Hormone Actions and Insulin Receptors By: Netee Papneja, PGY5

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Hormone Actions and Insulin ReceptorsBy
Netee Papneja, PGY5

• Netee Papneja
• PGY 5, Endocrinology

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Objectives

• Introduction to hormones
• Definition
• Classification
• Structure
• Synthesis and release
• Function
• Hormone receptors
• Classification
• Brief overview of each class
• Insulin and insulin receptors
• General information
• Mechanism of action

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• What is a hormone?
• A hormone (from Greek ''??µ?'' - "impetus") is a
  chemical released by one or more cells that
  affects cells in other parts of the organism.
• a chemical messenger that transports a signal
  from one cell to another
• Only a small amount of hormone is required to
  alter cell metabolism

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• Endocrine hormones - secreted (released) directly
  into the bloodstream
• Exocrine hormones - secreted directly into a
  duct, and from the duct they either flow into the
  bloodstream or they flow from cell to cell by
  diffusion in a process known as paracrine
  signalling.

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Levels at which hormone actions are considered.
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Classification by Structure
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• The physiologic functions of hormones can be
  divided into three general areas
• Growth and differentiation
• Multiple hormones and nutritional factors
• Reproduction
• The stages of reproduction include
• Sex determination during fetal development
• Sexual maturation during puberty
• Conception, pregnancy, lactation, and
  child-rearing
• Cessation of reproductive capability at menopause

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• Maintenance of homeostasis
• T4 controls about 25 of basal metabolism in most
  tissues
• Cortisol exerts a permissive action for many
  hormones in addition to its own direct effects
• PTH regulates calcium and phosphorus levels
• Vasopressin regulates serum osmolality by
  controlling renal free water clearance
• Mineralocorticoids control vascular volume and
  serum electrolyte (Na, K) concentrations
• Insulin maintains euglycemia in the fed and
  fasted states

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Communication system

• Optimal coordination and communication between
  organ systems is required to sustain homeostasis
• A complete communication system needs 
• A cell that produces the signaling molecule (the
  hormone, which is sometimes called the ligand)
• A target cell with a specific receptor that can
  bind the signal with high affinity and produce a
  desired effect. 

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• Receptors
• molecules that hormones bind to in order to exert
  their effects
• Characteristics of receptors
• Proteins or glycoproteins
• Able to distinguish their hormone from other
  molecules that may have very similar structures

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Characteristics of receptors

• Bind to the hormone, or ligand, even at
  exceedingly low concentrations
• Undergo a conformational change when bound to the
  hormone
• Catalyze biochemical events or transmit changes
  in molecular conformation to adjacent molecules
  that produce a biochemical change

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General Classification

• Membrane receptors
• Nuclear receptors

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Nuclear Receptors

• Change the degree of gene expression
• Can be located in the cytoplasm or in the nucleus
  
• Steroid receptors
• In the cytoplasm
• steroid diffuses through the membrane and binds
  to its receptor
• It then dissociates from proteins and
  translocates to the nucleus, where another
  steroid-receptor complex binds to it to form a
  dimer of steroid-receptor complexes, which
  exposes the DNA-binding site and, at this point,
  becomes active.
• Thyroid hormone receptors
• Are already in the nucleus and bound to the
  target genes
• There are inactivating thyroid binding proteins
  that dissociate once the hormone has bound,
  allowing the hormone-receptor complexes to cause
  changes in gene expression

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Membrane Receptors

• Activated through the binding of peptide hormones
  and catecholamines
• The ligand (hormone), or first messenger, binds
  to its receptor and causes activation of a second
  messenger system, which is mediated with
  intracellular signalling molecules (through
  phosphorylation reactions)

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• Membrane receptors can be classified according to
  the molecular mechanisms by which they accomplish
  their signaling function 
• Ligand-gated ion channels (e.g., nicotinic
  acetylcholine receptor)
• Receptor tyrosine kinases (e.g., receptors for
  insulin and insulin-like growth factor I IGF-I)
• Receptor serine/threonine kinases (e.g.,
  receptors for activins and inhibins)
• G proteincoupled receptors (e.g., receptors for
  adrenergic agents, muscarinic cholinergic agents,
  glycoprotein hormones, glucagon, and parathyroid
  hormone)
• Cytokine receptors (e.g., receptors for growth
  hormone, prolactin, and leptin)

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• Ligand-gated ion channels
• Receptor tyrosine kinases
• Receptor serine/threonine kinases
• Receptor guanylate cyclase
• Bifunctional molecules that can bind hormone as
  well as serve as effectors by functioning either
  as ion channels or as enzymes.

