Neuroendocrinology Flashcards
Hormones - definition
- highly potent specialised organic molecules which control biological functions by interacting with specific receptors on/in responsive cells
- traditional definition: hormones are synthetised within specialised endocrine glands, secreted into circulation and to act at their target cells
- more recent, broader, definition: hormones are synthetised in tissues, secreted into circulation and to act at their target cells
Hormones - two different principles
• involvement in homeostatic control (e.g. uptake and release of carbohydrates, proteins, fatty acids, electrolytes, water in tissues)
• morphogenetic actions (e.g. control of changes during growth, differentiation, development)
Example of hormone actions
• control of enzyme activity
• control of gene expression
• regulation of transport across membranes
• control of the secretion of other hormones
Classification based on chemical nature/structure
- protein- and peptide hormones
- aminoacid-derivatives
- steroids
- fatty acid derivatives, retinoid acid…
“Classical” definitions
Hormones
• synthesis in endocrine cells
• release into circulation
• act at specific receptors
Neurotransmitters
• synthesis in neurons
• localized in presynapsis
• release triggered by neuronal activity
• act at specific receptors (post- or presynaptic)
BUT NEURONS CAN ALSO HAVE ENDOCRINE ACTIONS
Peptide hormones
• synthesis in a great number and variety of cells, including neurons
• short biological half life
- some are also neurotransmitters
Different ways of secretion
- autocrine action
- paracrine action
- Neuroendocrine action -> neuron secretes peptide hormone into bloodstream
- endocrine action: neurosecretory cell -> hormone -> endocrine tissue -> hormone -> target cell (can inhibit neurosecretory cell via negative feedback)
“Classical” neurotransmitters vs Peptide neurotransmitters (neuropeptides)
“Classical” neurotransmitters
• acetylcholine
• aminoacids
• monoamines
• noradrenaline
• adrenaline
• dopamine
• serotonine (5-HT)
Peptide neurotransmitters (neuropeptides)
• neuropeptide Y (NPY)
• opiods
• hypothalamicreleasinghormones (CRF, TRH, LHRH…)
•…
Neuroendocrinology
• many physiological processes are controlled by both nervous and endocrine systems
• interactions serve to coordinate both systems
• occasionally, the same substances are involved in both systems
Hypothalamus - pituitary - organ systems
• central endocrine system in vertebrates
• controlsmanyphysiologicalprocesses:
- reproduction
- growth
- intermediary metabolism
- metamorphosis
- behaviour
- osmotic balance
- stress reaction
- blood pressure
- immune response
- …
Hormones of the hypothalamus-pituitary system
Hypothalamus
• thyreotropin releasing h. (TRH)
• gonadotropin releasing h. (GnRH, LHRH)
• corticotropin releasing h. (CRH, CRF)
• growth hormone releasing h. (GHRH)
• somatostatin (SS,GHIH)
• PROLACTIN RELEASING H. (PRH)
• prolactin inhibiting H (PIH)
Pituitary, anterior lobe (adenohypophysis)
• adrenocorticotropic h. (ACTH)
• thyroid-stimulating h. (TSH)
• follicle-stimulating h. (FSH)
• luteinizing h. (LH)
• growth h. (GH)
• prolactin (PRL)
pituitary, posterior lobe (neurohypophysis)
• oxytocin
• (arginine) vasopressin, antidiuretic h. (AVP,ADH)
Hypothalamus
• afferent and efferent connections with various brain structures
• control of pituitary hormones
• feedback regulation
• integration of internal and external input
Pituitary (hypophysis)
• best investigated neurosecretory system
• consists of two functional and structural differing parts
neural ectoderm -> neurohypophysis = posterior lobe = eminentia mediana
outer/oral ectoderm (German: epidermal) -> adenohypophysis = anterior lobe
DEVELOPS FROM BOTH NEURAL AND ECTODERMAL TISSUE
Endocrine hypothalamus (magnocellular neurons)
• cell bodies in n. supraopticus (SON) and n. paraventricularis (PVN)
• mainly projecting to the posterior lobe of the pituitary (neurohypophysis)
• contain antidiuretic hormone (ADH) and oxytocin
Neurohypophysis - how does it work?
