes PKC (eta) and PKC (lambda)
have role in insulin mediated glucose uptake
and GLUT4 translocation to cell membrane

diverges at the level of the insulin receptor
kinase,
tyrosine phosphorylation of the Cbl proto -
oncogene through a process that does not
involve IRSs but requirs recruitment of the
adapter protein APS

CAP/Cbl bind to flotillin in caveolin containing
lipid raft
Once the CAP/Cbl complex is bound to
flotillin, Cbl presents a recognition site for
recruitment of the CrkII C3G complex to the
lipid raft
C3G is a guanyl nucleotide exchange factor
for TC10 and other small molecular weight
GTP - binding proteins.
TC10 is a member of the Rho family of
GTPases, with capacity to undergo post -
translational modification by farnesylation
and palmitoylation
TC10 regulate the actin cytoskeleton and
have a role in the translocation of GLUT - 4.

Has critical role in cell growth and mitogenesis
Grb - 2, a small cytosolic adapter protein
docks with IRS
SOS (mammalian homolog of the Drosophila
son - of - sevenless protein), a GDP/GTP
exchange factor binds Grb 2
SOS facilitates GTP activation of membrane
bound small molecular weight GTPase Ras
GTP - bound Ras bind toand activates Raf - 1
kinase



Raf - 1 kinase then initiates a cascade leading to
sequential phosphorylation and activation of the
dual - specificity kinase MEK (MAPK/ERK
kinase), which in turn phosphorylates
extracellular regulated kinases (ERK1 and ERK2)
Activated ERKs phosphorylate multiple targets
that mediate the mitogenic actions of the
Ras/MAPK pathway and the growth promoting
effects of insulin
Insulin receptors can also mediate activation
of the Ras/MAPK pathway through another
substrate docking molecule, SHC
Independent of IRS
SHC can also activate the Ras/Raf - 1/MEK
phosphorylation cascade by forming a
complex with Grb - 2/SOS in response to
insulin.

At level of receptor
Decrease number of receptors
1. Enhance internalization and lysosomal
degradation
2. Decrease receptor gene expretion
PC 1 ( plasma difrentiation factor ) an
intrinsic inhibitor of insulin receptor tyrosine
kinase


Grb proteins
Grb10 and Grb14 bind to receptor and
decrease receptor activity
At level of receptor substrate proteins
Dephosphorylation by protein
phosphotyrosine phosphatases
i. ( PTP- 1B ) and
ii. ( LAR )
Serine- thrionine phosphorylation
The major mechanism
By several kinases
PKC isoforms
TNF
JNK
NFB
I b kinase

Increase catabolism of PIP3 by
phosphoinositide phosphatases
PTEN and SHIP
At level of protein protein interactions
Supressors of cytokins signalings SOCS
Negative feed back loop
Inhibit tyrosine phosphorylated receptors by
Competitive binding to substrate or decrease
receptor kinase activity
Tribles
Pseudokinases
Can bind to kinase substrate and inhibit their
phosphorylation
Definition
Inability of insulin to produce its usual
biological action at circulating concentrations
that are effective in normal subject

Genetic
Physiological variation
lack of exercise
pregnancy
puperity
Dietry
overfeeding
alcohol
diet fat content
starvation
Metabolic disturbances
High level of circulatory free fatty acids
Chronic hyperglycaemia
Infection inflamatory conditions and cell
stress



Insulin Resistance
Overeating
Truncal obesity
High blood sugar
Hunger Craving carbohydrates
Excessive
insulin production
by reduced number
of beta cells
Beta cell
burnout
Two separate, but likely, complementary
mechanisms have recently emerged as a potential
explanations for Insulin resistance.
First, changes in IRS-1 either due to mutations or
serine phosphorylation of IRS proteins can
reduce their ability to attract PI 3-kinase,
thereby minimizing its activation.
Second , increased expression of p85 subunit
of PI3K and disrubtion of P85 \ P110 balance
other mechanisms linked with excess
circulatory fatty acid and excess glucose
roles in insulin resistance not related to the
former two mechanisms
Adipose tissue play central and crucial role in
insulin resistant
rare mutations of the IRS-1 protein are
associated with insulin resistance
disruption of the IRS-1 results in insulin
resistance mainly of skeletal muscle and
adipose tissue
genetic analysis of the IRS-1 gene has
revealed several base-pair changes that result
in amino acid substitutions
The most common amino acid change is a
glycine to arginine substitution at codon
972 (G972R), which has an overall
frequency of 6% in the general
population
Expression of this variant is associated with a
significant (20-30%) impairment of insulin-
stimulated PI3- kinase activity, as well as
reduced binding of IRS-1 to the p85
regulatory subunit of PI3- kinase
there may be an interaction between the
mutant allele and obesity, such that, in the
presence of obesity, the mutant variant may
aggravate the obesity-associated insulin
resistance
Moreover, the presence of a mutated IRS-1
gene is associated with dyslipidemia,
IRS-2 gene defects not only show insulin resistance of
muscle, fat and liver, but also manifest diabetes as
a result of cell failure
a number of polymorphisms have been identified in
the IRS-2 gene Among those, the amino acid
substitution Gly1057Asp has been found in various
populations with a prevalence sufficiently high
Although the polymorphism was associated with
decreased insulin sensitivity and impaired glucose
tolerance in women with polycystic ovary
syndrome it showed no association with insulin
sensitivity in other studies
As discussed later
Recent studies have demonstrated hyper-
serine phosphorylation of IRS-1 on
Ser302, Ser307, Ser612, and Ser632 in
several insulin-resistant rodent models