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• G proteincoupled receptors
• Cytokine receptors
• Have the ability to bind the hormone but must
  recruit a separate molecule to catalyze the
  effector function.

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G protein-coupled receptors

• Span the membrane seven times, with the receptor
  extracellularly and regions that activate a G
  protein intracellularly
• Three components a, ß and ? subunits 
• Ligand binding to the receptor results in a
  conformational change that causes the a subunit
  to exchange a GDP molecule for that of a GTP
  molecule
• This causes the GTP-bound a subunit to dissociate
  from the ß? complex and act at an effector
  (usually an enzyme, but sometimes an ion channel
  or other protein).
• The a subunit hydrolyses GTP back to GDP to
  terminate the process.

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• Receptor Tyrosine Kinases
• Have several structural features in common
• an extracellular domain containing the
  ligand-binding site
• a single transmembrane domain
• an intracellular portion that includes the
  tyrosine kinase catalytic domain

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• 100 receptor tyrosine kinases sequenced in the
  human genome
• Classified into 16 subfamilies based on the
  extracellular domain
• Mediate the actions of many ligands
• insulin,
• epidermal growth factor (EGF),
• platelet-derived growth factor (PDGF)
• vascular endothelial cellderived growth factor.

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Receptor activation Role of Dimerization

• Plays a central role in the activation of most
  receptor tyrosine kinases.
• The molecular mechanisms differ from receptor to
  receptor

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• Examples of mechanisms of dimerization
• Ligand has two subunits, each binds to a receptor
  (dimeric ligand)
• Two receptor-binding sites on a single molecule
  of ligand
• Pre-existing receptor dimers, undergo
  conformational change and become activated when
  bound to ligand

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• After dimerization, receptor tyrosine kinases
  undergo conformational change in the kinase
  domain
• Mainly due to autophosphorylation of the
  activation loop
• Ultimately leads to activation of the receptor to
  phosphorylate other proteins

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Termination of signal

• Receptor-Mediated Endocytosis
• Protein Tyrosine Phosphatases
• Serine/Threonine Kinases

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Receptors that Signal through Associated Tyrosine
Kinases

• Members of the cytokine family of receptors
  resemble receptor tyrosine kinases in their
  mechanism of action
• Instead of the tyrosine kinase being intrinsic to
  the receptor, enzymatic activity resides in a
  protein that associates with the cytokine
  receptor.
• Ligand binding to the cytokine receptor activates
  the associated kinase.

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Insulin Receptors and action
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Insulin Discovery and Evolution

• 1980 - von Mering and Minkowski identified
  diabetic phenotype in dogs after pancreatectomy
  -link between pancrease and diabetes
• 1916 -Nicolae Paulescu- developed a pancreatic
  extract which normalizes blood sugar levels when
  injected into dogs. Some claim Paulesco was the
  first to discover insulin.
• 1921 - Frederick G. Banting and student Charles
  Best, with supervision by John MacCleod at UFT,
  extracted insulin from animal pancreases.
• J. B. Collip- extracted a more pure formula of
  insulin from the pancreas of cattle
• 1922 first therapeutic use of regular human
  insulin on 14-year-old Leonard Thompson, Toronto
  General Hospital

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Early containers of insulin from the University
of Toronto
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Frederick Banting's lab at the University of
Toronto
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Frederick Banting, right, and his assistant
Charles Best Frederick G. Banting and John
Macleod are awarded the Nobel Prize in Physiology
or Medicine for their discovery of insulin
treatment for diabetes.
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Evolution

• Since 1992, Insulin therapy has significantly
  evolved
• major improvements in insulin purification,
  production, formulation, regimens, and delivery
  systems

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Insulin Physiology

• Insulin key regulator of glucose, protein, fat
  homeostasis
• 51-amio acid anabolic hormone made of 2 peptide
  chains connected by 2 disulfide chains

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Insulin Biosynthesis

• Pre-proinsulin(PPI) synthesized from the
• Insulin gene

• PPI into the endoplasmic reticulum
• by the signal sequence, where it folds into
• a proper conformation that is stabilized
• by three disulfide bonds (SS)

• The signal sequence is removed
• and proinsulin is further processed in the
• Golgi apparatus, where the C-peptide is
• removed and packaged with insulin
• for secretion