similar to neurons
• neurons in the hypothalamus (SON und PVN) produce ADH and oxytocin
• peptides are packaged into vesicles
• axonal transport to the posterior lobe of the pituitary
• release from axon terminals
Oxytocin
• induceslabour
• elicits milk ejection reflex
• affects behaviour (social interaction, reproduction, food intake)
Antidiuretic hormone (ADH)
• most import antantidiuretic hormone
• increases water reabsorption in the kidneys
• hereby participates in osmoregulation
• also known as (arginine)-vasopressin (AVP)
Diabetes insipidus (centralis)
• production and/o rrelease of ADH is reduced
• symptoms:
- polyuria->polydipsia
- serum Na+ upregulated, osmolality upregulated
- urine Na+ downregulated, osmolality downregulated
DI renalis (rare): kidneys do not respond to ADH
Hypothalamus - adenohypophysis system
Adenohypophysis is under hypothalamic control
• hypothalamic neurons synthetize releasing and inhibiting factors/hormones
• release factors from axons into capillaries of the pars tuberalis in the pituitary
• capillaries lead to portal veins
• transport via portal veins to adenohypophysis
• control of pituitary hormone release
Endocrine hypothalamus (parvocellular neurons)
• cells mainly contain hypophysiotropic hormones, which control adenohypophysis
• releases hormones into the portal system
• cell bodies are located in PVN and SON (like the magnocellular neurons)
Other neuroendocrine nuclei of the hypothalamus
• anteriorhypothalamus
• N. periventricularis
• N. arcuatus(ARC)
• N. preopticusmedialis
Prolactin-inhibiting hormone = dopamine
- neurotransmitter and (here:) hormone
- inhibition of prolactin secretion
Prolactin (PRL)
• controls the development of mammary glands
• stimulation of milk production
Gonadotropin-releasing hormone (GnRH)
• 10 AA, multiple isoforms
• stimulates luteinizing (LH) and follicle stimulating hormone (FSH) release
• mainly synthetized in n. preopt. and n. arcuatus (ARC)
• aka gonadoliberin, gonadorelin, luteinizing hormone releasing hormone (LHRH )
Luteinizing hormone (LH)
• stimulates testosterone synthesis in Leydig cells (together with FSH) (♂)
• triggers ovulation and development of corpus luteum (♀)
Follicle stimulating hormone (FSH)
• initiates follicular growth (♀)
• sensitizes Leydig-cells for LH (♂)
• stimulates spermatogenesis together with LH (♂)
Growth hormone-releasing hormone (GHRH)
• 44 or 45 AA, is processed into shorter forms
• crucial for activity: AA 1-29
• pulsatile secretion
• stimulates GH-secretion (but also LH and FSH)
• belongs to the VIP-family of peptides
• mainly in ventromedial hypothalamus (VMN) and ARC
• outside CNS: in placenta and duodenum (amongst others)
• stimulation through GABA, Opioids
• inhibition through somatostatin and GH
Somatostatin (SS, GHIH)
• inhibitsGH-secretion
• 14 AA, processed from a prohormone (92 AA)
• synthetized also outside the brain (bowels)
• inhibits TRH-stimulation of TSH
Growth hormone (GH)
• episodic secretion, circadian rhythm
• 191 AA
secretion stimuli:
• youth
• starvation
• stress
• activity
• all energy-expending processes
direct actions:
• hyperglycemic
- promotion of gluconeogenesis
- inhibition of glycogen synthesis in the liver
• lipolytic
• protein-anabolic
- stimulation of aminoacid uptake and protein synthesis in muscle, liver, bone
-> GH facilitates the use of products from intermediary metabolism for growth and sustainment
indirect actions:
Indirect actions through increased production of IGF-I in the liver
• stimulates the growth of bones
• activates mitosis of chondrocytes and proliferation of cartilage
• bones lose sensitivity for IGF-1 during puberty
Growth hormone - diseases
- short stature, normal proportions - growth hormone deficiency during childhood
- gigantism - hypersecretion in children
- acromegaly- hypersecretion in adults; growth of tissue and bones of the face, hands, feet
Thyrotropin-Releasing Hormon (TRH)
• tripeptide, N- und C-terminal modified
• multiple copies in one pro-hormone sequence
• identified in all parts of brain and spinal cord
• stimulates thyrotropin (thyreocyte stimulating hormone (TSH)) secretion
• stimulates also prolactin- and growth hormone secretion
Thyreocyte stimulating hormone (TSH)
• control of thyroid hormone synthesis
Thyroid hormones (T3, Thyroxin (T4)) - actions