More than 20 Ser residues of IRS-1 were found
to undergo insulin-stimulated phosphorylation
in human muscle biopsies, three of which were
newly identified sites: Thr495, Ser527, and
Ser1005
Serine phosphorylation of IRS proteins can
occur in response to a number of
intracellular serine kinases

The causes of IRS-1 serine phosphorylation
are-
1. mTOR- p70S6 kinase, Amino acids,
Hyperinsulinemia
2. JNK- Stress, Hyperlipidemia, Inflammation
3. IKK- Inflammation
4. TNF- Obesity, Inflammation
5. Mitochondrial dysfunction stress
6. PKC - Hyperglycemia, Diacylglycerol,
Inflammation
Ser318 of IRS-1 is a potential target for PKC
JNK, and kinases along the PI3K-mTOR
pathway
It is located in close proximity to the PTB
domain. Therefore, its phosphorylation
presumably disrupts the interaction between
IR and IRS-1.
Phosphorylation of Ser318 is not restricted to
insulin stimulation. Elevated plasma levels of
leptin, also stimulates the phosphorylation of
Ser318 This down regulates insulin-stimulated
Tyr phosphorylation of IRS-1 and glucose
uptake.
phosphorylation of Ser312 and Ser636 of
IRS-1 is mediated by the mTOR-S6K pathway
((Mammalian target of rapamycin signaling
pathway))
is a central signal integrator that receives signals
arising from growth factors, nutrients and cellular
energy metabolism, and then activates pathways
that control cell growth, proliferation and survival
glucose and amino acids, especially leucine,
leads to an increase in mTOR activity
Oxidative stress represents an important
factor in the development of insulin
resistance
A considerable portion of total body oxidant
stress and ROS production are generated by
dysfunctional mitochondria
mitochondrial dysfunction accompanied by a
decreased mitochondrial fatty acid oxidation
and accumulation of fatty acid acyl CoA
and diacylglycerol
The mechanism of insulin resistance in these
cases has been suggested to involve
activation of a novel PKC that either by
itself or via IKK or JNK-1 could lead to
increased serine phosphorylation of IRS-1.
disruption in the balance between the
amounts of the PI 3-kinase subunits
PI 3-kinase belongs to the class 1a 3-kinases
which exist as heterodimers, consisting of a
regulatory subunit p85, which is tightly
associated with a catalytic subunit, p110
Normally, the regulatory subunit exists in
excess to the catalytic one, resulting in a pool
of freep85 monomers not associated with the
p110 catalytic subunit.
However, there exists a balance between the
free p85 monomer and the p85-p110
heterodimer, with the latter being
responsible for the PI 3-kinase activity
the p85 monomer and the p85-p110
heterodimer compete for the same binding
sites on the tyrosine-phosphorylated IRS
proteins
an imbalance that cause Increase in
expression of p 85 would result in a shift in
the balance in the favour of free p85
mismatch between free p85 and p85-p110
complexes has been recently supported by
studies in insulin-resistant states induced
by
human placental growth hormone
obesity,
type 2 diabetes
short-term overfeeding

insulin resistance of pregnancy is likely due
to increased expression of skeletal muscle
p85 in response to increasing
concentrations of human placental
growth hormone
increased expression of p85 may be an
early molecular step in the pathogenesis
of the nutritionally induced insulin
resistance
Insulin receptor is one of the major targets
in FFA-induced impairment of insulin
activity
accumulation of intracellular fatty acids or
their metabolites results in an impairment of
signaling through IRS/PI 3-kinase
PDK1 can directly phosphorylate all PKCs
including nPKCs
The PKC is nPKCs has recently been shown to
be related to insulin resistance
Insulin stimulation of PDK1 phosphorylation
is inhibited by FFA
FFA in of skeletal muscle cells and
adipocytes inhibit PDK1 phosphorylation but
suprisingly Increased PKC phosphorylation

