molecular and cellular mechanism of action of insulin

1. Dr Rajendra Gode Institute of Pharmacy
Molecular & Cellular mechanism of action of
insulin & Pharmacology of oral
hypoglycaemic drug
Guide: Dr P.V. Ajmire
Subject: Advance Pharmacology II
Presented by: Mansi P. Nikhade
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2. Content
• Diabetes Mellitus
• Introduction of insulin
• History
• Biosynthesis of insulin
• Insulin receptors
• Mechanism of insulin secretion
• Mechanism of action of insulin
• Insulin analogue
• Hypoglycaemia
• Oral hypoglycaemic agents
• Classification
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3. Diabetes Mellitus
• Diabetes mellitus is a metabolic disorder characterized by
hyperglycaemia, glucosourea, hyperlipaemia, negative nitrogen
balance and sometimes ketonaemia.
• Two major types of diabetes mellitus:
• i) Insulin-dependent diabetes mellitus (IDDM)
• ii) Non-Insulin-dependent diabetes mellitus (NIDDM)
Various symptoms are:
Feeling very thirsty and tired, high level of glucose in urine, constant hunger
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4. • Insulin-dependent diabetes mellitus:
There is ẞ cell destruction inpancreatic islets; majority of cases are autoimmune
antibodies that destroy ẞ cell are detectable in blood, but some are idiopathic,
there are no ẞ cell antibody are found. In all cases circulating insulin levels are low.
• Non insulin-dependent diabetes mellitus:
There is no loss or moderatereduction in ẞ cell mass, insulin in circulation is low,
normal or even high, no anti-ẞ-cell antibody is demonstrable; has a high degree of
genetic predisposition, generally has a late onset.
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5. • IDDM: insulin must be injected or inhaled.
• NIDDM: Food control , exercise, medicines.
i. Which increase insulin secretion.
ii. Which increase the sensitivity of target organs to insulin .
iii. Which decrease glucose absorption.
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6. Introduction of insulin
• Insulin is a peptide hormone secreted by B-cells in the pancreatic
islets of Langerhans.
• The main function of insulin is to lower serum glucose and promote
anabolism.
• Insulin is an essential growth factor required for normal
development.
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7. Structure of insulin
• Human insulin consists of 51aa in two chains
connected by 2 disulfide bridges (a single gene
product cleaved into 2 chains during post-
translational modification).
• T1/2 5-10 minutes, degraded glutathione-insulin
by glutathione insulin transhydrogenase
(insulinase) which cleaves the disulfide links.
• Bovine insulin differs by 3aa, pork insulin differs by
1aa.
• Insulin is stored in complex with Zn2+ions.
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8. History
• Insulin is a polypeptide hormone which was the hormone to be discovered by
"Fredric Banting and Dr. Charles best" from the pancreas of Dog, The two scientist
were awarded the Nobel prize.
• The molecular formula of human insulin is C254H377O75N65S6 which consist of 51
amino acid. In 1952 "Fredrick Sangar "examined that insulin was made up of two
chain of amino acid A B. The two chain are parallel to each other and bounded by
disulfide bond.
• In 1977 "Rosalyn sussman Yalow "developed Radioimmunoassay for Insulin for
which she received the novel prize. The first commercial insulin came from Cow
(Bovine) and Pig (Porcine).
• In 1977 "Herbert Boyer" using "Escherichia coli "produce the first genetically
engineering synthetic human insulin in the laboratory
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9. Biosynthesis of insulin
• There are mainly 3 major steps :
1. Synthesis of Preproinsulin.
2. Conversion of preproinsulin to proinsulin.
3. Conversion of proinsulin to insulin
Insulin is synthesized as preproinsulin in pancreatic B-cells. It contains a
signal peptide which directs the nascent polypeptide chain to the rough
endoplasmic reticulum. Then it is cleaved as the polypeptide is
translocated into lumen of the RER, forming proinsulin. Proinsulin is
transported to the trans-Golgi network (TGN) where immature granules are
formed,
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10. Proinsulin undergoes maturation into active insulin through action
of cellular endopeptidases known as prohormone convertases (PC1
and PC2), as well as the exoprotease carboxypeptidase E. The
endopeptidases cleave at 2 positions, releasing a fragment called
the C-peptide, and leaving 2 peptide chains, the B- and A- chains,
linked by 2 disulfide bonds. The cleavage sites are each located after
a pair of basic residues and after cleavage these 2 pairs of basic
residues are removed by the carboxypeptidase. The C-peptide is the
central portion of proinsulin, and the primary sequence of
proinsulin goes in the order "B-C-A.