regulation of basal cell metabolism via control of ATP-production and utilisation
• hyperglycemic:
• stimulates gluconeogenesis in the liver
• stimulates glycogenolysis in the liver
• inhibits lipogenesis
• essential for normal growth and development, in particular for the nervous system
Corticotropin-Releasing hormon (CRH, CRF)
• 41 AA
• stimulates ACTH secretion
• widely spread in central nervous system (CNS)
• in CNS, existence of CRF-related peptides, e.g. urocortin
ACTH (Corticotropin) - actions
• 39 AAs
• is processed (together with other peptides) from precursor protein proopiomelanocortin (POMC)
• stimulates synthesis of steroids from the adrenal cortex
Glucocorticoid - actions
- increased in stress
- hyperglycemic
- inhibit synthesis of proteins and fat
- inhibit immune system
Cortisol related diseases
Cushing syndrom
• primary cause: excessive CRF secretion
-> ACTH upregulation + cortisol upregulation
• primary cause: excessive ACTH secretion
-> cortisol upregulation, CRF down regulation
• primary cause: excessive cortisol secretion
-> CRF down regulation + ACTH down regulation
primary adrenal cortex insufficiency (Morbus Addison)
• primary cause: reduced cortisol secretion
-> CRF upregulation + ACTH upregulation
secondary adrenal cortex insufficiency
• primary cause: reduced ACTH secretion
-> cortisol down regulation, CRF upregulation
Cortisol feedback
• CRF–release in hypothalamus
• transport to anterior pituitary via portal system
• inanterior pituitary, release of ACTH
• ACTH stimulates cortisol-release in adrenal cortex
• cortisol inhibits ACTH-release in pituitary (short loop feedback)
• cortisolinhibitsCRF-releasein hypothalamus (long loop)
CRF - physiological functions
• HPA-axis
• cardiovascular system
• respiration
• cognitive and locomotor behavior
• food intake
• gastrointestinal system
• reproduction and growth
• immune system
CRF - peripheral distribution
• pituitary (anterior and intermediate lobe)
• adrenal cortex
• lung
• gastrointestinal organs
• skin
• Leydig cells, spermatocytes, ovaries
• endometrium, placenta, amniotic fluid
CRF - centralnervous distribution
• hypothalamus
- hypophysiotropic structures, mainly parvocellular part of the PVN
- but also neurons projecting to brain stem and spinal cord
• cortex
• subcortical structures associated with the regulation of autonomic functions
CRF - Distribution in subcortical structures
• diencephalon
- med. preopt. area (MPOA), DMN, N. ARCTUS (ARC), post. hypothalamus, mamill. nuclei,
med. nuclei of the thalamus
• telencephalon
- CeN and other n. of the AMYGDALA, striaterminalis, substantia innominata, some hippocampal areas…
• brainstem and medulla
- E.g. LOCUS COERULEUS, Kölliker-Fuse nucleus, N. TRACTUS SOLITARIUS, oculomotorius neurons, catecholaminergic cell group A7, n. parabrachialis, raphe nuclei, n. vestibularis medialis, n. paragigantocellularis, periaqueductal grey, dors. vagal complex
• spinal cord
CRF - system
• afferences and efferences (examples)
- efferences from amygdala to hypothalamus
- reciprocal connections between Locus coeruleus (LC) and hypothalamus
-reciprocal connections between amygdala and autonomic centers and brainstem (N. parabrachialis, dorsal n. of the vagus)
• significance
-presences of CRF in brainstem and spinal cord (N. tractus solitarius (NTS), LC …) indicates its role in the regulation of autonomic nervous system activity (respiration, blood pressure etc.)
CRF - types of receptors
• 2 subtypes, 70% homology
• 7 putative transmembrane helices
• 5 putative extracellular glycosylation sites
• multiple phosphorylation sites
• gs-protein coupled -> adenylate-cyclase
• can also interact with other g-proteins
CRF - receptors: central nervous distribution
• dense
- hypothalamus (n. paraventricularis (PVN), n. suprachiasmaticus ((SCN)), bed nucleus of
stria terminalis
• moderate
- cortex
• low
- thalamus, basal ganglia, hippocampus, amygdala, septum, brainstem, spinal cord
CRF - receptor-subtypes
• CRF1–widelydistributed
- several subtypes (CRF1α-h)
- e.g.neuroendocrine regulation of pituitary function
• CRF2α-distinct distribution
- neuroendocrine actions in hypothalamus, regulation of behavior (e.g. food intake) and autonomic nervous system
• CRF2β – peripheral and central nervous (in CNS in non-neuronal structures)
- endothelial actions
• CRF2γ-?
