2. Introduction: Insulin
Insulin is a peptide hormone produced by β-cells
of the pancreas in response to nutritional stimuli.
Main sites of action of Insulin in the body include
Liver, Adipose Tissue, Muscles & Brain.
Insulin is secreted by β-cells in response to an
increase in Blood glucose levels.
Insulin binds to the insulin receptor and facilitates
uptake of glucose by the cell.
Insulin’s interaction with the cell triggers both
metabolic and mitogenic cellular responses.
Insulin-like Growth Factor, IGF-1 is a mediator of
cell growth and differentiation.
http://www.jbc.org/content/271/48/30625.ful
http://www.abcam.com/pathways/overview-of-insulin-signaling-pathways l
3. Introduction: Insulin Receptor
Insulin receptor belongs to a family of receptor-
tyrosine kinases (RTKs), which phosphorylate
their substrate proteins on tyrosine residues.
The insulin receptor comprises of two subunits:
the extracellular α-subunit and the
transmembrane β-subunit. The functional receptor
exists as a dimer/heterotetrameric complex: α2β2
The α-subunit is contains the ligand binding site,
as it is the only subunit identified by affinity
labelling protocols.
The β subunit is involved in intracellular signalling.
4.
5. When insulin interacts with the receptor, dimerisation of the receptor takes
place. Due to this, the cytoplasmic domains of the receptor come close
together faciliating autophosphorylation and triggering the signal
transduction pathway.
Insulin Receptor
6. Types of Signaling Mechanisms
Insulin's interaction with its cell surface receptor triggers both
metabolic and mitogenic cellular responses.
In order to do this, Insulin employs two kinds of pathways: Ras-
dependant and Ras-independent.
Ras is a member of a large family of small molecular weight GTP
binding proteins.
Many RTKs are known to activate the Ras protein in order to
mediate signals.
Ras-dependant Pathway: Upon insulin binding, the Ras protein is
phosphorylated leading to activation of Mitogen Activated Protein
Kinase (MAPK) pathway which is involved in cellular growth and
proliferation.
Ras-independent Pathway: In this case, another pathway known
as the PI-3/AKT signaling pathway is activated by a series of
events facilitating glucose uptake by the cell.
http://www.ncbi.nlm.nih.gov/books/NBK21659/
http://link.springer.com/article/10.1023/A%3A1006819008507#page-1
http://www.jbc.org/content/271/48/30625.full
10. Role of Proteins Involved in PI-3/AKT
Signaling
Insulin & Insulin Receptor: When blood glucose levels rise, insulin
released by the beta-cells of the pancreas binds to the Insulin Receptor on
muscle cells and/or adipocytes and facilitated dimerisation of the receptor.
As a result of this dimerisation, the receptor is autophosphorylated at specific
residues.
IRS: Insulin receptor substrate family are proteins that are primarily activated
as a result of autophosphorylation. The Ser/Thr residues of these proteins
are known to be phosphorylated. There are two types of IRS: IRS-1 and IRS-
2. IRS-1 is known to function in glucose metabolism, while IRS-2 functions in
lipid metabolism. However, both of these are recruited by the insulin receptor
as well as IGF-R to exert different kinds of effects.
PI-3 Kinase: Phosphatidylinositol kinase is activated by IRS-1 and acts as
an adaptor protein. Activated PI-3Ks serve to phosphorylate phosphatidyl
inositol on the D3 position of the inositol ring generating PtdIns-3,4,5-P3.
These specialised lipids recruit PH-domain containing proteins such as Akt
to the plasma membrane.
Protein Kinase B or Akt: Akt helps in translocation of GLUT-4 storage
vesicles to the plasma membrane by a series of events leading to activation
of Rab protein. As GLUT-4 vesicles fuse with the plasma membrane, no. of
receptors increase thereby increasing glucose uptake.
11. Insulin binding to the receptor leads to a conformational change that induces
autophosphorylation, similar to activation of other RTKs. After IRS1 binds to a
phosphotyrosine residue through a PTB domain, the activated kinase in the
receptor’s cytosolic domain phosphorylates IRS1. One subunit of PI-3 kinase binds
to the receptor-bound IRS1 via its SH2 domain, and the other subunit then
phosphorylates PI 4,5-bisphosphate and PI 4-phosphate to PI 3,4,5- trisphosphate
and PI 3,4-biphosphate, respectively.
IRS-
1
AK
T
PI-
3K
12. AKT
The phosphoinositides bind the PH domain of protein kinase B (PKB), thereby
recruiting it to the membrane. Two membrane-bound kinases, in turn,
phosphorylate membrane-associated PKB (AKT) and activate it.