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Proinsulin

• A single chain of 86 amino acids
• Small amount escapes from pancreas uncleaved and
  circulates in serum
• Metabolized in kidney instead of liver therefore
  3 to 4-times the half life of insulin
• 12-20 of circulating insulin that we measure
  in the fasting state
• Has 7-8 of insulins biologic activity

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C-Peptide

• 31 amino acid peptide
• No known biologic activity
• Excreted by kidney
• Half-life 3-4-times that of insulin

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Insulin

• Processed and secreted by pancreatic B cells of
  the islets of Langerhans
• Transported via portal circulation to hepatic
  vein to the liver
• 50 of insulin is removed by a single pass
  through the liver, remainder of it by kidneys
• Circulatory half-life of 3-5 minutes

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Insulin Secretion

• Pancreas secretes about 30 units per day
• Release of insulin occurs at a basal rate and in
  short lived large bursts with glucose load
• Basal insulin secretion occurs during
  fasting/resting states to inhibit hepatic
  glycogenolysis, ketogenesis, and gluconeogenesis
• 40 of total insulin output/24h

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Insulin Secretion

• Bolus insulin occurs when plasma glucose levels
  are gt4.4-5.6mmol/L after meals
• Bolus insulin released in 2 phases
• First phase initial transient surge
• Second phase prolonged steady increase
• Increased levels start 8-10 minutes after eating
  and peaks in 30-45 minutes

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Glucose transport

• A family of specialized glucose-transporter
  (GLUT) proteins carry glucose through the
  membrane into cells
• GLUT-1 enables basal non-insulin-stimulated
  glucose uptake into many cells
• GLUT-2 transports glucose into the beta-cell a
  prerequisite for glucose sensing
• GLUT-3 enables non-insulin-mediated glucose
  uptake into brain neurons and placenta
• GLUT-4 enables much of the peripheral action of
  insulin. It is the channel through which glucose
  is taken up into muscle and adipose tissue cells
  following stimulation of the insulin receptor

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• Glucose into pancreatic cell via GLUT-2
  transporters
• Glucose metabolism generates ATP
• Glucokinase key enzyme, glucose concentration
  dependent, catalyzes the conversion of Glucose to
  G6P
• ATP displaces ADP from open ATP sensitive K
  channels
• Bound ATP causes causes channels to close,
  restricting efflux of K deploarizing the cell
• Depolarlization opens Voltage gated calcium
  channels triggering exocytosis of insulin
  vesicles

INSULIN RELEASE
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Insulin action on a target cell
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Insulin receptor

• Most body cells (hepatocytes, fat, muscle cells)
  have insulin receptors
• Composed of
• Two alpha subunits and two beta subunits linked
  by disulfide bonds
• It is a transmembrane receptor that is activated
  by insulin, IGF-1, IGF-II, and belongs to class
  of tyrosin kinase receptors
• a kinase is a type of enzyme that transfers
  phosphate groups from high-energy donor
  molecules, such as ATP to specific target
  molecules ? phosphorylation.
• Kinase enzymes that specifically phosphorylate
  tyrosine amino acids are termed tyrosine kinases.

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• Mitogenic functions mediated via the
  mitogen-activated protein kinase (MAP kinase)
  pathway.
• Metabolic actions mediatedby phosphatidylinositol
  -3-kinase (PI-3K) pathway

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• PI-3K-signaling pathway is responsible for
• Translocation of GLUT-4 containing vesicles to
  the surface
• Increasing GLUT-4 density on the membrane and
  rate of glucose influx
• Promoting glycogen synthesis via activation of
  glycogen synthase
• Promoting protein synthesis and lipogenesis,
  while inhibiting lipolysis

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Insulin Receptors

• Insulin binds to a subunits of insulin receptor
  tyrosine kinase and causes shape changes ?
  communicated to the intracellular ß subunits and
  cause it to bind ATP and autophosphorylate
• This then allows other intracellular proteins to
  bind to the intracellular domain of the receptor,
  and become phosphorylated and generate their
  actions.
• A cascade of phosphorylations and shape/activity
  changes START  

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• Signal transduction pathways
• IRS (insulin receptor substrates) ? binding sites
  for PI3K (phosphatidylinositol-3-kinase)?
  activates Akt/PKB (Protein Kinase B) and the aPKC
  (Protein Kinase C) cascades

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• Activated Akt/PKB induces glycogen synthesis
  through inhibition of GSK-3 (Glycogen synthase
  kinase 3) protein synthesis via mTOR (mammalian
  target of rapamycin) and gluconeogenesis via
  FOXO-1 (Forkhead box protein O1)