CRF - receptor subtypes: central nervous distribution
• CRF1
- cortex, septum, amygdala
- pituitary (anterior and intermediate lobe)
• CRF2α
- lateral septum, PVN, ventromedial hypothalamus (VMN), n. supraopticus (SON), limbic system, bulbus olfactorius, raphe nuclei, n. tractus solitarius (NTS)
• CRF2β
- plexus chorioideus
- cerebral arterioles
• CRF2γ
- limbic system, cortex
CRF - gastrointestinal effects
- down regulation gastric acid secretion
- down regulation gastric emptying
- down regulation gastrointestinal motility
CRF - cardiovascular effects
- upregulation heart rate
- upregulation blood pressure
- downregulation peripheral resistance
-> uncoupling of the baroreceptor-reflex
action via
- blockade of preganglionic vagal nuclei
- stimulationof preganglionic sympathetic neurons
- ! Independent from HPA!
!only in awake animals - maybe result of general arousal?
CRF - metabolic effects
- upregulation physical activity
- upregulation O2 consumption
- upregulation plasma-glucose
- upregulation glucagon
- mediated via sympathetic nerve
- !independent from HPA!
CRF - behavior
- upregulation locomotor activity - independent from dopamine!
- antigenic effects (participation of LC and amygdala)
- downregulation food and water intake
- alterations of sleep architecture (down regulation slow wave sleep)
- down regulation reproduction
- !independent from HPA!
CRF - pathophysiological significance
• anorexia, bulimia
• depression and anxiety disorders
• Alzheimer’s disease
Pathological DEX/CRF test in depression
• Reduced sensitivity of the glucocorticoid-receptors (GR) lead to increased release of CRF and loss of the suppressing action of dexamethasone at the pituitary
CRF - related peptides
• can possess different/distinct actions
dependent on
- distribution
- receptor affinity
• sauvagin
- philomedusa sauvagii
• urotensin I, urotensin II
- teleosteer
• urocortin, urocortin II
- mammalia
Locus coerulus
• contains the largest number of noradrenergic neurons in the CNS
• function
- attention
- orientation
- stress
• a little comment
- locus coeruleus (latin: ‚dark blue place‘) contains melanin (at least in humans)
Locus coerulus - neuroanatomy
• compact nucleus in the pons
• largest number of noradrenergic perikarya in the CNS
• widespread, mostly ipsilateral efferences, innervating e.g.
- cortex, cerebellum, various nuclei of the hypothalamus, spinal cord and nuclei of the brainstem (involved in autonomic functions)
• noradrenergic afferences of neocortex and hippocampus are exclusively LC-projections
Locus coerulus - anatomy/function
• LC is innervated by CRF-ergic afferences (amongst others), e.g. from n. paragigantocellularis, n. prepositus hypoglossi)
• CRF-axon-terminals possess synapses on dendrites of catecholaminergic neurons
• release of CRF in or in vicinity of the LC activates noradrenergic neurons
• integration of autonomic functions, behaviour and anxiety
Locus coerulus - projections
• afferences
- nucleus praepositus hypoglossi
- nucleus gigantocellularis
• efferences
- brainstem, both mesencephalon and rhombencephalon
- cortex
- cerebellum
- thalamus
- hippocampus
- hypothalamus
- spinal cord
Model systems
• immortalised cell lines
• primary cells
• organotypical tissue culture
• explants (e.g. hypothalamus)
• isolated organs
• intact organism
Cell culture
immortalised cells
• ‚unlimited‘ cultivation
• results might not be applicable to ‚normal‘ cells
primary cell culture
• usually fetal tissue
• enzymatic digestion of tissues, cells become dispersed
• three dimensional structure is lost
• artificial medium
Organotypic tissue culture, explants
organotypic tissue culture
• stable for weeks
• three dimensional structure is kept intact
explants
• stable for hours or days under static conditions or perifusion
• three dimensional structure is kept intact
Designing an animal experiment
Set a hypothesis and design an experiment/experiments to test it
• what parameters need to be measured?
• how will that be done?
- species, strain, genetic modification
- number(statistics!)