13. Activated PKB is released from the membrane and promotes glucose uptake by
the GLUT4 transporter and glycogen synthesis. The former effect results from
translocation of the GLUT4 glucose transporter from intracellular vesicles to
the plasma membrane. The latter effect occurs by PKB-catalyzed
phosphorylation of glycogen synthase kinase 3 (GSK3), converting it from its
active to inactive form. As a result, GSK3-mediated inhibition of glycogen synthase
is relieved, promoting glycogen synthesis.
14. GLUT-4 Translocation: A key
step
In the basal state, a dynamic trafficking process ensures that the bulk of GLUT4 is sequestered into
intracellular vesicles resulting in a low level of GLUT4 at the cell surface.
Insulin stimulates GLUT4 translocation to the plasma membrane predominantly by releasing GLUT4
from this specialised intracellular pool resulting in a pronounced increase in glucose uptake.
Key mediators: PI-3K, PtdIns-3,4,5-P3, Atk and Rab proteins.
Key evidences:
Rat adipocytes were pre-incubated with insulin and [3H]2-deoxy-D-glucose, followed by centrifugation
and measurements. Adipocytes pre-incubated with insulin and wortmannin were exposed to [3H]2-
deoxy-D-glucose followed by centrifugation and radioactivity measurements. Glucose uptake was
found to decrease significantly upon treatment of cells with wortmannin. Since, wortmannin is a
specific inhibitor of PI-3k, this study establishes that PI-3k is important for insulin-induced glucose
uptake.
Cells were transfected with either wild type (Akt-WT), constitutively active (Akt-myr), or dominant
inhibitory (Akt-K179A) forms of Akt, and effects of overexpression of these constructs on insulin-
stimulated translocation of a cotransfected epitope-tagged GLUT4 were studied. Overexpression of
Akt-WT resulted in significant translocation of GLUT4 to the cell surface even in the absence of
insulin. Interestingly, overexpression of Akt-myr resulted in an even larger effect that was independent
of insulin. More importantly, overexpression of Akt-K179A (kinase-inactive mutant) significantly
inhibited insulin-stimulated translocation of GLUT4. Taken together, our data suggest that Akt is not
only capable of stimulating the translocation of GLUT4 but that endogenous Akt is likely to play a
significant physiological role in insulin-stimulated glucose uptake in insulin targets such as muscle and
adipose tissue.
17. General Representation of MAPK Signaling:
When a peptide hormone/growth factor binds to its receptor and causes
subsequent dimerisation, a set of signaling mechanisms are initiated. One such
pathway involved mitogen-activated protein kinases (MAPK proteins) that are
known to function in cell growth and proliferation. The general mechanism as
represented in the figure is similar for all ligands. However, some proteins may
18. The pathway
Insulin binds to its receptor and causes dimerisation.
This event facilitates autophosphorylation at the specific tyrosine residues within the
cytoplasmic domain of beta-subunit of the receptor.
The phosphorylated tyrosine residues are docking sites for a number of proteins. In
this case primarily two kinds of proteins are activated: Insulin Receptor Substrate
(IRS) and Shc family of proteins.
Both these proteins contain the PTB domain that interacts with the phosphorylated
residues on the activated receptor. In case of signaling via insulin, both IRS and Shc
are important as indicated by this experiment.
A CHO cell line overexpressing the mutant receptor substituting Ala960 for TyrS6O,
which was known to show the severely impaired insulin-dependent tyrosine
phosphorylation of IRS-1 showed severely reduced insulin-dependent tyrosine
phosphorylation of Shc and moderately impaired Ras activation. Using a CHO cell
line overexpressing the mutant insulin receptor lacking 82 amino acids of the C
terminus of the P-subunit, which was recently reported to exhibit severely impaired
receptor kinase activity but retain normal insulin-dependent tyrosine phosphorylation
of IRS-1 it was found that insulin induced the submaximal activation of Ras while
tyrosine-phosphorylation of Shc protein was severely reduced.
Interference with PTB domains of both IRS and Shc blocks the ability of insulin to
elicit cellular responses along both the branches of insulin signaling.
19. Contd..
IRS proteins contain PH domain that facilitates interaction
with specific plasma membrane sites adjacent to the
receptor.
Phosphorylated IRS and Shc recruit the SH2 domain
containing proteins such as PI-3K or Grb2 respectively. Grb2
is a growth factor receptor binding protein that also binds the
Guanine nucleotide exchange factor, Sos.
Shc-Grb2-Sos activity promotes the dissociation of Ras
proteins allowing them to bind to GTP instead. This serves to
activate the Ras proteins initiating a cascade of
phosphorylation and activation of several Ser/Thr kinases.
https://books.google.co.in/books?id=hgiuDHVUuT4C&pg=PA
421&lpg=PA421&dq=ras+dependant+insulin+pathway&sourc
e=bl&ots=icjLmRRyDF&sig=k8b2p1ZCghRoIGmoGID6nE8C
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