Insulin

•    Polypeptide hormone secreted by beta cells of islet of Langerhans in pancreas

–  First hormone to be identified, isolated, purified and produced by recombinant DNA technology

•    Anabolic hormone; influences the metabolism of

–  Carbohydrate

–  Fats and 

–  Proteins

 

•    Chemistry:

•    Heterodimeric protein

–  Two different polypeptide chains (A & B chains) linked together by 2 disulfide bonds

•    Connecting A₇ to B₇ and A₂₀ to B₁₉ – A chain:

•    21 amino acids

•    An additional disulfide bond A₆ and A₁₁ – B chain:

•    30 aminoacids

•    Terminal sequences of aminoacids, 3 disulfide bonds, and 3D stuructures is similar in different species

– Insulin from one animal is very likely to biologically active in other species

 

Fig: Structure of Insulin (A and B Chains) [Showing interchain and intrachain disulfide bonds]

•    Biosynthesis of Insulin:

•    Insulin is synthesized by β-cells of islets of

Langerhans of pancreas

– Pre-pro-insulin (108 AA) is formed, converted to proinsulin (86 AA) that is ultimately converted to insulin

•    Sequential degradation to insulin and connecting peptide (C-peptide)

•    C-peptide is itself biologically inactive but serves as a useful index for endogenous insulin production

–  Pre-Pro-insulin is synthesized in polysomes, attached to rough ER in β-cells

•     Transferred to lumen of rough ER cisternae

•     There Signal peptidase enzyme split 23 AA peptide from Nterminal of pre-pro-insulin

•     Pro-insulin with 86 amino acids is formed

•     Pro-insulin is transferred from ER to golgi bodies

–  Trypsin-like-protease hydrolyses it at two sides

–  2 peptides; A-chain (21AA) and B-chain (30AA) and 1 connecting C-peptide (31AA) is formed

•    Insulin (and also pro-insulin) combines with two Zn²⁺ to form hexamer complex and stored in cytosol secretory granules

–  Released by exocytosis in response to various stimuli

•    Regulation of Insulin Release:

•    40-50 Units of insulin is secreted daily

–  Normal plasma insulin concentration is 20-30μU/mL

•    Various factors stimulate or inhibit the release of insulin from β-cells

•    Factors stimulating insulin secretion are:

–  Glucose (most important stimulus)

•    Carbohydrate rich meal and elevated BSL

–  Aminoacid

•    Rise in plasma amino acid concentration

•    Arginine, leucine are potent stimulator of insulin release

–  GI hormones (Secretin, gastrin etc.)

•     Release after the ingestion of food

•     Epinephrine from adrenal medulla suppresses insulin release

–  promotes energy metabolism by mobilizing energyyielding compounds

•     Glucose from liver and fatty acids from adipose tissue

Target Tissues:

•     Major target tissues are Muscles, liver, heart and adipose tissue

Mechanism of Insulin Action:

•     Act by binding to specific insulin receptors

–  Tetramer glycoprotein receptors consist of 2 alpha and 2 beta subunits

•   Insulin binds to extracellular alpha subunit

•   Beta subunit is a transmembrane tyrosine specific protein kinase enzyme, activated by insulin molecule

–  Up to 20,000 receptors are found on target plasma membrane

•     Being constantly synthesized and degraded daily

 

•     Signal transduction in insulin receptors:

–  Binding to alpha unit induce conformational changes in it

–  This activates tyrosin kinase (β-subunit)

• Autophosphorylation of tyrosine residues on β-subunits and insulin receptor substrate (IRS)

–  Phosphorylated IRS promotes activity of other protein kinases and phosphatases that regulate biological activity

•     An overall fall in cAMP and rise in cGMP levels

•     Both function in a reciprocal relationship

•     Insulin also act by affecting the rate of transcription of specific genes: Increase proteins and enzymes

–  Decrease the synthesis of phosphoenol pyruvate carboxy kinase (PEPCK) (Key enzyme of gluconeogenesis)

–  Induce synthesis of Phosphofructokinase and pyruvate kinase (Key enzymes of glycolysis)

•    Biological Function of Insulin:

