Molecular Mechanisms of Insulin Action
Cellular action of insulin:
Insulin elicits a remarkable array of biological responses. The important target tissues for
regulation of glucose homeostasis by insulin are liver, muscle, and fat, but insulin exerts
potent regulatory effects on other cell types as well.
One should bear in mind that insulin is the primary hormone responsible for controlling the
uptake, utilization, and storage of cellular nutrients. Insulin’s anabolic actions include the
stimulation of intracellular utilization and storage of glucose, amino acids, and fatty acids,
while it inhibits catabolic processes, such as the breakdown of glycogen, fat, and protein.
It accomplishes these general purposes by stimulating the transport of substrates and ions
into cells, promoting the translocation of proteins between cellular compartments,
activating and inactivating specific enzymes, and changing the amounts of proteins by
altering the rate of transcription of specific genes.
Some effects of insulin occur within seconds or minutes, including the activation of
glucose and ion transport systems, the covalent modification (i.e. phosphorylation or
dephosphorylation) of enzymes, and some effects on gene transcription (i.e. inhibition of
the phosphoenolpyruvate carboxykinase gene). Other effects, such as those on protein
synthesis and gene transcription, may take a few hours. Effects of insulin on cell
proliferation and differentiation may take days. It is not clear whether these kinetic
differences result from the use of different mechanistic pathways or from the intrinsic
kinetics of the various processes.
Insulin binds to the α subunits of the insulin receptor and stimulates the tyrosine kinase
activity of β subunits. This initiates a complex cascade of biochemical interactions that
results in several physiological, biochemical and molecular events.
Regulation of glucose transport:
Stimulation of glucose transport into muscle and adipose tissue is a crucial component of
the physiological response to insulin. Glucose enters cells by facilitated diffusion through
one of a family of glucose transporters. To date, complementary DNAs that encode a family
of six distinct mammalian glucose transporters have been cloned and sequenced. Five of
these ( GluT1 through GluT5) are thought to be involved in Na+-independent facilitated
diffusion of glucose into cells; GluT6 is a pseudogene, and GluT7 is found in endoplasmic
reticulum and allows efflux of glucose after its dephosphorylation by glucose-6-
phosphatase.
The glucose transporters are integral membrane glycoproteins with molecular masses of
about 50 kDa, and each has 12 membrane-spanning α-helical domains. Insulin stimulates
glucose transport at least in part by promoting the energy-dependent translocation of
intracellular vesicles that contain the GluT4 and GluT1 glucose transporters to the plasma
membrane. This effect is reversible; the transporters return to the intracellular pool upon
removal of insulin. Insulin can also regulate the synthesis of GluT4 ; this phenomenon may
be particularly important when there is long-term insulin resistance, as in patients with
type II or non-insulin-dependent diabetes mellitus.
Regulation of glucose metabolism:
The facilitated diffusion of glucose into cells along a downhill gradient is assured by
glucose phosphorylation. This enzymatic reaction, the conversion of glucose to glucose 6-
phosphate (G6P), is accomplished by one of a family of hexokinases. The four hexokinases
(I through IV), like the glucose transporters, are distributed differently in tissues, and two
are regulated by insulin. Hexokinase IV, a 50-kDa enzyme more commonly known as
glucokinase, is found in association with GluT2 in liver and pancreatic β-cells.
There is one glucokinase gene, but different first exons and promoters are employed in
the two tissues. The liver glucokinase gene is regulated by insulin. Hexokinase II, a 100-kDa
enzyme, is found in association with GluT4 in skeletal and cardiac muscle and in adipose
tissue. Like GluT4, hexokinase II is regulated at the transcriptional level by insulin.
G6P is a branch-point substrate. It can enter the glycolytic pathway and lead to the
production of ATP through a series of enzymatic reactions,
many of which are promoted by insulin. The effects of insulin on this pathway are exerted
on gene transcription or through alteration of enzyme
activity by phosphorylation or dephosphorylation on serine and/or threonine residues.
Alternatively, G6P can be incorporated into glycogen after isomerization to glucose 1-
phosphate (G1P).
Insulin promotes glycogen deposition by stimulating the activity of glycogen synthase, the
rate-limiting enzyme in glycogen synthesis, and by inhibiting phosphorylase, the rate-
controlling enzyme in glycogen degradation. As in glycolysis, these effects of insulin are
mediated through changes in the phosphorylation state of the enzymes. Covalent
modification by phosphorylation/dephosphorylation is a major mechanism of action of
insulin. For example, phosphorylation increases the activity of acetyl-CoA carboxylase and
citrate lyase, whereas glycogen synthase and pyruvate dehydrogenase are activated by
dephosphorylation. The latter occurs as a result of the activation of phosphatases by
insulin. Dozens of protein are so modified, with resulting changes in their activity.
