Study Guide

1. Study Guide • How do hormones regulate adenylyl cyclase activity? PLC activity? • Describe the mechanism of regulation of PKA by cAMP • Contrast diabetes mellitus type I and type II • Describe the architecture of insulin and the insulin receptor • How does insulin activate the Raf-MEK-ERK pathway? • How does glucagon produce hyperglycemia? • How does one treat diabetic hypoglycemia?

2. How Do Hormones Regulate cAMP levels and PLC Activity? • Seven transmembrane segment receptors that interact with G-proteins • G-protein: GTPase activity • Gs stimulates adenylyl cyclase • Gi inhibits adenylyl cyclase • Gq activates phospholipase C (PLC) • Leads to generation of two messengers • Diacylglycerol, activates PKC • Inositol 1,4,5 trisphosphate, releases Ca2+ from intracellular stores in the ER

3. G-Protein Cycle (Fig. 19-10)

4. Regulation of Adenylyl Cyclase (Fig. 19-11) Gs activates adenylyl cyclase (12) Gi inhibits adenylyl cyclase

5. Cyclic AMP Metabolism Revisited (Fig. 10-13)

6. How Does Glucagon Lead to an Acute Rise in Blood Glucose? • Earl W. Sutherland, Jr. asked how does epinephrine injection in dog lead to hyperglycemia? • Epinephrine in dogs uses the beta adrenergic receptor and the cAMP second messenger system (Sutherland’s system) • Epinephrine in rats, mice, and humans works via the alpha receptor and not by the cAMP protein kinase A cascade • In liver, glucagon activates its receptor, Gs, and adenylyl cyclase to increase cAMP and activate PKA; glucagon in humans works the same as epinephrine in the dog • This leads to a cascade that activates glycogen phosphorylase • This leads to the inhibition of glycogen synthase • Review Daniel Stewart’s presentation on 11 February 2004

7. The Protein Kinase Reaction • ATP + protein  phosphoprotein + ADP • PKA is a serine/threonine kinase • It is a broad specificity enzyme with many substrates

8. Fig 10-8: Overview of Glycogen Metabolism

9. Regulation of Glycogen Metabolism (Fig. 10-14) cAMP activates PKA; this illustrates the actions of PKA

10. Phospholipase C and Inositol (Fig. 19-13)

11. Diabetes Mellitus • A relative or absolute deficiency of insulin • Chronic hyperglycemia and disturbances of carbohydrate, lipid, and protein metabolism • Incidence • 16 Million Americans aged 20 years and older and the incidence is increasing • 60-70 patients per thousand dental patients; 50% are not diagnosed • Increases with obesity • Polydipsia, polyphagia, polyuria is the classic triad; understand the mechanisms • Hyperglycemia leads to polyuria as glucose transport maximum is exceeded • Polyuria leads to polydipsia • Loss of energy (calories) leads to excessive food intake, or polyphagia • Type I: insulin-dependent, juvenile, immunologic destruction of the beta cells of the islets of Langerhans; 10% • Type II: Adult onset; 90%

12. Comparison of Type I and II Diabetes Mellitus

13. Metabolic Disorders Associated with Type II Diabetes • Hyperglycemia • Dyslipidemia • Elevated triglycerides • Decreased HDL (Good Cholesterol)

14. Diabetes Mellitus: Complications • Retinopathy • Vision changes • Most common cause of blindness in the US • Nephropathy (renal failure) • Neuropathy • Sensory, loss of sensation in hands, feet, legs • Autonomic • Change in cardiac rate, rhythm, conduction • Impotence • Accelerated cardiovascular disease and atherosclerosis • Peripheral vascular disease (amputations) • Coronary artery disease • Stroke • Hypertension • Dental complications • Alterations in wound healing • Increased incidence of infections • Xerostomia • Increased incidence of oral candidiasis (controversial)

15. Diabetes and Periodontal Health • Risk factor for prevalence and severity of gingivitis and periodontitis • Altered host defense secondary to diabetes may contribute • Increased collagen breakdown owing to increased collagenase production • Not only does diabetes promote periodontal disease, but periodontal disease can make the diabetes more difficult to control (any inflammatory flare up can increase insulin requirement) • Possible findings in an undiagnosed diabetic • Severe, progressive periodontitis • Enlarged gingiva that bleed easily when manipulated • Multiple periodontal abscesses