- technique(s)
- surgery
- methods to measure parameters of interest
- pharmacological intervention
Performing an animal experiment (here in particular: involving surgery)
• best possible design!!!
• least invasive technique; optimised pain control
• best equipment available for surgery
• decent technical execution
- experience of the experimenter
- habituation of the animals to the experiment
The “3R” by Russel and Burch (1959)
guiding principles for more ethical use of animals in testing
• replacement: methods which avoid or replace the use of animals in research
• reduction: use of methods that enable researchers to obtain comparable levels of information from fewer animals, or to obtain more information from the same number of animals
• refinement: use of methods that alleviate or minimize potential pain, suffering or distress, and enhance animal welfare for the animals used
Selection of the appropriate animal model
• species
- comparability
- availability
- size
- costs
• strain
- ‚healthy‘ or with metabolic/endocrine defect
- ability to learn
- …
• transgenic
- tissue specific or general
- inducible
- …
Manipulations of the endocrine system - Applications, stimulations
• techniques
- microinjections into the central nervous system
- other ways of application (i.v., i.p., s.c., oral, implantation of drug-carriers/dispensers …)
- electrical stimulation of neurons
• substances
- receptor agonists or antagonists (important for physicians: function tests)
- hormone antibodies
- antisense Oligonucleotides
- RNA Interference (RNAi), shRNA
- other drugs
Surgery/other interventions
• examples: lesions, food / water restriction, sleep deprivation, stress, …
Pituitary function test (example): CRF test
• tests whether ACTH (and consequently release) can be induced
• performed to test for anterior pituitary insufficiency
Hypothalamus function test: insulin-hypoglycemia test
• tests whether ACTH (and cortisol), growth hormone and prolactin can be stimulated
Human chorionic gondaotropin (HCG) - bioassay
former pregnancy test:
• HCG in urine of pregnant women induces release of spermatocytes in frogs and ovulation in rabbits
Animal experimental methods
• measurement of metabolic and endocrine parameters
- hormones
- body weight, food- and water intake
- body temperature, heat production
- …
• measurement of cardiovascular and respiratory parameters
- blood pressure
- heart rate
- …
• monitoring of neuronal activity
- microdialysis
- electrophysiology
• behavioral experiments
- motivation
- learning, memory
- anxiety
- locomotion
- …
Direct calorimetry
• measurement of heat dissipation
-> determination of heat production → energy expenditure
with the help of
• Thermonetics Seebeck Envelope Calorimeters(SEC)
• or the ‚PMC‘
Surgery - anesthesia
• sufficient anesthesia and analgesia
• inhalation or injection anesthesia
• not longer than necessary
• in neuroendocrinology usually experiments in awake (freely moving) animals
Microinjection
Guide cannula, Dummy canula, Internal cannula
Microdialysis
• possible in awake (freely moving) animals
• little trauma
• frequent sampling possible
- monitoring of time courses
- reduction of animal numbers
• drug application through microdialysis probe possible
• dialysis of interstitial fluid
• monitoring of neurotransmitter release
• analysis by
- high performance liquid chromatography (HPLC) (e.g. catecholamines)
- immunoassays(e.g.peptides)
Cre/loxP-recombination system
• Cre-Recombinase (cyclization recombination) from bacteriophage P1
• Cre catalyses recombination between two loxP- sequences (locus of X-over P1) (‚flanked by loxP‘ = ‚floxed‘)
• same direction/orientation loxP: excision
• opposite direction/orientation loxP: inversion
Tissue specific knockout
Mouseline 1
• Cre is under the control of a cell/tissue specific promotor Mouseline 2
• target gene is floxed
Crossing mouseline 1 x mouseline 1
-> floxed gene is excised by Cre
Conditional knockout
ligand-inducible Cre-recombinase
• e.g. by fusion of Cre with a modified ligand binding domain for the estrogen receptor; this domain is binding tamoxifen (synthetic anti-estrogen)
• only upon binding of tamoxifen modified estrogen receptors translocate cytoplasm to nucleus
• estrogen receptor associated Cre excises floxed gene
Optogenetics to study the involvement of nesfatin-1 in “liking”
• DA neurons in the VTA will express a light-sensitive protein → the blue light-activated cation channel-rhodopsin-2 (ChR2)
• light stimulation through a laser connected to an implanted optic fiber will determine dopamine release into the Nucleus accumbens
DREADD-system
• receptors which can only become activated by a synthetic substance (Designer Receptor Exclusively Activated by Designer Drugs (DREADD))
