Berthold Endocrine Experiment

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Berthold Endocrine Experiment Remove-
replacement-injection
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Objectives
1. Chemical classes of hormones 2. Biosynthesis
of a particular hormone 3.Transport of the
hormones 4.Recognition signaling of the
hormone 5.Functions of the hormones. 6.Degradation
of the hormone.
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Endocrine Methods

• Remove-replacement-injection
• Purification and cloning
• Synthesis and production of hormone
• Test biological activity with pure hormones
• Development of antibodies
• Localization by immunocytochemistry
• Establish assays (RIA)
• Microarray,deep sequencing, proteomics
• Knock-out/Knock down/mutants

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Hormone Types/Functions
Three structural divisions 1) Amines--H2O sol.
(small--AA) catecholamines and thyroid
hormones 2) Steroids--lipid sol. cyclic
hydrocarbon derivatives from cholesterol
3) Peptide/protein -- H2O sol. largest, complex
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Amines

• Hormones derived from tyrosine and tryptophan.
• Include hormones secreted by adrenal medulla,
  thyroid, and pineal glands.

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Thyroid Hormones

• Tyrosine derivatives bound together.
• Contain 4 iodine atoms (T4).
• Contain 3 iodine atoms (T3).
• Small, non-polar molecules.
• Soluble in plasma membranes.

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Steroids

• Lipids derived from cholesterol.
• Are lipophilic hormones.
• Testosterone
• Estradiol
• Cortisol
• Progesterone

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Peptides/Proteins

• Chains of amino acids (lt 100 amino acids in
  length).
• ADH
• Insulin
• Long polypeptides (gt100) bound to one or more
  carbohydrate groups.
• FSH
• LH

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Biosynthesis of Peptides and Protein Hormones
DNA (The gene)
RNA (Primary transcript)
RNA processing
mRNA
translation
Pre-(Pro)-hormone
Proteolysis via signal peptide cleavage
Pro-hormone
Proteolysis via second modification
Glycosylation phosphorylation
Hormone
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Classification of chemical communication
systems 1) Autocrine--secretion that affects the
same cell which the secretion originated Ex
Adrenergic nerve endings 2) Paracrine--secretion
that affects neighboring cells Ex Inflammatory
response 3) Endocrine--a secretion of a chemical
substance that is released into the blood and
affects a distant target
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4) Exocrine--secretion of a substance that is
released onto surface of animal--including
internal structures
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Exocrine Glands/tissues --possess
ducts --salivary glands, intestinal epithelium,
secretory cells in stomach, and secretory cells
of the liver and pancreas Endocrine
Glands/tissues --lack a definite duct --Adrenal
gland, GI tract, heart, kidney, ovary, pancreas,
thyroid, pituitary, placenta, testes, and thymus
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Hypothalamus
Bone
Hypothalamus
Optic chiasm
Connecting stalk (infundibulum)
Anterior lobe of pituitary
(b)
Posterior lobe of pituitary
Posterior pituitary
Anterior pituitary
(a)
Fig. 7-8, p.265
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Anterior and posterior pituitary glands.
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Posterior Pituitary

• Also called the neurohypophysis.
• Formed by down growth of the brain during fetal
  development.
• Is in contact with the infundibulum.
• Nerve fibers extend through the infundibulum.

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Anterior Pituitary

• Master gland (also called adenohypophysis).
• Derived from a pouch of epithelial tissue that
  migrates upward from the mouth.
• Consists of 2 parts
• Pars distalis anterior pituitary.
• Pars tuberalis thin extension in contact with
  the infundibulum.

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Hypothalamic Control of Posterior Pituitary

• Hypothalamus produces
• ADH supraoptic nuclei.
• Oxytocin paraventricular nuclei.
• Hormones transported along the hypothalamo-hypophy
  seal tract.
• Stored in posterior pituitary.
• Release controlled by neuroendocrine reflexes.

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Posterior Pituitary (neurohypophysis)
--releases neurohormones 1) antidiuretic hormone
(vasopressin) 2) oxcytocin
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Larhammar et al, Ann. N.Y. Acad. Sci. 1163
201208 (2009)
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Hypothalamic Control of the Anterior Pituitary

• Hormonal control rather than neural.
• Hypothalamus synthesizes releasing hormones and
  inhibiting hormones.
• Hormones are transported to axon endings of
  median eminence.
• Delivers blood and hormones to anterior pituitary
  via portal system.

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Hypothalamic Control of the Anterior Pituitary

• Hormones secreted into the hypothalamo-hypophyseal
  portal system regulate the secretions of the
  anterior pituitary.

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Feedback Control of the Anterior Pituitary

• Anterior pituitary and hypothalamic secretions
  are controlled by the target organs they
  regulate.
• Negative feedback inhibition by target gland
  hormones.

