Mechanism of action of insulin

insulin-History, Pancreas anatomy,Structure of insulin, Biosynthesis of insulin, Regulation of insulin secretion, metabolic effects, mechanism of action, insulin receptor, insulin signalling pathyways- PI3-K/Akt, mTOR-TSC, Ras-MAPK pathways

1. Akash Mahadev Iyer S4 M Sc Biochemistry Department of Biochemistry Karyavattom campus Mechanism of Action of Insulin

2. Outline… Introduction to Insulin History Structure of Insulin Biosynthesis of Insulin Degradation of Insulin Regulation of secretion Biological actions Effect on Carbohydrate, lipid and protein metabolism Insulin Receptor Mechanism of action Insulin signaling pathways Disorders

3. Introduction Insulin is a peptide hormone secreted by β-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.

4. In 1869, a German medical student, Paul Langerhans , noted that the pancreas contains two distinct groups of cells the acinar cells, which secrete digestive enzymes, and cells that are clustered in islands, or islets, which he suggested served a second function. In 1889, Oskar Minkowski and Joseph von Mering observed that total pancreatectomy in experimental animals leads to the development of severe diabetes mellitus In 1920, Frederick banting managed to isolate the fluid extract-that he called ‘ isletin ’- from Islets of Langerhans and injected into diabetic dogs. The dogs abnormally high sugar level in the blood lowered and they survived for long a they received the extract. The Nobel Prize in medicine and physiology was awarded to Banting and Macleod with in 1923. Insulin is sequenced by British biochemist Frederick Sanger, and is the first protein to be fully sequenced. In 1958 Sanger receives the Nobel Prize in Chemistry. Dorothy Hodgkin won the 1964 Nobel Prize in Chemistry for her work in protein crystallography/ x-ray crystallography.  1978, Biotechnology firm Genentech ( Califor .) used rDNA technology to produce synthetic “human” insulin- first human protein to be manufactured through biotechnology.

5. Insulin should have been named “Protein of the 20 th century” Insulin was: the first protein shown to have hormonal action the first protein to be crystallized in pure form (Abel, 1926 ) the first protein to be fully sequenced (Sanger et al , 1955 )  the first protein to be synthesized chemically (Du et al , Zahn , 1964) the first protein to be synthesized as a large precursor molecule (Steiner et al , 1967) the first protein synthesized for commercial use by DNA recombinant  technology (1979)

6. In adult humans, the pancreas weighs about 80 g. Located behind the stomach Derived from endoderm The pancreas is a retroperitoneal organ and does not have a capsule. The spleen is adjacent to the pancreatic tail. The regions of the pancreas are the head, body, tail and uncinate process . The distal end of the common bile duct passes through the head of the pancreas and joins the pancreatic duct entering the duodenum. For this reason, pathologic processes of the pancreas, such as a cancer at the head of the pancreas or swelling and/or scarring of the head of the pancreas due to pancreatitis, can lead to biliary system obstruction and injury. Because of its posterior position, the pancreas is usually protected from trauma Pancreas; a dual gland

7. Pancreas -both an endocrine gland and an exocrine gland The pancreas provides both The enzymes that digest the food in the gut (The ductal and acinar cells of the exocrine compartment -80%) and The hormones that control utilization of the nutrients supplied by that digested food (the islets of Langerhans of the endocrine portion are scattered throughout the exocrine matrix- 2%)

8. The human pancreas has 1 to 2 million islets of Langerhans , each only about 0.3 millimeter in diameter and organized around small capillaries into which its cells secrete their hormones. The islets contain three major types of cells, alpha , beta , and delta cells, which are distinguished from one another by their morphological and staining characteristics .

9. 3.) The acini 2.) The Islets 1.) Pancreas

10. Structure of Insulin Human insulin- 51 aa Mw; 5.7 kDa Comprises of 2 polypeptide chains A (with 21 amino acid residues) and B (with 30 amino acid residues) The two chains are linked by two interchain disulfide bridges that connect A7 to B7 and A20 to B19 A third intrachain disulfide bridge connects residues 6 and 11 of A chain -

11. Insulin exists primarily as a monomer at low concentrations (~10–6 M) and forms a dimer at higher concentrations at neutral pH. At high concentrations and in the presence of zinc ions, insulin aggregates further to form hexameric complexes. Monomers and dimers readily diffuse into blood, whereas hexamers diffuse poorly. Hence , absorption of insulin preparations containing a high proportion of hexamers is delayed and slow . The three conserved regions in insulin — amino terminal A-chain ( GlyA1-IleA2- ValA3-GluA4 or AspA4), carboxyl terminal A-chain (TyrA19-CysA20-AsnA21 ), and carboxyl terminal B chain (GlyB23-PheB24-PheB25-TyrB26 )—are located at or near the surface of insulin and therefore may interact with the insulin receptor .