• allows activation of non-natural (designed) G-protein receptors (developed by mutagenesis) with a high spacial and temporal resolution
• activation by low molecular weight synthetic, but not natural, ligands (e.g. Clozapine N- oxid (CNO) in designed muscarinergic receptors)
DREADD - example
Expression of a stimulatory DREADD (hM3Dq) in Agouti related protein (Agrp) expressing cells
(hM3Dq depolarises neurons via Gq pathway)
• Agrp-IRES-Cremice
• Cre-recombinase–dependent adeno-associated virus (AAV) with hM3Dq–construct (fusioned with mCherry) (AAV-hM3Dq-mCherry)
• stereotaxic injection of AAV-hM3Dq-mCherry in the ARC of AgRP-Ires-Cre mice
Direct vs indirect calorimetry
direct calorimetry
• determination of dry heat loss
indirect calorimetry
• determination of oxygen consumption of an organism
• calculation of energy expenditure:
energy expenditure = oxygen consumption × caloric equivalent
• caloric equivalent can be calculated from respiratory quotient (RQ) (necessary when food composition is unknown)
IDEALLY, BOTH METHODS ARE USED SIMULTANEOUSLY
Direct calorimetry - advantages and disadvantages
Advantage
• direct und exact method
• no need to make assumptions
• if no heat storage takes place: heat production = metabolic rate
Disadvantage
• elaborate experimental setting, e.g. to keep environmental temperature constant
• equipment is expensive (and for mice and rats not commercially available anymore)
• evaporative heat loss is not measured
Heat loss - different means of “transportation”
• Radiation (R)
dependent on temperature difference between animal and calorimeter and surface characteristics
• Convection(C)
transfer via movement of liquid or gas
• Conduction (K)
by direct contact – of little importance in calorimetric systems, often avoided by construction details
• Evaporation(E)
heat loss by evaporation of water (evaporative heat loss, EHL) (~ 2,43 kJ/g = ~580 cal/g)
• R + C + K = DHL (dry heat loss)
• DHL + EHL = THL (total heat loss)
Determination of metabolic rate in a calorimeter
• if (like usually in a calorimeter) no physical work is taking place (basal metabolic rate), heat production (HP) equals metabolic rate (MR)
HP = MR
• according to the First law of thermodynamics, when no heat is stored: MR = HP = THL
Measuring principles of calorimeters
two general principles
• compensation of the measuring effect
- phase transformation (Lavoisier and Laplace)
- thermoelectric compensation (Peltier elements)
• measuring of a difference in temperature (temporal or spacial)
- heat flow from the calorimeter is conveyed through a solid body
Solid body consists of thermoelements or they are attached/part of it (spacial) (our system)
- temperature change over time (temporal) (tea cup calorimeter)
Thermoelectric effect, thermopile
• a temperature difference between two dissimilar electrical conductors or semiconductors produces a voltage difference at the junctions of the two substances (Seebeck effect)
• a thermopile consists of some/many (thermo)couples of semiconductors or metals with different thermoelectric coefficient
• thermocouples are connected usually in series
Calibration with an electric resistor
R= U / I
P =U * I
-> P = U2 / R
Metabolic energy expenditure and heat loss can be related to
• total body mass
• lean body mass
• a function of body mass (e.g. 3⁄4 power of body mass)
• regression analysis of energy expenditure against body composition
•…
Kleiber’s law
• to eliminate influences of body size variation on energy expenditure, data can be related to the 3⁄4 power of BW
• appropriate for many species incl. rats
• however only, when animals are adult and have a normal body composition (the
assumption is made, that all tissues have an equivalent metabolic demand)
• better, but only feasible when a large number of animals (and experiments) is available: regression analysis of energy expenditure against body composition data
Morbus Cushing
- Core body adiposity (Hyperglycaemic action)
- Diabetes-like metabolism (Hyperglycaemic action)
- Arterial hypertony (Hypertonic action)
- Moon face, central obesity (Hypertonic action)
- Immune suppression (immunosuppressive action)
- Skin atrophies, striae rubrae (Proteolytic & feedback effects)
- Osteoporosis (Proteolytic & feedback effects)
- Muscle fatigue (Proteolytic & feedback effects)
- Depression
Morbus Addison
- Anorexia (Hyperglycaemic action)
- Spontaneous hypoglycaemia (Hyperglycaemic action)
- Hypotonia (Hypertonic action)
- Osteoporosis (Hypertonic action)
- Feaver / autoimmune diseases (immunosuppressive action)
- Skin pigmentation (also of mucosa) (Proteolytic & feedback effects)
- Muscle and joint pain (Proteolytic & feedback effects)
- Fatigue, loss of appetite
Steroids - lipophil oder lipophob?