HOW
This phosphorylation totally independent of
PDK1
palmitate IN skeletal muscle cells or
adipocytes may affect palmitoylation of
PKC resulting in constitutive
phosphorylation of PKC Taken together, it is
clear that FFA causes PDK1-independent
phosphorylation of PKC which in turn
translocates to the nucleus, and its time of
entry into the nucleus coincides with inhibition
of IR gene transcription

It is a possibility that activated PKC
phosphorylates HMGA1 which inhibits HMGA1
mobilization to the promoter region IR gene.
phosphorylation of HMGA1 protein reduces its
DNA-binding ability Phosphorylated HMGA1
protein would preferentially interact with positively
charged histones as there would be an additional
negative charge, while dephosphorylated HMGA1
proteins are expected to interact with negatively
charged DNA
one important mechanism underlying glucose -
induced insulin resistance involves glucose
metabolism through a minor intracellular
pathway, the hexosamine biosynthetic pathway.
Increased glucose flux through the hexosamine
biosynthetic pathway results in impaired ability
of insulin to stimulate the glucose transport
effector system in adipocytes and muscle cells,
HOW

The mechanisms by which products of the
hexosamine biosynthetic pathway induce insulin
resistance, however, have not been fully
elucidated.
Increased metabolic flux through thispathway
results in increased O - glycosylation of the
insulin receptor, IRS1, Akt/PKB, GLUT - 4 and
GSK3 , and this can impair the functional
capabilities of these proteins or accelerate
their proteasomal degradation.
Glycosylation can also affect activities of nuclear
transcription factors.
O - GlcNAc adducts can decrease transcriptional
activity of Sp1 but increase DNA binding and
transcriptional regulation by Foxo1.
O glycosylation can impair phosphorylation and
DNA binding of C/EBP , and decrease NK - B
binding with I B freeing NF B for relocation
to the nucleus thereby including transcription of
proinflammatory genes.
Adipose tissue can modulate whole body
glucose metabolism by regulating levels of
circulating free fatty acids (FFA) and also
by secreting adipokines
resistance to the antilipolytic action of insulin
in adipose tissue resulting in excessive release
of FFA and glycerol would have deleterious
effects on glucose homeostasis.
Patients suffering from insulin resistance
and type 2 diabetes frequently display
signs of abnormal lipid metabolism,
increased circulatory concentration and
elevated deposition of lipids in the skeletal
muscle
The molecular mechanism underlying defective
insulin- stimulated glucose transport activity can
be attributed to increases in intracellular lipid
metabolites such as fatty acyl CoAs and
diacylglycerol and ceramides, which in turn
activate a serine/threonine kinase cascade, thus
leading to defects in insulin signaling through
Ser/Thr phosphorylation of insulin receptor
substrate 1
Recent reports also demonstrate a link
between nPKCs( , , , ) and FFA induced
insulin resistance
Diacylglycerol is an attractive trigger for
fat-induced insulin resistance in skeletal
muscle, it is a known activator of novel
protein kinase C (PKC) isoforms
accumulation of intracellular lipid
metabolites DAG and ceramides activate a
serine kinase cascade involving PKC-,
leading to decreased insulin receptor kinase
activity
Mitochondrial glycerol-3-phosphate
acyltransferase (mtGPAT) is a key enzyme in
de novo fat synthesis in liver
Defects in this enzyme are related with
intracellular accumulation of diacylglycerol
and fat-induced insulin resistance in liver
through activation of PKC-
Adipose tissue also acts as an endocrine
organ producing adipokines and other
hormones
TNF increase serine phosphorylation of
IRS-1 and down-regulates GLUT4
expression, thereby contributing to insulin
resistance
the action of PPAR on adipocytes
normally keeps the cells ready to synthesize
and store triacylglycerols
in obese individuals the adipocytes become
filled with TAG, and the adipose tissue cannot
meet any increased demand for TAG storage
Adipocytes and their precursors,
preadipocytes, become less sensitive to
insulin
Gene expression normally associated with the
development of new adipocytes (genes for the
transcription factors SREBP1 and PPAR , for
example) is downregulated in the adipocytes of
obese individuals, but is upregulated in other
tissues, including skeletal muscle and liver, which
begin to store TAGs
Substantial quantities of triacylglycerols are now
stored ectopically in abnormal locations
excess stored fatty acids and TAGs are toxic to liver
and muscle.