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
  (Sutherlands 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 Stewarts 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
Type I Type II
Age of onset lt20 gt30
Ketosis Common Rare
Body weight Non-obese Obese
Prevalence 0.5 5-6
Islet cell antibodies 65-85 lt10
Insulin Rx Necessary Usually not required
Complications Frequent Frequent
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 lt 110 mg/dL diabetes gt 126 mg/dL
• 2-hour serum glucose after 75 g of glucose PO
• lt140 mg/dL diabetes gt 200 mg/dL
• Hemoglobin A1c
• Normal lt6 diabetes gt7 (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
  a-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-bisphos
  phatase
• 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
Fig 7-11

• Glucagon increases cAMP and PKA activity
• PKA increases Frc 2,6 BPase activity and
  decreases Frc 2,6 BP
• Glycolysis decreased, gluconeogenesis increased

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!!!