- are highly lipophilic (Poor solubility in blood / good membrane passage)
Adrenal anatomy
- Capsule
- Cortex – produces steroids
- Medulla – produces catecholamines
Cytochrome P450 oxidoreductase -> Cofactor?
Co-factor (anabolic) - NADPH
The HPA axis
CRH: Corticotropin releasing hormone; corticoliberin
- 41 AA long peptide
- made in the PVN of the hypothalamus (pulsatile circadian and/or stress-induced)
ACTH: Adrenocorticotropic hormone; adrenocorticotropin
- 39 AA long peptide
- Cleavage product of proopiomelanocortin (POMC)
- made in the frontal lobe of the pituitary (pulsatile circadian and/or stress induced)
Cortisol
- binds in blood to transcortin
- acts similar to glucagon and opposite to insulin
- anabolic on the heart (mehr Gluconeogenesis, mehr Glycogenolysis und weniger Glucose uptake)
- catabolic on the muscle (mehr Proteolysis, weniger Glucose uptake)
- catabolic on fat cells (mehr Lypolysis, weniger Glucose uptake)
- increases the addiction risk
- a multi-rythmic hormone
Melatonin
- biosynthesis in the pineal gland (epiphysis)
- “Hormon mit Taktgefühl” -> SCN controls the rythmic secretion
-acute suppression of melatonin levels by lights
Corticotropin-releasing hormone/ CRH (or CRF)
- 41aa long
- Cleavage product of a 196-aa prepro-hormone
- Origin: parvocellular neuroendocrine cells of the PVN
- Target: eminentia mediana
- But also: production in the placenta Target: labor induction at the end of pregnagncy
Thyreotropin-releasing hormone/ TRH (or TRF)
- (pyro)Glu-His-Pro-NH2
- Cleavage product of a 242-aa prepro-hormone
- Origin: medial neurons of the PVN
- Target: anterior pituitary
- But also: production in gastrointestinal system and pancreas
Target: modulation of epithelial transport and ß-cell maintenance
Gonadotropin-releasing hormone/GnRH (or LHRH)
- pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro- Gly-NH2
- Cleavage product of a 92-aa prepro-hormone
- Origin: nucleus preopticus
- Target: eminentia mediana
- But also: production in placenta and gonads Target: regulation of tumor development (mammary, ovary, prostata, endometrium)
The flipflop model of sleep
Wake phase
- Off: VLPO, eVLPO
- On: LC, TMN, Raphe
Sleep phase
- On: VLPO, eVLPO
- Off: LC, TMN, Raphe
Orexin / hypocretin
Orexin A - 33 aa
Orexin B - 28 aa
Targets: Appetite, Thermogenesis, Wakefulness
-> Orexin deficiency causes narcolepsy
-> Orexin acts as a sleep switch
Hormones -> Summary
• Many hormones show multiple rhythms (e.g. cortisol).
• The circadian rhythm center is the SCN.
• Melatonin is SCN- and light-regulated.
• The main hormonal axes show circadian and pulsatile
rhythmicity.
• Sleep/wake centers of the CNS: VLPO, LC and Orexin
neurons.
• Orexin deficiency causes narcolepsy.
Leptin
- in contrast to cortisol a primarily behavior-driven hormone
Symptoms of Hyperthyroidism
→ weight loss
→ hair loss
→ diarrhea
→ heat intolerance
→ tachycardia
→ HAND TREMOR
→ ANXIETY
→ INSOMNIA
Symptoms of Hypothyroidism
→ weight gain
→ dry brittle hair
→ dry skin
→ cold intolerance
→ muscle cramps
→ constipation
→ irregular cycles
→ DEPRESSION
→ MEMORY PROBLEMS
-> Usually all reversible after normalization of thyroid hormone levels.
Target Tissues -> Why are there so many symptoms?