The resulting mature insulin is packaged inside mature granules
waiting for metabolic signals (such as leucine, arginine, glucose and
mannose) and vagal nerve stimulation to be exocytosed from the
cell into the circulation.
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11. Insulin Receptor
• Insulin is released from the islet into the bloodstream
and its actions are mediated by the insulin receptor (IR)
on the surface of target cells.
• Cell-surface receptor with tyrosine-kinase activity-
Phosphorylate substrate proteins on Tyrosine residues
• Heterotetrameric glycoprotein 2 extracellular alpha and
2 transmembrane ẞ subunits linked together by disulfide
bonds – alpha2 beta 2
• Molecular weight 300 kDa
• alpha subunits - insulin binding site
• ẞ subunits -tyrosine kinase activity, involved in
intracellular signalling.
• Expressed in most mammalian tissues adipose & liver
have highest IR density (>300,000 receptor/cell)
Tyrosine kinase activity
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12. Mechanism of Insulin Secretion
• Glucose enters into the beta cells through the GluT 2
transporter (insulin independent).
• Glucose is phosphorylated into Glucose-6-phosphate by
glucokinase.
• Glucose-6-phosohate undergoes oxidation to form ATP.
• ATP causes closure of ATP sensitive potassium channels
found on the membrane of the beta cells .
• This leads to the depolarization of the cell membrane
thereby opening voltage gated calcium channels .
• Influx of calcium leads to docking of insulin. containing
vesicles on the cell membrane.
• Thereby leading to the secretion of insulin into the
extracellular fluid.
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13. Mechanism of Action
Insulin
Bind and activate alpha subunit of insulin receptor
stimulate tyrosine kinase activity in beta subunit
activate cascade of reaction
(Phosphorylation and Dephosphorylation)
stimulate or inhibit enzyme
Response
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14. Biological action of insulin
• Biological action of action of insulin , initiated by its binding to INS-R and an have
short , intermediate and long term effect on cellular functions.
Short term effect
Intermediate effect
Long term effect
Short term effects:
• Immediate effects-occur within seconds after receptor activation .
• Activation of glucose and ion-transport systems and
• Covalent modifications (Phosphorylation and Dephosphorylation) of pre- existing enzymes
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15. Intermediate effect:
• Minutes to hours
• Induction of genes and expression of certain proteins
Long term effect:
• Hours to several days
• Stimulate DNA synthesis, cell proliferation cell differentiation and some gene
expression events.
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16. Insulin analogue
• Recombinant DNA technology, modified pharmacokinetic - greater stability
and consistency
• Insulin lispro: Reversing Proline and lysine at B 28 and B 29 position - quick
acting. just before meals
• Insulin aspart: B 28 is replaced by aspartic acid - mimics physiological
insulin
• Insulin glulisine: Replacing aspartic acid at B 23 by lysine and glutamic acid
replacing lysine at B 29 continuous SC insulin infusion (CSII)
• Insulin glargine: Long- acting precipitates at neutral pH on SC injection -
depot created slow dissociation - 24 hours low blood level usually at bed
time
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17. Reaction and drug interaction
HYPOGLYCAEMIA: Labile diabetics
• Causes: Injection of large doses, missing a meal after injection, vigorous exercise
• Symptoms: Sweating, anxiety, palpitation, tremor - counter regulatory; dizziness,
headache, behavioural changes, visual disturbances, hunger, fatigue, weakness,
muscular incoordination etc. due to deprivation - Below < 40 mg/dl - seizure and
coma
• Treatment: Glucose orally and IV - Glucagon - 0.5 to 1 mg IV
• Local reactions : (swelling), lipodystrophy, Allergy, Oedema
• Drug Interactions: Beta blockers (beta-2; prolong hypoglycaemia), Thiazides,
diuretics. steroids, OCPs (raises blood sugar), acute alcohol ingestion
(hypoglycaemia - glycogen depletion), Lithium and aspirin (hypoglycaemia -
enhance insulin secretion )
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18. Oral Hypoglycaemic Agents
• Agents that are given orally to reduce the blood glucose level in
diabetic patients.
• The oral antidiabetic drug are of value only in the treatment of
patients with type 2 (NIDDM) diabetes mellitus whose condition
cannot be controlled by diet alone.