12. Insulin molecules have a tendency to form dimers in solution due to hydrogen-bonding between the C-termini of B chains. Additionally, in the presence of zinc ions, insulin dimers associate into hexamers . Monomers and dimers readily diffuse into blood, whereas hexamers diffuse poorly. Hence , absorption of insulin preparations containing a high proportion of hexamers is delayed and somewhat slow. This phenomenon, among others, has stimulated development of a number of recombinant insulin analogs. The first of these molecules to be marketed - called insulin lispro - is engineered such that lysine and proline residues on the C-terminal end of the B chain are reversed; this modification does not alter receptor binding, but minimizes the tendency to form dimers and hexamers .

13. Site; β - cells of Islets Synthesis of Preproinsulin . Conversion of preproinsulin to proinsulin . Conversion of proinsulin to insulin. Insulin is synthesized by ribosomes of the rough ER as a larger precursor peptide that is then converted to the mature hormone prior to secretion Biosynthesis of Insulin – 3 major steps Biosynthesis of Insulin contd …..

14. Insulin is synthesized in the form of a hmw precursor, preproinsulin (110 aa ), processed via an intermediate precursor, proinsulin (86 aa ) , to the mature insulin (51 aa ) molecule Biosynthesis of Insulin contd …..

15. Conversion of proinsulin to insulin Proinsulin undergoes maturation into active insulin through action of cellular endopeptidases known as prohormone convertases ( PC1 and PC2 ), and 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 In the β cells, insulin combines with zinc to form complexes In this form, insulin is stored in granules of the cytosol which is released in response to various stimuli.

16. Collapsed cavity Insulin hexamer ;Top view Two Zn  2+  axial atoms per hexameric unit Monomer is the biologically active form of insulin, hexamer serves as the storehouse of the hormone. Stable cavity with water Water molecules Zn ² +  We find that these water molecules are dynamically slower than the bulk and weave an intricate hydrogen bond network among themselves and with neighboring protein residues to generate a robust backbone at the center of the hexamer that holds the association strongly from inside and maintains the barrel shape.

17. Peptide Hormone Synthesis, Packaging, and Release ECF Cytoplasm Plasma Capillary endothelium Messenger RNA on the ribosomes binds amino acids into a peptide chain called a preprohormone . The chain is directed into the ER lumen by a signal sequence of amino acids. mRNA Ribosome Endoplasmic reticulum (ER) Preprohormone 1 1

18. ECF Cytoplasm Plasma Capillary endothelium Messenger RNA on the ribosomes binds amino acids into a peptide chain called a preprohormone . The chain is directed into the ER lumen by a signal sequence of amino acids. Enzymes in the ER chop off the signal sequence, creating an inactive prohormone . mRNA Ribosome Prohormone Signal sequence Endoplasmic reticulum (ER) Preprohormone 1 2 1 2 Peptide Hormone Synthesis, Packaging, and Release

19. Golgi complex ECF Cytoplasm Plasma Capillary endothelium Messenger RNA on the ribosomes binds amino acids into a peptide chain called a preprohormone . The chain is directed into the ER lumen by a signal sequence of amino acids. Enzymes in the ER chop off the signal sequence, creating an inactive prohormone . The prohormone passes from the ER through the Golgi complex. mRNA Ribosome Prohormone Signal sequence Transport vesicle Endoplasmic reticulum (ER) Preprohormone 1 2 3 1 2 3 Peptide Hormone Synthesis, Packaging, and Release

20. 4 Active hormone Golgi complex Secretory vesicle ECF Cytoplasm Plasma Peptide fragment Capillary endothelium Messenger RNA on the ribosomes binds amino acids into a peptide chain called a preprohormone . The chain is directed into the ER lumen by a signal sequence of amino acids. Enzymes in the ER chop off the signal sequence, creating an inactive prohormone . The prohormone passes from the ER through the Golgi complex. Secretory vesicles containing enzymes and prohormone bud off the Golgi. The enzymes chop the prohormone into one or more active peptides plus additional peptide fragments. mRNA Ribosome Prohormone Signal sequence Transport vesicle Endoplasmic reticulum (ER) Preprohormone 1 2 3 1 2 3 4 Peptide Hormone Synthesis, Packaging, and Release

21. 4 5 Active hormone Golgi complex Secretory vesicle ECF Cytoplasm Plasma Peptide fragment Release signal Capillary endothelium Messenger RNA on the ribosomes binds amino acids into a peptide chain called a preprohormone . The chain is directed into the ER lumen by a signal sequence of amino acids. The secretory vesicle releases its contents by exocytosis into the extracellular space. Enzymes in the ER chop off the signal sequence, creating an inactive prohormone . The prohormone passes from the ER through the Golgi complex. Secretory vesicles containing enzymes and prohormone bud off the Golgi. The enzymes chop the prohormone into one or more active peptides plus additional peptide fragments. mRNA Ribosome Prohormone Signal sequence Transport vesicle Endoplasmic reticulum (ER) Preprohormone 1 2 3 1 2 3 4 5 Peptide Hormone Synthesis, Packaging, and Release