16. Abscesses in Diabetes

17. Periodontitis in Diabetes

18. What do I do with a patient suspected of having diabetes? • Ask whether the patient has experienced polydipsia, polyphagia, polyuria • Probably will be negative, but you have to ask • This classical triad is associated with type I diabetes more often than type II diabetes • Symptoms for type II diabetes include lethargy and fatigue • Recent weight loss (paradoxical in an obese person) • Family history, i.e., a parent or sibling with diabetes • Refer to your sister-in-law, the internist • Diagnosis • Fasting blood glucose • Normal < 110 mg/dL; diabetes > 126 mg/dL • 2-hour serum glucose after 75 g of glucose PO • <140 mg/dL; diabetes > 200 mg/dL • Hemoglobin A1c • Normal <6%; diabetes >7% (usually 10-15%) • Glucosuria; this was noted by Dr. Thomas Willis (of the circle of Willis) • The urine of the diabetic patient….the spirits of honey

19. Formation of Hb A1c (Fig. 7-5)

20. Insulin • 51 residues • Two chains • 3 Disulfide bonds • What happens when you remove Asn21? • Produced in which cells of the pancreas? • Hyperglycemia  increased secretion • First protein to be sequenced: Fred Sanger

21. Insulin Receptor Protein-Tyrosine Kinase • Insulin stimulates glucose uptake in muscle and fat, glycogen synthesis, lipogenesis, and protein synthesis, and insulin inhibits lipolysis, proteolysis, and glycogenolysis • Insulin receptor undergoes autophosphorylation and phosphorylates IRS1-4 (Insulin receptor substrates 1-4), PI3 kinase binding protein, and Shc • Expressed in almost all cells, but at much higher levels in liver, fat, and muscle • Insulin does not increase glucose transport into the liver

22. Protein-Tyrosine Kinase (PTK) Cascades • Initial step represents the activation of a PTK • The enzyme is not active as a monomer; it must dimerize • There is transphosphorylation: A phosphorylates A’, and A’ phosphorylates A to achieve activation • These phosphotyrosines can function as docking sites • Attraction of proteins to the docking sites can be regulatory • The PTK may phosphorylate other proteins that can serve as docking sites, or they may activate or inhibit activity

23. Insulin Receptor • It is a protein-tyrosine kinase • It autophosphorylates itself and insulin substrates • The resulting phosphotyrosines serve as docking proteins that attract Grb2 and Shc • These attract Sos, a GEF, and Ras to start the signal transduction cascade

24. Insulin Receptor Architecture • Insulin binds to the N-terminal half of the α-subunit • Human autoantibodies recognize 450-601 • Y965, Y972 yields sites for PTB (phosphotyrosine binding) domains that are found in IRS1-4 and Shc • After IRS binds to pY972, it can be phosphorylated • pY1334 binds SH2 domains of p85 regulatory subunit of PI3 kinase

25. Ras GTP-Cycle (Fig. 20-3) • Ras is a GTPase • It is on one pathway for insulin action • It is on many other pathways that lead to cell growth and division • Ras is frequently mutated in cancer (25% of all human cancers)

26. Grb2, Sos, and Ras • pY of IRS binds SH2 of Grb2 • SH3 of Grb2 binds to Sos (son of sevenless, a GEF) • Sos mediates the exchange

27. Ras-Raf-MEK-ERK Overview • Raf-Mek-ERK is associated with cell growth and cell division • MEK is a dual specificity kinase • However, it can lead to apoptosis • The final result depends upon the conditions, or context • It is not clearly understood • SOS = GEF

28. Docking Sites and Activation

29. Insulin Receptor and PI3 Kinase

30. The PI-3 Kinase Pathway • Activated allosterically by binding to protein-tyrosine phosphate • Catalyzes the phosphorylation of PIP2 to form PIP3 • PIP3 activates phosphoinositide-dependent protein kinase (PDK) allosterically • PDK phosphorylates S6K, PKB (AKT), and PKC • PKB phosphorylates glycogen synthase kinase 3 (GSK3)

31. PI3 Kinase Cascade and Insulin

32. Phosphoprotein Phosphatase-1 • Insulin stimulates glycogenesis in muscle, but epinephrine stimulates glycogenolysis • Glycogenolyis (breakdown) is associated with phosphorylation (the cascade) • Glycogenesis (build up) is associated with dephosphorylation • Insulin promotes the dephosphorylation of glycogen synthase and phosphorylase • These reactions are catalyzed by the catalytic subunit of PPase-1 • Insulin leads to the phosphorylation and activation of PPase-1 • Epinephrine leads to the phosphorylation and inactivation of PPase-1