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19. Classification of oral hypoglycaemic agents
A. Enhance insulin secretion:
1. Sulfonylureas:
• First generation: Tolbutamide
• Second generation: glibenclamide, glipizide, gliclazide, glimepride
2. Meglitinide/ phenylalanine analogues
• Repaglinide, Nateglinide
3. Glucagon-like peptide-1 (GLP-1) receptor agonists → injectable drugs
• Exenatide, Liraglutide
4. Dipeptidyl peptidase-4 (DPP-4) inhibitors
• Sitagliptin, Vildagliptin, Saxagliptin, Alogliptin, Linagliptin
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20. B. Overcome insulin resistance
• Biguanides : Metformin
• Thiazolidinediones : Pioglitazone
C. Miscellaneous antidiabetic drugs:
1. Alpha –Glucosidase inhibitors : Acarbose , Miglitol, Voglibose
2. Amylin analogue : Pramlintide
3. Dopamine D2 receptor agonist : Bromocriptine
4. Sodium glucose cotransport-2(SGLT-2) Inhibitors : Dapagliflozin
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21. Sulfonylurease
These agents are classified as insulin secretagogues, because
they promote insulin release from the ẞ cells of the pancreas.
The primary drugs used today are tolbutamide and the second-
generation derivatives, glyburide, glipizide, and glimepiride.
Mechanism of action :
1. stimulation of insulin release from the ẞ cells of the
pancreas by blocking the ATP-dependent K+ channels,
resulting in depolarization and Ca2+ influx
2. reduction in hepatic glucose production
3. increase in peripheral insulin sensitivity.
Pharmacokinetic :
• Given orally, these drugs bind to serum proteins
• Metabolized by the liver
• Excreted by the liver or kidney
• Tolbutamide has the shortest duration of action (6-12 hours),
whereas the second-generation agents last about 24 hours
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22. Adverse drug reaction
• Weight gain
• Hyperinsulinemia
• Hypoglycemia
• These drugs should be used with caution in patients with hepatic or renal
insufficiency, because delayed excretion of the drug-resulting in its
accumulation-may cause hypoglycemia.
• Renal impairment is a particular problem in the case of those agents that
are metabolized to active compounds, such as glyburide.
• Glyburide has minimal transfer across the placenta and may be a
reasonably safe alternative to insulin therapy for diabetes in pregnancy.
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23. Meglitinide analogue
This class of agents includes repaglinide and nateglinide. Although they are not sulfonylureas, they
have common actions.
Mechanism of action:
• Their action is dependent on functioning pancreatic ẞ cells .
• They bind to a distinct site on the sulfonylurea receptor of ATP-sensitive potassium channels,
thereby initiating a series of reactions culminating in the release of insulin .
• However, in contrast to the sulfonylureas, the meglitinides have a rapid onset and a short
duration of action.
• They are are categorized as postprandial glucose regulators
• Meglitinides should not be used in combination with sulfonylureas due to overlapping
mechanisms of action
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24. • Pharmacokinetic:
• These drugs are well absorbed orally after being taken 1 to 30 minutes before meals .
• Both meglitinides are metabolized to inactive products by CYP3A4 in the liver .
• Excreted through the bile.
Adverse drug reaction:
• Incidence of hypoglycemia is lower than that of the sulfonylureas.
• Repaglinide has been reported to cause severe hypoglycemia in patients who are also taking the
lipid-lowering drug gemfibrozil.
• Weight gain is less of a problem with the meglitinides than with the sulfonylureas.
• Must be used with caution in patients with hepatic impairment.
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25. Biguanides
• Metformin (glucophage), the only currently available biguanide
• it increases glucose uptake and utilization by target tissues, thereby
decreasing insulin resistance.
• Requires insulin for its action, but it does not promote insulin secretion
.
• Hyperinsulinemia is not a problem. Thus, the risk of hypoglycemia is far
less than that with sulfonylureas.
Mechanism of action :
• reduction of hepatic glucose output, largely by inhibiting hepatic
gluconeogenesis .
• Slowing intestinal absorption of sugars
• Improves peripheral glucose uptake and utilization.
• Metformin may be used alone or in combination with one of the other
agents, as well as with insulin.
• Hypoglycemia occurred when metformin was taken in combination.
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26. Pharmacokinetic
• Metformin is well absorbed orally, is not bound to serum proteins
• It is not metabolized
• Excretion is via the urine.
Adverse drug reaction:
• These are largely gastrointestinal.
• Contraindicated in diabetics with renal and/or hepatic disease, acute myocardial
infarction, severe infection, or diabetic ketoacidosis.
• It should be used with caution in patients greater than 80 years of age or in those
with a history of congestive heart failure or alcohol abuse.
• Long-term use may interfere with vitamin B12 absorption.
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27. Thiazolinediones
• Another group of agents that are insulin sensitizers are the
thiazolidinediones (TZDs) or, more familiarly the glitazones.
• Although insulin is required for their action, these drugs do not
promote its release from the pancreatic β cells thus,
hyperinsulinemia does not result.
• Troglitazone was the first of these to be approved for the treatment
of Type 2 diabetic, but was withdrawn after a number of deaths due
to hepatotoxicity were reported. Presently, two members of this class
are available, pioglitazone and rosiglitazone.