22. 4 5 To target Active hormone Golgi complex Secretory vesicle ECF Cytoplasm Plasma Peptide fragment Release signal Capillary endothelium Messenger RNA on the ribosomes binds amino acids into a peptide chain called a preprohormone . The chain is directed into the ER lumen by a signal sequence of amino acids. The secretory vesicle releases its contents by exocytosis into the extracellular space. The hormone moves into the circulation for transport to its target. Enzymes in the ER chop off the signal sequence, creating an inactive prohormone . The prohormone passes from the ER through the Golgi complex. Secretory vesicles containing enzymes and prohormone bud off the Golgi. The enzymes chop the prohormone into one or more active peptides plus additional peptide fragments. mRNA Ribosome Prohormone Signal sequence Transport vesicle Endoplasmic reticulum (ER) Preprohormone 1 2 3 6 1 2 3 4 5 6 Peptide Hormone Synthesis, Packaging, and Release

23. Leptin Fasting

24. Regulation of insulin secretion by glucose in pancreatic β cells .

25. Similar to other neuroendocrine cells, insulin-secreting cells are electrically excitable. This means that insulin secretion in response to glucose is dependent on initiation of electrical activity from a basal electronegative resting state.. In the normal course of glucose excitation coupling to insulin secretion, glucose enters the cell via GLUT2 (and GLUT1) glucose transporters and is trapped in the cell by phosphorylation via glucokinase , a specialized high Km hexokinase . The high Km of glucokinase for glucose underlies its role as the “glucose sensor” of the β cell, in that its enzyme activity reflects physiologic circulating glucose concentrations .

26. Following glucose phosphorylation , further metabolism via glycolysis , the Krebs ( tricarboxycylic acid, TCA) cycle, and oxidative phosphorylation in the mitochondria leads to an increase in the ATP-to-ADP ratio in the cytoplasm. Interestingly , while glucose is the key physiologic stimulator of the insulin-secreting system, it can be replaced or enhanced by other energy-providing metabolites (e.g., leucine , and succinic acid monomethyl ester) that either feed into glycolysis , the TCA cycle, or related oxidative phosphorylation pathways . The ATP/ADP ratio increase is driven by metabolism that leads to ATP-dependent potassium channel (KATP) channel inhibition, in turn leading to accumulation of positive charge (K+ and Na+) inside the cell and causing the cell membrane to depolarize. As the membrane potential reaches about –20mV from the resting level of about –70mV, voltage-dependent calcium channels open, allowing entry of Ca2+.

27. Interaction of numerous K+, Ca2+, and Na+ voltage-dependent channels contributes to the appearance of tonic or periodic transients of both membrane potential and Ca2+. Once KATP closes and Ca2+ entry begins via the opening of voltage-dependent calcium channels, a variety of mechanisms control intracellular Ca2+, which is a key regulatory factor for insulin release . These include regulation of the plasma membrane potential by other voltage- and Ca2+-dependent K+ channels that serve to help repolarize the plasma membrane, and voltage dependent Na+ channels that accelerate plasma membrane depolarization especially in human, canine, and porcine islets .

28. Inside of the cell, Ca2+ can be sequestered by the action of the sarcoplasmic /endoplasmic reticulum adenosine triphosphatase (SERCA) pump. Release of intracellular Ca2+ from the endoplasmic reticulum through action of inositol triphosphate (IP3) on IP3 receptors, as well as via ryanodine receptors that can be activated by additional messengers, may all play key roles in regulating intracellular Ca2+ transients. Spatial and temporal control of Ca2+ signals may be highly regulated in plasma membrane Ca2+ microdomains where insulin granules fuse. Mitochondria are also important stores of calcium and take up Ca2+ during metabolic activity in part to regulate the key dehydrogenases in the TCA cycle

29. Other anaplerotic molecules coming from the mitochondria, glutamate in particular, can be implicated in regulation of secretion. The elevation in intracellular free Ca2+ activates multiple proteins (e.g., small G proteins such as Rabs , and soluble N- ethylmaleimide attachment protein receptor [SNARE] pathways) regulating the Ca2+-triggered fusion of the ( predocked ) insulin-containing granules with the cell membrane, resulting in the first phase of insulin secretion .