Regulation of gene transcription:
It is now clear that a major action of insulin is the regulation of transcription of specific
genes. The first example of this activity to be identified was the inhibition of
phosphoenolpyruvate carboxykinase transcription by insulin. This finding helped explain
how insulin inhibits gluconeogenesis and may explain why the liver overproduces glucose
in the insulin-resistant state that is characteristic of non-insulin-dependent diabetes
mellitus.
There are now nearly 100 examples of genes that are regulated by insulin, and the list
continues to grow. The exact mechanism by which these effects are accomplished is not
known.
The insulin receptor:
Insulin initiates its actions by binding to a cell-surface receptor. Such receptors are
present in virtually all mammalian cells, including not only the classic targets for insulin
action (liver, muscle, and fat) but also such non-classic targets as circulating blood cells,
brain cells, and gonadal cells. The number of receptors varies from as few as 40 per cell on
erythrocytes to 300,000 per cell on adipocytes and hepatocytes.
The insulin receptor is a large transmembrane glycoprotein composed of two 135-kDa α
subunits (719 or 731 amino acids, depending on whether a 12-amino-acid insertion has
occurred through alternate splicing of mRNA) and two 95-kDa β subunits (620 amino acids);
the subunits are linked by disulfide bonds to form a β-α-α-β heterotetramer (Figure: 10).
Both subunits are derived from a single-chain precursor molecule that contains the entire
sequence of the α and β subunits, separated by a processing site consisting of four basic
amino acid residues.
These two subunits are specialized to perform the two functions of the receptor. The α
subunits are entirely extracellular and contain the insulin-binding domain, while the β
subunits are transmembrane proteins that possess tyrosine protein kinase activity. After
insulin is bound, receptors aggregate and are rapidly internalized. Since bivalent (but not
monovalent) anti-insulin receptor antibodies cross-link adjacent receptors and mimic the
rapid actions of insulin, it has been suggested that aggregation of the receptor is essential
for signal transduction. After internalization, the receptor may be degraded or recycled
back to the cell surface.
A heterotetramer consisting of two extracellular insulin-binding subunits linked by a
disulfide bonds to tow transmembrane beta subunits. The beta subunits contain an
intrinsic tyrosine kinase activity that is activated upon insulin binding to the alpha
subunit. "
Insulin Therapy:
Insulin is the mainstay for treatment of virtually all IDDM and many NIDDM patients. When
necessary, insulin may be administered intravenously or intramuscularly; however, long-
term treatment relies predominantly on subcutaneous injection of the hormone.
Subcutaneous administration of insulin differs from physiological secretion of insulin in at
least two major ways: The kinetics do not mimic the normal rapid rise and decline of
insulin secretion in response to ingestion of nutrients, and the insulin diffuses into the
peripheral circulation instead of being
released into the portal circulation; the preferential effect of secreted insulin on hepatic
metabolic processes is thus eliminated. Nonetheless,
when such treatment is performed carefully, considerable success is obviously achieved.
Preparations of insulin can be classified according to their duration of action into short-,
intermediate-, and long-acting, and by their species of origin-human, porcine, bovine, or a
mixture of bovine and porcine. Human insulin (Humulin, Novolin) is now widely available as
a result of its production by recombinant DNA techniques. Porcine insulin differs from
human insulin by one amino acid (alanine instead of threonine at the carboxy terminal of
the B chain, i.e. in position B 30), and bovine insulin differs by two additional alterations
of the A chain (threonine and isoleucine in positions A8 and A10 are replaced by alanine
and valine, respectively).
Prior to the mid-1970s, commercially available insulin preparations contained proinsulin or
glucagons-like substances, pancreatic polypeptide, somatostatin, and vasoactive intestinal
peptides. These contaminants were avoided with the advent of monocomponent porcine
insulins. During the late 1970s, intense work was carried out on the development of
biosynthetic human insulin. This resulted in the first doses of human insulin being
administered to normal volunteers during the summer of 1980.
The physicochemical properties of human, porcine, and bovine insulins differ owing to
their different amino acid sequences. Human insulin, produced using recombinant DNA
technology, is more soluble than porcine insulin in aqueous solutions, owing to the
presence of threonine (instead of alanine), with its extra hydroxyl group. All preparations
are now supplied at neutral pH, which improves stability and permits storage for several
days at a time at room temperature.
Insulin preparations:
1. Short-acting insulins:
a) Soluble insulin.
b) Insulin lispro.
c) Insulin aspart.
2. Intermediate-and long-acting insulins:
a) Insulin zinc suspension.
b) Isophane insulin.
c) Protamine zinc insulin.
3. Biphasic insulin:
a) Biphasic insulin lispro.
b) Biphasic isophane insulin.
1. Short-acting insulin:
Soluble insulin is a short-acting form of insulin, for maintenance regimens it is usual to
inject it 15 to 30 minutes before meals.
Soluble insulin is the only appropriate form of insulin for use in diabetic emergencies and
at the time of surgery. It can be given intravenously and intramuscularly, as well as
subcutaneously. When injected subcutaneously, soluble insulin has a rapid onset of action
(after 30 to 60 minutes), a peak action between 2 and 4 hours, and a duration of action of
up to 8 hours. Human sequence preparations tend to have a more rapid onset and a
shorter overall duration. When injected intravenously, soluble insulin has a very short half-
life of only about 5 minutes and its effect disappears within 30 minutes.
