Hormone Actions and Insulin Receptors

By: Netee Papneja, PGY5

Netee PapnejaPGY 5, Endocrinology

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 ''ὁρμή'' - "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.

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:

1. Growth and differentiation:• Multiple hormones and nutritional factors

2. Reproduction:• The stages of reproduction include:

1. Sex determination during fetal development

2. Sexual maturation during puberty

3. Conception, pregnancy, lactation, and child-rearing

4. Cessation of reproductive capability at menopause

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3. 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

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

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)

• Membrane receptors can be classified according to the molecular mechanisms by which they accomplish their signaling function:

1. Ligand-gated ion channels (e.g., nicotinic acetylcholine receptor)

2. Receptor tyrosine kinases (e.g., receptors for insulin and insulin-like growth factor I [IGF-I])

3. Receptor serine/threonine kinases (e.g., receptors for activins and inhibins)

1. G protein–coupled receptors (e.g., receptors for adrenergic agents, muscarinic cholinergic agents, glycoprotein hormones, glucagon, and parathyroid hormone)

2. 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 protein–coupled 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: α, β and γ subunits • Ligand binding to the receptor results in a conformational

change that causes the α subunit to exchange a GDP molecule for that of a GTP molecule

• This causes the GTP-bound α subunit to dissociate from the βγ complex and act at an effector (usually an enzyme, but sometimes an ion channel or other protein).

• The α subunit hydrolyses GTP back to GDP to terminate the process.

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 cell–derived 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

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

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

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

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

Insulin Biosynthesis

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 insulin’s biologic activity

C-Peptide

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

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

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

Insulin Secretion

• Bolus insulin occurs when plasma glucose levels are >4.4-5.6mmol/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

• 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

Insulin action on a target cell

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.

• Mitogenic functions: mediated via the mitogen-activated protein kinase (MAP kinase) pathway.

• Metabolic actions: mediatedby phosphatidylinositol-3-kinase (PI-3K) pathway

• 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

Insulin Receptors • Insulin binds to α subunits of insulin

receptor tyrosine kinase and causes shape changes communicated to the intracellular β 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

• 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

• 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)

Glucose uptake… • Insulin stimulates glucose

uptake via translocation of GLUT4 vesicles to the plasma membrane

• Activation of PKB and PKC-λ lead to translocation of GLUT4 molecules to the cell surface resulting in increased glucose uptake

• 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

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, PKCζ, p70 S6K, and the MAPK cascades results in serine phosphorylation and inactivation of IRS signaling

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

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

References:

• Companion site for Basic Medical Endocrinology, 4th Edition, by Dr. Goodman

• Basic Medical Endocrinology, Dr Goodman• Williams Textbook of Endocrinology • Harrison’s Textbook of Medicine

• Henderson, J. J Endocrinol 2005;184: 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