•    It increases 

–  carbohydrate metabolism, glycogenesis and glycogen storage

–  FA synthesis, Triglyceride storage

–  Aminoacid uptake and protein synthesis

i) Action on carbohydrate metabolism:

• Net effect is

–  Lowering blood glucose

–  Increase glycogen stores

a)   increase glucose uptake

–  Directly or by stimulating glucose transporters GLUT4

–  Induce synthesis of glucokinase in hepatocytes

•   Phosphorylate glucose: lowering intracellular concentration

b)   increase glycolysis

–  Induce enzymes phosphofructokinase and pyruvate kinase

–  Increase utilization of glucose for providing energy

c)    increase pyruvate to acetyl Co-A conversion

–  Activates pyruvate dehydrogenase enzyme

•   Increases aerobic oxidative decarboxylations of pyruvate to acetyl Co-A

d)   Promote glycogenesis

–  Activates glycogen synthase

•   Key and rate limiting enzyme of glycogenesis

e)   increase HMP-shunt pathway

–  Activates Glucose-6-Phospho Dehydrogenase and 6phosphogluconate dehydrogenase

–  Stimulate HMP shunt pathway and produce NADPH

f)     Decrease Gluconeogenesis

–  Decrease rate of transcription of PEP-carboxykinase

(PEPCK) 

•   Key rate limiting enzyme of gluconeogenesis

–  Inactivates fructose-2,6-biphosphatase that allosterically inhibit fructose-1,6-bi-phosphatase

•   Increase concentration of fructose-2,6-bi phosphate in cells

g)   Decrease glycogenolysis

–  Inactivates glycogen phosphorylase and glucoe-6phosphatase enzymes

 

ii) Action on Lipids metabolism:

• Overall action of insulin on lipid metabolism is decreased release of fatty acids from stored fats

–  Lowering of free fatty acid levels (↓ FFA level)

–  Increase triglyceride stores (↑ TG store)

a)   decreases lipolysis in adipose cells

–  Activates phosphoprotein phosphatase

•    Dephosphorylates triacyl glyceol lipase

–  Activates phosphodiesterase enzyme

•    Deactivates cAMP, prevents rephosphorylation and reactivation of TG lipase

b)   increases the synthesis of triglycerides from

glucose in adipose tissue

 

–  Provide more raw material

•    glycerol-3-phosphate (from glycolysis)

•    NADPH (from HMP shunt pathway)

–  Induce the synthesis of lipoprotein lipase enzyme

•    Increased hydrolysis of VLDL and TG of circulating chylomicrones to provide FFA for the synthesis of triglycerides in adipocytes

c)    increases fatty acid synthesis (for TG synthesis)

–  Provide more acetyl Co-A through various mechanisms

–  Increase activity of acetyl Co-A carboxylase

•    Key enzyme in fatty acid synthesis

d)   decreases ketogenesis

–  Formation of ketone bodies (Ketone group containing water soluble molecules) from fatty acids by the liver

•    Ketone bodies are formed from acetyl CoA condensation to acetoacetyl CoA and finally 3-hydroxy-3-methyl glutarylCoA (HMG-CoA)

–  Insulin decreases

•    FFA level

•    β- oxidation of fatty acids to produce acetyl-CoA

•    The activity of HMG-CoA synthetase enzyme

–  Increases the utilization of acetyl-CoA for 

•     Oxidation

•     Lipogenesis

•     The overall effect is the reduced availability of acetyl-CoA for ketogenesis

iii) Action on Protein Metabolism:

• Net effect is increased protein synthesis and decreased protein degradation

a)   increases aminoacid uptake by the tissues

–  Stimulate the synthesis of AA transporter proteins

b)   Effect gene transcription at nuclear level

–  Regulate specific mRNA synthesis

c)    Effect translation at ribosomal level iv) Action on Mineral metabolism: • Decrease K⁺ and inorganic phosphate (P_i)

–  Enhanced glycogenesis and phosphorylation of glucose

v) Action on Growth and cell replication:

•    Stimulate cell growth and replication

– Regulated through various growth factors

•    Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) etc.