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Feedback Control of the Anterior Pituitary

• Negative feedback at 2 levels
• The target gland hormone can act on the
  hypothalamus and inhibit releasing hormones.
• The target gland hormone can act on the anterior
  pituitary and inhibit response to the releasing
  hormone.

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Fig. 7-11, p.269
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Fig. 7-12, p.270
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Water vs. Lipid
Water soluble Lipid
soluble hydrophilic
hydrophobic external (2nd mess.)
internal external receptors
cytoplasmic rec. short half-life
long half-life intermediary resp.
Long-term resp. protein activation
gene activation
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Mechanisms of Hormone Action

• Hormones of same chemical class have similar
  mechanisms of action.
• Location of cellular receptor proteins.
• Target cell must have specific receptors for that
  hormone (specificity).
• Hormones bind to receptors with high bond
  strength (affinity).
• Low capacity of receptors (saturation).

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Hormones That Bind to Nuclear Receptor Proteins

• Lipophilic steroid and thyroid hormones bound to
  plasma carrier proteins.
• Hormones dissociate from carrier proteins to pass
  through lipid component of the target cell
  membrane.
• Receptors for the lipophilic hormones are known
  as nuclear hormone receptors.

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Nuclear Hormone Receptors

• Function within cell to activate genetic
  transcription.
• mRNA directs synthesis of specific enzyme
  proteins that change metabolism.
• Receptor must be activated by binding to hormone
  before binding to specific region of DNA called
  HRE (hormone responsive element).
• Located adjacent to gene that will be transcribed.

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Mechanisms of Steroid Hormone Action

• Steroid receptors located in cytoplasm.
• Bind to steroid hormone.
• Translocates to nucleus.
• DNA-binding domain binds to specific HRE of the
  DNA.
• Dimerization occurs.
• Stimulates transcription.

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Mechanism of Thyroid Hormone Action

• Receptor proteins located in nucleus.
• T3 binds to ligand-binding domain.
• DNA-binding domain can then bind to the half-site
  of the HRE.
• Other half-site is vitamin A derivative
  9-cis-retinoic acid.
• Two partners can bind to the DNA to activate HRE.

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Hormones That Use 2nd Messengers

• Cannot pass through plasma membrane.
• Catecholamines, polypeptides, and glycoproteins
  bind to receptor proteins on the target cell
  membrane.
• Actions are mediated by 2nd messengers
  (signal-transduction mechanisms).
• Extracellular hormones are transduced into
  intracellular second messengers.

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Hormones That Use 2nd Messengers

• 2nd messenger systems
• Adenylate cyclase
• Phospholipase C
• Tyrosine kinase
• NO
• various kinases

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Adenylate Cyclase-cAMP

• Hormone binds to receptor protein.
• Dissociation of a subunit of G-protein.
• G-protein binds and activates adenylate cyclase.
• ATP cAMP PPi
• cAMP attaches to inhibitory subunit of protein
  kinase.

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Adenylate Cyclase-cAMP

• Activates protein kinase.
• Phosphorylates enzymes within the cell to produce
  hormones effects.
• Modulates activity of enzymes present in the
  cell.
• Alters metabolism of the cell.
• cAMP inactivated by phosphodiesterase.
• Hydrolyzes cAMP to inactive fragments.

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Phospholipase-C-Ca

• Binding of epinephrine to alpha-adrenergic
  receptor activates a G-protein, (phospholipase
  C).
• Phospholipase C splits phospholipid into inositol
  triphosphate (IP3) and diacylglycerol (DAG).
• Both derivatives serve as second messengers.

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Phospholipase-C-Ca

• IP3 diffuses through cytoplasm to ER.
• Binding of IP3 to receptor protein in ER causes
  Ca channels to open.
• Ca diffuses into the cytoplasm.
• Ca binds to calmodulin.
• Calmodulin activates specific protein kinase
  enzymes.
• Alters the metabolism of the cell, producing the
  hormones effects.

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Catecholamines TRH LHRH Oxytocin ADH
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Two ways to increase cytosol free Ca 2
1)By release of Ca2 from intracellular Calcium
storages 2)By influx of Calcium from cell
exterior Ca2 channel
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Tyrosine Kinase

• Receptor protein on cell membrane is tyrosine
  kinase.
• Insulin receptor consists of 2 units that
  dimerize when they bind with insulin.
• Insulin binds to ligandbinding site, activating
  enzymatic site.
• Autophosphorylation occurs, increasing tyrosine
  kinase.
• Activates signaling molecules, altering the
  metabolism of the cell.

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GH/PRL signaling mechanism
PRLR alpha
Extra-cellular
Circulation
P
P
Intra-cellular
PI3K
Src
Fyn
MAPK
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OVERVIEW

• Physiological Effects of Hormones
• Reproduction (Gonads)
• Stress Steroids (Adrenal)
• Metabolism (Pancreas and Thyroid)
• Electrolyte/Water Balance

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Gonads and Placenta

• Gonads (testes and ovaries)
• Secrete sex hormones.
• Testosterone.
• Estradiol.
• Progesterone.
• Placenta
• Secretes large amounts of estrogen and
  progesterone.