The recently introduced human insulin analogues, insulin lispro and insulin aspart, have a
faster onset and shorter duration of action than soluble insulin; as a result, compared to
soluble insulin, fasting and preprandial blood-glucose concentration is a little higher,
postprandial
blood-glucose concentration is a little lower, and hypoglycaemia occurs slightly less
frequently. Subcutaneous injection of insulin lispro or of insulin aspart may prove
convenient to those who wish to inject shortly before or, when necessary, shortly after a
meal. They may also help those prone to pre-lunch hypoglycaemia and those who eat late
in the evening and are prone to early nocturnal hypoglycaemia. Insulin aspart and insulin
lispro may also be administered by subcutaneous infusion.
a) Soluble insulin:
A sterile solution of insulin (i.e. bovine or porcine) or of human insulin; ( pH 6.6_8.0 ).
Indications:
Diabetes mellitus; diabetic ketoacidosis.
Rote of administration:
By subcutaneous, intramuscular, or intravenous injection or intravenous infusion,
according to requirements.
Trade names:
Hypurin®( Bovine Neutral, Porcine Neutral), Pork Actrapid®, HumalinS®.
b) Insulin aspart:
Indications:
Diabetes mellitus.
Rote of administration:
By subcutaneous injection, immediately before meals or when necessary shortly after
meals according to requirements.
By subcutaneous infusion, according to requirements.
Trade name:
NovoRapid®.
c) Insulin lispro:
Indications:
Diabetes mellitus.
Rote of administration:
By subcutaneous injection, shortly before meals or when necessary shortlyafter meals,
according to requirements.
By subcutaneous infusion, according to requirements.
Trade name:
humalog®.
2. Intermediate- and long- acting insulin:
When given by subcutaneous injection, intermediate- and long-acting insulin have an
onset of action of approximately 1-2 hours, a maximal effect at 4-12 hours, and a duration
of 16-35 hours. Some are given twice daily in conjunction with short-acting (soluble)
insulin, and others are given once daily, particularly in elderly patients. They can be mixed
with soluble insulin in the syringe, essentially retaining the properties of the two
components, although there may be some blunting of the initial effect of the soluble
insulin component (especially on mixing with protamine zinc insulin).
Isophane insulin is a suspension of insulin with protamine which is of particular value for
initiation of twice-daily insulin regimens. Patients usually mix isophane with soluble insulin
but ready-mixed preparations may be appropriate (biphasic isophane insulin or biphasic
insulin lispro).
Insulin zinc suspension (crystalline) has a more prolonged duration of action; it may be
used independently or in insulin zinc suspension (30% amorphous, 70% crystalline).
Protamine zinc insulin is usually given once daily with short-acting (soluble) insulin. It has
the drawback of binding with the soluble insulin when mixed in the same syringe, and is
now rarely used.
Dose of intermediate- and long-acting insulin:
By subcutaneous injection, according to requirements.
a) Insulin zinc suspension:
A sterile neutral suspension of bovine and/or porcine insulin or of human insulin in the
form of a complex obtained by the addition of a suitable zinc salt; consists of
rhombohedral crystals (10-40 microns) and of particles of no uniform shape (not exceeding
2 microns).
Indications:
Diabetes mellitus (long acting)
Trade names:
Insulin zinc suspension :
Lentard MC®, Human Monotard®, Humulin Lente®.
Insulin zinc suspension (crystalline):
Human Ultratard®, Humalin Zn®.
b) Isophane insulin:
A sterile suspension of bovine or porcine insulin or of human insulin in the form of a
complex obtained by the addition of protamine sulphate or another suitable protamine.
Indications:
Diabetes mellitus (intermediate acting).
Side effects:
Protamine may cause allergic reactions.
Trade names:
Pork Insulatard®, Hypurin®Procine Isophane, Humulin I®.
c) Protamine zinc insulin:
A sterile suspension of insulin in the form of a complex obtained by the addition of a
suitable protamine and zinc chloride.
Indications:
Diabetes mellitus (long acting).
Side effects:
Protamine may cause allergic reactions.
Trade name:
Hypurin® Bovine Protamine Zinc.
3. Biphasic insulin:
Indications:
Diabetes mellitus (intermediate acting).
Side effects:
Protamine may cause allergic reactions.
Rote of administration:
By subcutaneous injection, according to requirements.
Trade names:
Biphasic insulin lispro: Humalog®Mix 25, Humalog®Mix 50.
Biphasic isophane insulin: Hypurin® Porcine 30/70 Mix,
Pork Mixtard 30, Human Mixtard® 10.
Cautions:
Hypoglycemia is the primary adverse effect of insulin and may be serious if it is not
detected early and treated appropriately.Skin reactions may also occur but are less
frequent with human source insulins.
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Diabetes Mellitus