30. As in neurotransmitter release, β cell granule fusion depends on interactions of synaptosome -associated proteins v-SNAREs (VAMP2 and synaptotagmin ) with plasma membrane receptors such as SNAP-25, a t-SNARE (target localized SNAP receptor), and syntaxins . Second-phase insulin secretion refers to the continued release of insulin following the initial peak. In parallel with fusion of the docked insulin containing granules, the granules located farther away of plasmalemma (termed resting newcomers ) can be recruited via microtubules and associated kinases , chaperones, and small GTP-binding proteins (syntaxin4, Munc18) through the actin network until they too can dock at t-SNARE sites and fuse to the plasma membrane.158

31. Increased ATP closes the ATP-sensitive K + channels K + efflux Depolarizes the cell membrane Open voltage sensitive calcium channels Calcium enters the cell Intracellular Ca 2+ Triggers insulin secretion by exocytosis Glucose ;key regulator ( aa , ketones , various nutrients, GIPs, and NTs also influence insulin secretion.) Glucose levels > 3.9 mmol /L (70 mg/ dL ) stimulate insulin synthesis, primarily by enhancing protein translation and processing

32. Degradation of Insulin Half-life; 4-6 minutes. Liver -principal site of insulin degradation Hepatic glutathione insulin transhydrogenase – Reduces the disulphide bonds and then individual A and B chains are rapidly degraded IDE – Cleaves B-chain at Tyr16- Leu17

33. Insulin promotes the anabolic state by channeling metabolism towards the storage of carbohydrates and lipids, and towards protein synthesis. Insulin promotes energy storage by stimulating the synthesis of fatty acids and triglycerides in the liver and adipose tissue, glycogen in liver and skeletal muscle, and protein synthesis in muscles. At the same time, it opposes the catabolism by inhibiting the breakdown of proteins, triglycerides and glycogen, and by suppressing gluconeogenesis by the liver. Metabolic effects of insulin

34. Insulin acts on three main target tissues:

35. Different organs and tissues handle fuels differently At rest, the brain uses approximately 20% of all oxygen consumed by the body. Glucose is normally its only fuel: during starvation, however, the brain adapts to the use of ketone bodies as an alternative energy source. The two pathways that provide glucose are glycogenolysis and gluconeogenesis . When glucose concentration in the extracellular fluid decreases, it is first replenished by degrading liver glycogen. However, when the fasting period extends gluconeogenesis is initiated. Gluconeogenesis takes place mostly in the liver, and the kidneys also contribute during prolonged fast. Its main substrates are lactate (from anaerobic glycolysis ), alanine (from the amino acids released during breakdown of muscle protein) and glycerol (from the breakdown of triacylglycerols in the adipose tissue.

36. Insulin Action on Carbohydrate Metabolism: Liver: Stimulates glycolysis Promotes glucose storage as glycogen Inhibits glycogenolysis Inhibits gluconeogenesis Muscle: Stimulates glucose uptake (GLUT4) Promotes glucose storage as glycogen Adipose Tissue: Stimulates glucose transport into adipocytes Promotes the conversion of glucose into triglycerides and fatty acids

37. Insulin promotes glycolysis

38. In liver, insulin decrease gluconeogenesis by suppressing the enzymes pyruvate carboxylase , phosphoenol pyruvate carboxykinase and glucose-6-phosphatase In liver and muscles insulin increases Glycolysis and glycogen synthesis

39. Effect on lipogenesis : Insulin favours the synthesis of triacylglycerols from glucose by providing more glycerol 3-phosphate & NADPH. Insulin increases the activity of acetyl CoA carboxylase , a key enzyme in fatty acid synthesis. Effect on lipolysis : Insulin decreases the activity of enzyme -hormone- sensitive lipase & reduces the release of fatty acids from stored fat. Effect on ketogenesis : Insulin reduces ketogenesis by decreasing the activity of HMG CoA synthase . Effects on lipid metabolism

40. Insulin regulates glucose uptake by adipocytes and triggers fatty acid transport protein translocation as well as fatty acid uptake by fat cells. Binding of insulin to its specific cell-surface receptor produces tyrosine phosphorylation and activation of the insulin receptor, which leads to the interaction with the insulin receptor substrates (IRS-1 and IRS-2), in turn activating the p hosphatidyl inositol 3-kinase (PI3K) complex. Insulin powerfully inhibits basal and catecholamine-induced lipolysis through phosphorylation (via a PKB/ Akt -dependent action) and activation of phosphodiesterase-3B (PDE-3B). The phosphodiesterase catalyses the breakdown of cAMP to its inactive form, thereby decreasing cAMP levels, which in turn reduces PKA activation and, therefore, also translates into preventing HSL stimulation. Insulin may also suppress lipolysis through phosphorylation of the regulatory subunit of protein phosphatase-1 (PP-1), which once activated rapidly dephosphorylates and deactivates HSL, thus decreasing the lipolytic rate.

41. Effect on lipogenesis : ACC Effect on lipolysis :

42. Stimulates the entry of amino acids into the cells, Enhances Protein Synthesis and Reduces protein degradation In the liver, insulin depresses the rate of gluconeogenesis conserves the amino acids Effects on protein metabolism Insulin and Growth hormone interact synergistically to promote growth

43. Important role in Potassium homeostasis Stimulates K+ uptake by the cells  Inflammation and Vasodilation Insulin’s actions within endothelial cells and macrophages have an anti-inflammatory effect on the body. Within endothelial cells, insulin stimulates the expression of endothelial nitric oxide synthase ( eNOS ).  eNOS functions to release nitric oxide (NO), which leads to vasodilation .