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Glucose uptake

• Insulin stimulates glucose uptake via
  translocation of GLUT4 vesicles to the plasma
  membrane
• Activation of PKB and PKC-? lead to translocation
  of GLUT4 molecules to the cell surface resulting
  in increased glucose uptake

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• Insulin signaling also has growth and mitogenic
  effects, which are mostly mediated by the Akt
  cascade as well as by activation of the Ras/ MAPK
  pathway

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Other actions of Insulin

• insulin signaling inhibits gluconeogenesis in the
  liver, through disruption of CREB/CBP/Torc2
  binding
• promotes fatty acid synthesis through activation
  of SREBP-1C, USF1, and LXR
• A negative feedback signal from Akt/PKB, PKC?,
  p70 S6K, and the MAPK cascades results in serine
  phosphorylation and inactivation of IRS signaling

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Regulators of Insulin Release

• Stimulates release
• glucose, vagal stimulation, sulfoylureas,
  meglitinides
• Amplifies release
• GLP-1, GIP, cholecystokinin, gastrin, secretin,
  beta-adrenergic, arginine, GLP-1 agonists
• Inhibits release
• Catecholamines, somatostatin, diazoxide,
  phenytoin, vinblastine, colchicine

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Insulin receptor

• Down-regulation
• obesity, high carb intake, too much exogenous
  insulin
• Up-regulation
• exercise, fasting
• Cortisol
• decreased insulin binding
• exact mechanism of insulin resistance unknown
• Post-receptor defects
• cause of most clinically relevant insulin
  resistance

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Mutations of the Insulin Receptor

• Rare forms of severe insulin resistance
• Type A syndrome
• Particular form of polycystic ovary syndrome with
  severe hyperandrogenism, acanthosis nigricans,
  and marked insulin resistance
• Leprechaunism (Donohue syndrome)
• Growth retardation, multiple developmental
  defects, lipoatrophy, severely elevated insulin
  levels, and hyperglycemia
• Very early death (or miscarriage) is the norm
• 31 reported cases

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• Rabson-Mendenhall syndrome
• growth retardation, dysmorphisms, lack of
  subcutaneous fat, acanthosis nigricans, enlarged
  genitalia, hirsutism, premature and dysplastic
  dentition, coarse facial features, paradoxical
  fasting hypoglycemia and post-prandial
  hyperglycemia, extreme hyperinsulinemia and
  pineal hyperplasia
• Lipoatrophic diabetes
• lack of subcutaneous fat, high blood sugar, and
  high blood insulin, hyperinsulinemia.

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Improving Insulin Sensitivity

• Weight Loss
• In obese patients, reduces hepatic glucose
  production, insulin resistance and fasting
  hyperinsulinemia
• ? by changing patterns of skeletal muscle
  metabolism of fatty acids and content of fat
  within muscles
• Exercise
• Acute increase in insulin-independent glucose
  transport
• Increase translocation of GLUT4 to cell surface
• Upregulation of insulin receptors

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Glucose Metabolism
Major Metabolic Effects of Insulin Consequences of Insulin Deficiency
Stimulates glucose uptake into muscle and adipose cells Inhibits hepatic glucose production Hyperglycemia? osmotic diuresis and dehydration
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Lipoprotein Metabolism
Major Metabolic Effects of Insulin Consequences of Insulin Deficiency
Inhibits breakdown of triglycerides (lipolysis) in adipose tissue Elevated FFA levels
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Ketone Metabolism
Major Metabolic Effects of Insulin Consequences of Insulin Deficiency
Inhibits ketogenesis Ketogenesis is the process by which ketone bodies are produced as a result of fatty acid breakdown Ketoacidosis
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Protein Metabolism
Major Metabolic Effects of Insulin Consequences of Insulin Deficiency
Stimulates amino acid uptake and protein synthesis Inhibits protein degradation Regulates gene transcription Muscle wasting
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References

• Companion site for Basic Medical Endocrinology,
  4th Edition, by Dr. Goodman
• Basic Medical Endocrinology, Dr Goodman
• Williams Textbook of Endocrinology
• Harrisons Textbook of Medicine
• Henderson, J. J Endocrinol 2005184 5-10   
• Nussey SS Whitehead SA. Endocrinology An
  Integrated Approach 2001
• Melmed S Conn PM (eds) Endocrinology Basic
  Clinical Principles 2nd Edition 2005

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THANK YOU