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Sertoli cells synthesis androgen-binding protein
and inhibin. Leydig cells produce and secrete
testosterone Testosterone key hormone for
produce sperm, develop male sex characters,
protein synthesis and general growth
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s
s
Cell type Ploidy/Chromosomes N Process Time of completion
Oogonium diploid/46 2N Oocytogenesis (mitosis) third trimester
primary Oocyte diploid/46 4N Meiosis I (Folliculogenesis) Dictyate in prophase I until ovulation
secondary Oocyte haploid/23 2N Meiosis II Halted in metaphase II until fertilization
Ovum haploid/23 1N
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1. hypothalamic GnRH control of FSH / LH release,
2. ovarian follicular development to ovulation
and subsequent corpus luteum formation 3. the
feedback control of FSH / LH secretion by ovarian
hormones.
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• the corpus luteum regresses,
• there is a rapid fall in the secretion of
  oestrogen and progesterone,
• the endometrium undergoes shrinkage due to
  extracellular fluid loss,
• the spiral arteries constrict,
• the endometrial blood flow decreases with cell
  death and destruction of blood vessels

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Adrenal Glands

• Paired organs that cap the kidneys.
• Each gland consists of an outer cortex and inner
  medulla.
• Adrenal medulla
• Derived from embryonic neural crest ectoderm
  (sympathetic ganglia).
• Synthesizes and secretes
• Catecholamines (mainly epinephrine but some
  norepinephrine).

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Adrenal Medulla

• Innervated by sympathetic nerve fibers.
• Increase respiratory rate.
• Increase heart rate, cardiac output and
  vasoconstrict blood vessels, thus increasing
  venous return.
• Stimulate glycogenolysis.
• Stimulate lipolysis.

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Adrenal Glands

• Adrenal cortex
• Does not receive neural innervation.
• Must be stimulated hormonally.
• Consists of 3 zones
• Zona glomerulosa
• Aldosterone regulate Na and K balance.
• Zona fasciculata
• Cortisol regulate glucose metabolism.
• Zona reticularis
• Androstenedione and DHEA supplement sex steroids.

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Pancreas

• Endocrine portion consists of islets of
  Langerhans.
• Alpha cells secrete glucagon.
• Stimulus is decrease in plasma glucose
  concentrations.
• Stimulates lipolysis.
• Beta cells secrete insulin.
• Stimulus is increase in plasma glucose
  concentrations.
• Promotes entry of glucose into cells.

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Regulating Blood Glucose
Two major hormones 1) insulin 2)
glucagon Originb cells, a cells-- islet of
Langerhans Control BG high--insulin, BG
low--glucagon
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Diabetes Mellitus
Two types 1) Type I (IDDM) --severe, insulin
dependant, juvenile onset, --loss of beta cell
mass 2) Type II (NIDDM) --less severe,
non-insulin-dependant, adult onset, more
common --defective insulin receptors untreated?f
at metabolism(ketones)? ketoacidosis ketoacidosis
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Thyroid Hormones

• Thyroid gland located just below the larynx.
• Thyroid is the largest of the pure endocrine
  glands.
• Follicular cells secrete thyroxine.
• Parafollicular cells secrete calcitonin.

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Production of Thyroid Hormones

• I- (iodide) actively transported into the
  follicle and secreted into the colloid.
• Oxidized to (Io) iodine.
• Iodine attached to tyrosine.
• Attachment of 1 iodine produces monoiodotyrosine
  (MIT).
• Attachment of 2 iodines produces diiodotyrosine
  (DIT).
• MIT and DIT or 2 DIT molecules coupled.

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Production of Thyroid Hormones

• T3 and T4 produced.
• TSH stimulates pinocytosis into the follicular
  cell.
• Enzymes hydrolyze to T3 and T4 from
  thyroglobulin.
• Attached to thyroid-binding protein and released
  into blood.

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T3 Effects

• Stimulates cellular respiration by
• Production of uncoupling proteins.
• Stimulate active transport Na/ K pumps.
• Lower cellular ATP.
• Increases metabolic heat.
• Increases metabolic rate.
• Stimulates increased consumption of glucose,
  fatty acids and other molecules.

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Hormonal Regulation H2O, Electrolytes
Major organs kidney, intestine, and
bone Antidiuretic hormone (ADH) also
vasopressin, regulates H2O turnover in kidney --
? premeability of H2O in duct (?
urine) Aldosterone ? Na reabsorption, ? blood
osmolarity Atrial natriuretic peptide (ANP) ?
Na , (? urine)
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