44. 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 α and 2 transmembrane β subunits linked together by disulfide bonds – α 2 β 2 Molecular weight 300  kDa α subunits - insulin binding site β subunits -tyrosine kinase activity, involved in intracelular signaling . Expressed in most mammalian tissues; adipose and liver have the highest IR density (>300,000 receptors/cell). Insulin receptor Tyrosine kinase activity ~

45. Activation of insulin signaling regulates the metabolisms of glucose and lipids, and protein synthesis.  Phosphorylation at Y972 of the β-subunit generates an NPXpY motif, leading to the increase in the affinity of the IRS binding to the IR and the tyrosine phosphorylation of IRS.

46. Binding of Insulin on α -subunit Phosphorylation of β -subunit Phosphorylation o f IRS Mechanism of action of insulin Insulin regulates both metabolic enzymes and gene expression. Does not enter cells, but initiates a signal that travels from the cell surface receptor to - cytosol and to the nucleus. The insulin receptor (INS-R) is a glycoprotein receptor with tyrosine kinase activity. Gene Expression Metabolism Growth CHANGES IN

47. Signaling through INS-R begins when -binding of insulin activates the Tyr kinase activity, and each β subunit phosphorylates three critical Tyr residues near the carboxyl terminus of the other β subunit . Binding of ligand causes a conformational change that promotes dimerization of the extracellular domains of RTKs, which brings their transmembrane segments—and therefore their cytosolic domains—close together. This autophosphorylation opens up the active site so that the enzyme can phosphorylate Tyr residues of other target proteins. a region of the cytoplasmic domain (an autoinhibitory sequence) that normally occludes the active site moves out of the active site after being phosphorylated , opening up the site for the binding of target proteins. Activation of the insulin receptor, which leads to the interaction with the insulin receptor substrates (IRS-1 and IRS-2), in turn activating the phosphatidyl inositol 3-kinase (PI3K) complex.

48. When INS-R is autophosphorylated and becomes an active Tyr. Kinase , it phosphorylates Insulin receptor substrate-1 (IRS-1) Once phosphorylated on several of its Tyr residues, IRS-1 becomes the point of nucleation for a complex of proteins , that carry the message from the insulin receptor to end targets in the cytosol and nucleus, through a long series of intermediate proteins.

49. Activation of the Tyr kinase allows each subunit to phosphorylate three Tyr residues (Tyr1158, Tyr1162, Tyr1163) on the other subunit. The introduction of three highly charged P –Tyr residues forces a 30 Å change in the position of the activation loop, away from the substrate-binding site, which becomes available to bind and phosphorylate a target protein.

50. Insulin signalling is one of the most important signalling network, which regulates some fundamental biological functions such as glucose and lipid metabolism, protein synthesis, cell proliferation, cell differentiation and apoptosis. These different biological responses are achieved by the insulin binding to its receptor and by the subsequent combined activation of three major pathways: The PI3K-AKT pathway , mostly responsible for the metabolic insulin action via the translocation of the glucose transporter type 4 (GLUT4) vesicles to the plasma membrane, which, in turn, allow the glucose uptake in muscle cells and adipocytes ; The TSC1/2-mTOR pathway , playing a critical role in protein synthesis since mammalian target of rapamycin ( mTOR ) is a central controller for several anabolic and catabolic processes including RNA translation, ribosome biogenesis and autophagy , in response not only to growth factors and hormones like insulin, but also to nutrients, energy and stress signals ; The RAS-MAPK pathway , promoting cell survival, division and motility via extracellular signal-regulated kinase 1/2 (ERK1/2) complex that, once phosphorylated , translocates into the nucleus activating many transcription factors, thus constituting an important connection between the cytoplasmic and nuclear events. Insulin signalling

51. Insulin powerfully inhibits basal and catecholamine-induced lipolysis through phosphorylation (via a PKB/ Akt -dependent action) and activation of phosphodiesterase-3B (PDE-3B). The phosphodiesterase catalyses the breakdown of cAMP to its inactive form, thereby decreasing cAMP levels, which in turn reduces PKA activation and, therefore, also translates into preventing HSL stimulation. Insulin may also suppress lipolysis through phosphorylation of the regulatory subunit of protein phosphatase-1 (PP-1), which once activated rapidly dephosphorylates and deactivates HSL, thus decreasing the lipolytic rate.