33. Phosphoprotein Phosphatase-1 (Fig. 20-5)

34. Diabetes: the Glucagon/Insulin Ratio • Glucagon • Produced by the alpha cells of the islets of Langerhans • Early preparations of “insulin” produced hyperglycemia followed by hypoglycemia • The hyperglycemic factor represented contamination • This factor was purified, characterized, and re-named glucagon • It produces hyperglycemia by at least three mechanisms • It promotes glycogen breakdown as noted above • It inhibits glycolysis and increases gluconeogenesis • cAMP activates PKA, which phosphorylates fructose-6-phosphate-2-kinase/fructose-2,6-bisphosphatase • This decreases [fructose-2,6-bisphosphate] • This removes a stimulant of glycolysis at the PFK step • This removes an inhibitor of gluconeogenesis at the fructose-1,6-bisphosphatase step • PKA promotes transcription of PEP carboxykinase, an important enzyme in gluconeogenesis • The high ratio of glucagon/insulin action promotes hyperglycemia

35. Regulation of [Fructose 2,6-BP] • Glucagon increases cAMP and PKA activity • PKA increases Frc 2,6 BPase activity and decreases [Frc 2,6 BP] • Glycolysis decreased, gluconeogenesis increased Fig 7-11

36. Reciprocal Regulation of Glycolysis and Gluconeogenesis (Fig. 25-2)

37. Insulin Action • Stimulates glucose transport into muscle, adipose tissue, and many other cells EXCEPT liver • This results from the recruitment of GLUT4 (of GLUT1-GLUT7) • Glucose transporters contains 12 transmembrane segments • Mechanism of recruitment is unclear • It does not rely on new transporter synthesis • GLUT4 associated with internal membranes fuses with the plasma membrane • Insulin promotes glycogen synthesis by inducing the production of glycogen synthase

38. Glucose Transporter with 12 TM Segments

39. GLUT Recyling

40. Diabetic Hypoglycemia • One of the five most common dental emergencies • Usually due to inadequate food intake • Ask every person receiving insulin whether they have eaten prior to Rx • If the answer is no, provide food before providing Rx • Characterized by confusion, agitation, anxiety, hostility (the previous four can be described as “acting weird”), dizziness, tachycardia, sweating, tremor • Severe: loss of consciousness • Make presumptive Dx of hypoglycemia • Rx • If conscious, give 15 g oral carbohydrate; 4-6 oz fruit juice or soda; hard candy; usually respond in a few minutes • If unable to take food by mouth, give 50% glucose IV (LSUHSC SOD) • If unable to take food by mouth, give 1 mg glucagon sq or im (This is not standard practice here.)

41. Angiotensin System • Renin, a proteolytic enzyme, is released from the juxtaglomerular (JG) cells of the kidney and converts angiotensinogen to angiotensin I • Angiotensin converting enzyme (ACE) catalyses the conversion of angiotensin I to angiotensin II • Angiotensin II is a potent vasoconstrictor and promotes the formation of aldosterone (increases Na+ reabsorption)

42. Angiotensin Metabolism

43. ACE Inhibitors • These compounds decrease peripheral vasoconstriction and decrease aldosterone synthesis • This class of drugs are widely used in the Rx of hypertension

44. Lipophilic First Messengers

45. Lipophilic Hormones • These hormones can diffuse through plasma and nuclear membranes • The intracellular receptors , which constitute the nuclear-receptor superfamily, function as transcription activators when bound to ligand • Receptor architecture • C-terminal variable segment • Middle DNA binding region with a C4 zinc finger segment • N-terminal hormone (ligand) binding domain • In some receptors, this domain functions as a repression domain in the absence of ligand

46. Lipophilic Hormones • The DNA binding sites, or response elements have been determined • Inverted repeats bind symmetric receptor homodimers: GRE, ERE • These are found in the cytoplasm in the absence of ligand bound to Hsp90 (heat shock protein of MW 90 kDa) • Binding of hormone releases the Hsp and allows nuclear translocation • After translocation and binding to its HRE, it activates transcription by interacting with chromatin-remodeling and histone acetylase complexes • Direct repeats bind with heterodimers with a common receptor called RXR: VDRE, TRE, RARE • The vitamin D3 response element is bound by the RXR-VDR heterodimer • Heterodimers are located exclusively in the nucleus • These repress transcription in the absence of ligand • They direct histone deacetylation at nearby nucleosomes • In the liganded state they direct hyperacetylation

47. Steroid Receptor Superfamily

48. Steroid Hormone Action

49. Hormone Response Elements (HREs)

50. The End Biochemistry is fun!!!