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28. Mechanism of action :
• Exact mechanism by which the TZDs lower insulin resistance remains to be elucidated
• They are known to target the peroxisome proliferator-activated receptor-y (PPARY)-a nuclear
hormone receptor. Ligands for PPARY regulate adipocyte production and secretion of fatty acids
as well as glucose metabolism, resulting in increased insulin sensitivity in adipose tissue, liver, and
skeletal muscle.
Pharmacokinetic :
• Both pioglitazone and rosiglitazone are absorbed very well after oral administration and are
extensively bound to serum albumin .
• Both undergo extensive metabolism by different cytochrome P450 isozymes.
• Pioglitazone : Renal elimination is negligible, with the majority of the active drug and metabolites
excreted in the bile and eliminated in the feces.
• Rosiglitazone: The metabolites are primarily excreted in the urine.
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29. Adverse Effects :
• Very few cases of liver toxicity have been reported with rosiglitazone or
pioglitazone.
• Weight increase can occur, possibly through the ability of TZDs to increase
subcutaneous fat or due to fluid retention.
• Glitazones have been associated with osteopenia and increased fracture risk.
• Other adverse effects include headache and anemia.
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30. Alpha glucosidase inhibitors
• Alpha-glucosidase inhibitors are oral anti- diabetic drugs used for diabetes mellitus type 2 that
work by preventing the digestion of carbohydrates (such as starch and table sugar).
Carbohydrates are normally converted into simple sugars (monosaccharides), which can be
absorbed through intestine. Hence, alpha-glucosidase inhibitors reduce the impact of
carbohydrates on blood sugar.
• Acarbose and miglitol are orally active drugs used for the treatment of patients with type 2
diabetes.
Mechanism of action :
These drugs are taken at the beginning of meals. They act by delaying the digestion of
carbohydrates, thereby resulting in lower postprandial glucose levels. Both drugs exert their effect
by reversibly inhibiting membrane- bound a-glucosidase in the intestinal brush border. This enzyme
is responsible for the hydrolysis of oligosaccharides to glucose and other sugars. Consequently, the
postprandial rise of blood glucose is blunted. Unlike the other oral hypoglycemic agents, these
drugs do not stimulate insulin release, nor do they increase insulin action in target tissues. Thus, as
monotherapy, they do not cause hypoglycemia. However, when used in combination with the
sulfonylureas or with insulin, hypoglycemia may develop.
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31. • Pharmacokinetic :
Acarbose is poorly absorbed. It is metabolized primarily by intestinal bacteria, and some of the
metabolites are absorbed and excreted into the urine. On the other hand, miglitol is very well
absorbed but has no systemic effects. It is excreted unchanged by the kidney.
Adverse effect:
The major side effect are flatulence ,diarrhea and abdominal cramping . Patients with inflammatory
bowel disease ,colonic ulceration or intestinal obstruction should not use these drugs.
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32. Dipeptidyl peptidase -4 inhibitors
• DPP-4 inhibitors or gliptins, s, are a class of oral hypoglycemics that block DPP-4. They can be
used to treat diabetes mellitus type 2
• The first agent of the class was sitagliptin approved by the FDA in 2006.
• Glucagon increases blood glucose levels, and DPP- 4 inhibitors reduce glucagon and blood
glucose levels. The mechanism of DPP-4 inhibitors is to increase incretin levels (GLP-1 and GIP),
which inhibit glucagon release, which in turn increases insulin secretion, decreases gastric
emptying, and decreases blood glucose levels.
• Sitagliptin is an orally active dipeptidyl peptidase –IV (DPP-IV) inhibitors used for the treatment of
patients with type 2 diabetes . other agents in this category are currently in development.
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33. • Mechanism of action
Sitagliptin inhibits the enzyme DPP- IV, which is responsible for the inactivation of incretin
hormones, such as glucagon-like peptide-1 (GLP-1). Prolonging the activity of incretin hormones
results in increased insulin release in response to meals and a reduction in inappropriate secretion
of glucagon. Sitagliptin may be used as monotherapy or in combination with a sulfonylurea,
metformin or a glitazone.
Pharmacokinetics
Sitagliptin is well absorbed after oral administration. Food does not affect the extent of absorption.
The majority of sitagliptin is excreted unchanged in the urine. Dosage adjustments are
recommended for patients with renal dysfunction.
Adverse effect
In general, sitagliptin is well tolerated, with the most common adverse effects being nasopharyngitis
and headache. Rates of hypoglycemia are comparable to those with placebo when sitagliptin is used
as monotherapy or in combination with metformin or pioglitazone.
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34. Thank you
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