52. STEPS Ligand reception Receptor Dimerizaton Autophosphorylation –Activation of Tyrosine kinase in INS-R Phosphorylation of target proteins ( eg ; IRS proteins) Tyrosine phosphorylated   IRS proteins  act as a binding site  for signaling molecules containing SH-2 (Src-homology-2) domains such as PI3’-kinase, and Grb2/ sos , SHP2. Insulin signaling

53. Biological actions of insulin, initiated by its binding to INS-R and an have short, intermediate, and long-term effects on cellular functions Short term effects Intermediate effects Long term effects Biological actions of insulin

54. 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 Intermediate effects Minutes to hours Induction of genes and expression of certain proteins

55. Long term effects Hours to several days Stimulate DNA synthesis, cell proliferation cell differentiation and some gene expression events. These effects are the results not of a simple linear pathway, but of the multiple diverging and converging pathways mediated by INS-R.

56. Insulin signalling is one of the most important signaling network, It regulates most fundamental biological functions such as; Glucose and lipid metabolism, Protein synthesis, Cell proliferation, Cell differentiation and Apoptosis. Insulin signaling

57. There are two major routes by which, insulin signals are transmitted

58. PI3K-Akt pathway The PI3K/AKT signaling pathway is a key regulator of normal cellular processes involved in cell growth, proliferation, metabolism, motility, survival, and apoptosis. Responsible for the metabolic effects of insulin Translocation of the glucose transporter type 4 (GLUT4) vesicles to the plasma membrane; allows glucose uptake in muscle cells and adipocytes Aberrant activation of the PI3K/AKT pathway promotes the survival and proliferation of tumor cells in many human cancers

59. The activated INS-R phosphorylates IRS. Phosphorylated IRS binds to the SH2 domains of PI3K. Binding of IRS, results in activation of the catalytic subunit of PI3K. The PI3K-Akt Signaling Pathway PIP3, which remains in the cytosolic leaflet of the plasma membrane recruits two protein kinases to the plasma membrane via their PH domains- Akt (PKB-Protein kinase B) and PDK1( Phosphoinositide -dependent protein kinase 1). Both are; Serine - threonine kinases

60. Recruitment of PDK1 to the plasma membrane, in close proximity to PKB, provides a setting in which PDK1 can phosphorylate and activate PKB . Phosphorylation by PDK1 (at Thr-308) is essential, but not sufficient for activation of PKB. Activation of PKB also depends on phosphorylation (at Ser-473) by a second kinase , mTOR . Remember Akt requires two phosphorylation events for activation! !! INS-R

61. AKT is activated by phosphorylation of Thr308 in its activation loop by the juxtaposed membrane-bound PDK1. AKT isoforms regulate phosphorylation of proteins that control cell survival, growth, proliferation, angiogenesis, metabolism, and migration. More than 100 AKT substrates are known and several are especially relevant to insulin signaling— including GSK3α/β (blocks inhibition of glycogen synthesis); AS160 (promotes GLUT4 translocation); BAD•BCL2 heterodimer (inhibits apoptosis); The FOXO transcription factors (regulates gene expression in liver, β-cells, hypothalamus, and other tissues); p21CIP1 and p27KIP1 (blocks cell-cycle inhibition); eNOS (stimulates NO synthesis and vasodilatation); PDE3b (hydrolyzes cAMP ); and TSC2 (tuberous sclerosis 2 tumor suppressor) that inhibits mTORC1 (mechanistic target of rapamycin complex 1)

62. Activated Akt phosphorylates various target proteins at the plasma membrane, as well as in the cytosol and nucleus. Four of the critical downstream substrates of Akt are mTOR , mammalian target of rapamycin , involved in the regulation of protein synthesis GSK3 (glycogen synthase kinase 3), involved in the regulation of glycogen synthesis FoxO ( forkhead box-containing protein, O subfamily) transcription factors, especially FoxO1, involved in the regulation of gluconeogenic and adipogenic genes and AS160 (AKT substrate of 160kDa), involved in glucose transport.

63. mTOR Cascade mTOR (mechanistic target of rapamycin ) is a Ser/ Thr kinase that is regulated through multiple mechanisms. It belongs to the PI3K-related kinase family and forms two large functionally distinct protein complexes; mTORC1 mTORC2 Composed of common and unique subunits. Both complexes are controlled by growth factors and insulin through the PI3K→AKT cascade, but they are recruited to different compartments and respond distinctly to nutrients, stress, hypoxia/energy status, and other stimuli to coordinate a diverse array of biological processes—including protein and lipid synthesis, liposome biogenesis, autophagy , and cell migration, growth, and proliferation.

64. In addition to the common catalytic subunit, mTORC1 and mTORC2 share mLST8 (mammalian lethal with sec-13 protein 8), DEPTOR (Disheveled, Egl - 10, and Pleckstrin domain containing mTOR -interacting protein), and Tti1 (Telomere maintenance 2 interacting protein 1). mTORC1 is distinguished by two specific components, including RAPTOR (RPTOR, regulatory associated protein of mTOR , complex 1) and AKT1S1 (PRAS40, AKT1 substrate 1 proline -rich). mTORC2 lacks the mTORC1-specific components, but includes RICTOR (RAPTOR-independent companion of mTOR , complex mSIN1 (SAPK interacting protein 1, or MEKK2 interacting protein 1), and PRR5 (protor1/2, protein observed with Rictor 1 and 2). mTORC1 is strongly regulated by nutrient concentration and inhibited by rapamycin , whereas mTORC2 is inhibited variably by rapamycin and is insensitive to nutrient levels.

65. The control of cell growth by the PI-3-kinase–Akt pathway depends in part on a large protein kinase called TOR (named as the target of rapamycin , a bacterial toxin that inactivates the kinase and is used clinically as both an immunosuppressant and anticancer drug). TOR was originally identified in yeasts in genetic screens for rapamycin resistance; in mammalian cells, it is called mTOR , which exists in cells in two functionally distinct multiprotein complexes. mTOR complex 1 contains the protein raptor; this complex is sensitive to rapamycin , and it stimulates cell growth—both by promoting ribosome production and protein synthesis and by inhibiting protein degradation. Complex 1 also promotes both cell growth and cell survival by stimulating nutrient uptake and metabolism. mTOR complex 2 contains the protein rictor and is insensitive to rapamycin ; it helps to activate Akt , and it regulates the actin cytoskeleton via Rho family GTPases .

66. The mTOR in complex 1 integrates inputs from various sources, including extracellular signal proteins referred to as growth factors and nutrients such as amino acids, both of which help activate mTOR and promote cell growth. The growth factors activate mTOR mainly via the PI-3-kinase–Akt pathway. Akt activates mTOR in complex 1 indirectly by phosphorylating , and thereby inhibiting, a GAP called Tsc2. Tsc2 acts on a monomeric Ras -related GTPase called Rheb . Rheb in its active form ( Rheb -GTP) activates mTOR in complex 1. The net result is that Akt activates mTOR and thereby promotes cell growth The mTORC1 complex is activated by Akt whereas mTORC2 is capable of activating AKT.  mTORC2, which can phosphorylate Akt at S473  in vitro  and  in vivo , thereby indicating that mTOR can act as both a substrate and effector of the Akt signaling pathway.

67. AKT regulates glucose and lipid metabolism. Activated AKT2, which is primarily expressed in insulin-responsive tissues, promotes translation of glucose transporter 4 (GLUT4). And the direct target of AKT is a substrate of 160 kDa (AS160), also known as TBC1D1  In intracellular compartments, AKT converts glucose to glucose 6-phosphate by stimulating hexokinase . AKT regulates two processes by glycolysis Glucose 6-phosphate and glycogen synthase kinase 3 (GSK3) to produce cellular energy via glycolysis and promotes glycogen production by inhibiting . FoxO proteins, particularly FoxO1, are the main target of AKT and affect energy homeostasis throughout body . FoxO1 and peroxisome proliferator -activated receptor- coactivator 1 α ( PGC1 α) coordinately regulate gene expression to increase gluconeogenesis and fatty acid oxidatio . On the other hand, FoxO1 induces the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase gene (G6PC), subsequently increases gluconeogenesis . AKT directly inhibits FoxO1, reducing glucose levels , FoxO1 simultaneously activates AKT to increase energy production and inhibits mTOR complex 1 (mTORC1) to reduce lipid and protein production . Finally, GSK3 inhibits glycogen synthase (GS), which promotes glycogen synthesis. AKT exerts an inhibitory effect on GSK3 by phosphorylation of GSK3 . AKT regulates lipid metabolism through sterol regulatory element-binding proteins (SREBP), which increases cholesterol and fatty acid accumulation, including SREBP-1c, SREBP-1a, and SREBP-2. Therefore, PI3K/AKT regulates glucose metabolism through FoxO1 and GSK-3 and lipid metabolism through mTORC1 and SREBP.

68. Forkhead box O (FOXO) ; transcription factors Regulate expression of target genes involved in DNA damage repair, apoptosis, metabolism, cellular proliferation, stress tolerance, and longevity. FOXO1 stimulates the synthesis of gluconeogenic enzymes and suppresses the synthesis of the enzymes of glycolysis , the HMP shunt, and TAG synthesis.

69. Ras MAP Kinase pathway The  Ras / MAPK pathway   regulates; cell differentiation, cell division, cell proliferation, inflammation, cell stress response, metabolism, and apoptosis. The pathway relays extracellular signals from the plasma membrane through the cytoplasm and into the nucleus. The MAPK pathway is involved in mediating long term effects of insulin- Growth and mitogenesis Players involved; Grb2; Growth factor receptor-bound protein 2- Adaptor Sos ; Son of Sevenless - GEF Ras ; GTPase switch proteins Raf ; a serine/ threonine kinase ; MAPKKK MEK, serine/tyrosine/ threonine kinase ; MAPKK ERK; serine/ threonine kinase ; MAPK

70. Ras Ras Ras superfamily of small GTPases help relay signals from RTKs The monomeric Ras protein belongs to the GTPase superfamily of intracellular switch proteins . Ras proteins are “switch” proteins that alternate between an active “on” state with a bound GTP and an inactive “off” state with a bound GDP’ Ras activation is accelerated by a guanine nucleotide exchange factor (GEF), which binds to the Ras∙GDP complex, causing dissociation of the bound GDP This kinase cascade culminates in activation of certain members of the MAP kinase family, which can translocate into the nucleus and phosphorylate many different proteins.

71. Recruitment of the Grb2 adaptor protein to PT residues of IRS via SH2 domains. (Grb2 brings IRS and Sos together) Grb2 adaptor, via its SH3 domain binds to proline -rich region of Sos .

72. STEPS Ligand induced dimerization and autophosphorylation of cytosolic domain of INS-R Phosphorylation of IRS by active INS-R Recruitment of the Grb2 adaptor IRS via SH2 domains. Grb2 binds to Sos via SH3 domains. When bound to Grb2, Sos acts as a GEF, replaces bound GDP with GTP on Ras . Active Ras -GTP recruits the protein Raf to the membrane, where it is activated. Raf-1 phosphorylates MEK on two Ser residues, activating it. MEK phosphorylates ERK on a Thr and a Tyr residue, activating it. Once activated, MAPK undergoes nuclear translocation, where it phosphorylates transcription factors.

73. Ras promotes the formation of a signal transduction complex, containing three sequentially acting protein kinases , at the cytosolic surface of the plasma membrane. Ras can exist in either the GTP-bound (active) or GDP-bound ( inactive) conformation . When GTP binds, Ras can activate a protein kinase , Raf-1 the first of three protein kinases—Raf-1, MEK, and ERK—that form a cascade in which each kinase activates the next by phosphorylation . The protein kinases MEK and ERK are activated by phosphorylation of both a Thr and a Tyr residue. When activated, ERK mediates some of the biological effects of insulin by entering the nucleus and phosphorylating transcription factors, such as Elk1, that modulate the transcription of about 100 insulin-regulated gene .

74. The proteins Raf-1, MEK, and ERK are members of three larger families, for which several nomenclatures are employed. ERK is in the MAPK family ( mitogen activated protein kinases ; mitogens are extracellular signals that induce mitosis and cell division). Kinases in the MAPK and MAPKKK families are specific for Ser or Thr residues, and MAPKKs (here, MEK) phosphorylate both a Ser and a Tyr residue in their substrate, a MAPK (here, ERK). The target of phosphorylation is often another protein kinase , which then phosphorylates a third protein kinase , and so on. The result is a cascade of reactions that amplifies the initial signal by many orders of magnitude

75. Regulation of Insulin signaling IR signaling is regulated in a variety of ways. Two protein tyrosine phosphatases ( PTPases ) dephosphorylate the IR, terminating insulin action without degrading the IR. Cell-surface IR density is regulated by addition from the Golgi apparatus and insulin-stimulated IR endocytosis . IR serine phosphorylation also contributes to the negative regulation of IR signaling.

76. Diabetes Mellitus The word ‘ diabetes ’ -derived from a Greek word, meaning to siphon and refers to the marked loss of water by urination, polyuria . The word ‘ mellitus’ derived from the Latin means sweet and thus diabetes mellitus is known as sweet urine disease. Group of metabolic diseases characterized by increased levels of glucose in the blood (hyperglycemia) resulting from defects in insulin secretion, insulin action, or both

77. Diabetes mellitus is classified into four broad categories: Type 1 Type 2 Gestational diabetes and Impaired glucose tolerance and pre diabetes Person has high blood sugar. This high blood sugar produces the classical symptoms of Glycosuria , Polyuria (frequent urination), Polydipsia (increased thirst) and Polyphagia (increased hunger).

78. Diabetic tissue damage includes ‘ Microvascular complications’ (e.g. Neuropathy, Retinopathy and nephropathy), ‘ Macrovascular complications’ (CHD, CVD, PVD, stroke and renal artery stenosis ) and Complications

79. Insulinoma Adenoma of islets of langerhans Increased insulin secretion Features :neuropsychiatric symptoms, nervousness, confusion and hypoglycemic attacks Increased Insulin Level

80. Insulin shock High level of insulin. Fall in blood glucose level. CNS depression. 50-70 mg/dl CNS excitability 20-50 mg/dl CONVULSION & COMA < 20 mg/dl COMA

81. What we have learnt…

82. References Adult and Pediatric Endocrinology, Volume 2- Larry Jameson, 7 th edition Biochemistry- U. Satyanarayana Human Endocrinology, Paul R. Gard Diabetes Mellitus: A Fundamental and Clinical Text- Derek LeRoith , Simeon I. Taylor, Jerrold M. Olefsky Principles of Biochemistry –Albert Lehninger , 7 th edition Principles of Mammalian Biochemistry- Abraham White, Emil Smith, Philip Handler. 7 th edition Slideshare

83. Thank You