Brain
- Development
- Function
- Motor Skills
- Hearing
- Color Vision
Pituitary
- Prolactin Regulation (Lactation)
- Growth Hormone Synthesis
Cardiovascular
- Heart Rate
- Contractile Force
Metabolism
- Temperature Regulation
- Basal Metabolic Rate
- Fat and Glucosemetabolism
Bone
- Strenght
Congenital Hypothyroidism
• prevalence of 1 in 3600 newborns
• if not detected and treated immediately, mental retarda1on (cretinism)
• reason for newborn screening since the 70s
Endemic Cretinism
• Special case of hypothyroidism: lack of iodine
• often in 3rd world countries
• extremely severe form of cretinism (as mother is also affected)
• severe and irreversible mental retardation
• muscle weakness
• growth problem
• deaf muteness
• around 7 Mio affected each year
• very tragic: could be completely avoided with iodinated table salt
Myxedemic Cretinism
Special case of hypothyroidism: iodine and selenium deficiency 3 deiodinases (selenoenzymes) regulate tissue T3/T4 levels
• iodine and selenium deficiency (Congo & Zaire)
• extremely severe case of cretinism
• selenium deficiency affects also other proteins, involved in redox-reactions (free radicals)
Animal model for Cretinism
• Pax8-/- Knockout: no thyroid gland
• no thyroid hormone after birth
• severely growth retarded
• deaf
• ataxic
• die after three weeks
• all reversible by T4 treatment
TR double KO mice
• TRα1TRβ double KO have enlarged thyroid glands
• clinical phenotype of hypothyroidism incl. growth retardation, despite high levels of T3/T4
• but in contrast to Pax8-/- viable and fertile
• the absence of the hormone is worse than the lack of the receptors!
• reason is the so called aporeceptor activity (apo-TR)
TR aporeceptor activity
basal level (no TRs)
-> activation (TR + T3)
-> suppression (app-TR) -> no hormone
Thyroid hormones -> Summary
• Thyroid hormones act in all tissues, especially the brain.
• The thyroid gland is tightly regulated by the HPT axis, thyroid hormone action is additionally fine-tuned by deiodinases and receptors.
• Adult hyper/hypothyroidism has many symptoms, however most of these normalize after correct treatment.
• Defects during development however can cause severe and irreversible damage (iodine deficiency and cretinism).
• Thyroid hormone are extremely important in fetal development, during the first half of pregnancy they are provided by the mother.
• Maternal hypothyroidism can be damaging for the offspring.
• The effects of thyroid hormone are mediated by 2 receptors, which stimulate or suppress gene expression. Mutations in the receptor lead to thyroid hormone resistance with tissue specific phenotypes.
• The cellular import of thyroid hormone occurs by specific thyroid hormone transporters. Defective transport e.g. in MCT8 can have severe consequences for affected individuals.
Maternal Hypothyroidism
• embryo starts own production of thyroid hormone in second half of pregnancy, before that the mother needs to provide the hormone
• pregnancy puts pressure on the maternal thyroid, as it needs to produce for 2
• if there is iodine deficiency or a preexisting hypothyroid condition (even if clinically not obvious), the thyroid might not be able to cope
• 12.3% of all pregnant women have hypothyroidism, 2.6% hyperthyroidism
• not routinely evaluated, so often undetected
• maternal hypothyroidism can have severe consequences for fetal development
• loss of few IQ points
• associated with autism and epilepsy
• often with vegan mothers that refuse fish or dietary supplements as non natural
T3/T4 Transporter
• passive diffusion through cell membrane = wrong concept!
• MCT8 is a very specific thyroid hormone transporter on
neurons
Allan Herndon Dudley
• X-linked mental retarda1on due to defective MCT8 allele
• no thyroid hormone import into brain possible
Resistance to TH (RTHbeta)
• over 1000 patients to date, mutations all over TRβ
• mutated TRβ does not bind T3 => permanent apo-TR
- high T3/T4, not suppressed TSH
- functional TRα but defective TRβ
- hyperthyroid in TRα tissues (tachycardia, high metabolic rate, high thyroid hormone in brain = ADHS, tremor, insomnia)
- hypothyroid in TRβ tissues (high TSH/TRH, high cholesterol)
- complex phenotype, often misdiagnosed
Resistance to TH (RTHalpha)
• first case in 2012, after long search
• to date over 30 patients identified
• growth retardation, large head & short legs
• cognitive and motoric defects
• severe constipation
All defects resemble hypothyroidism, but are limited to TRα tissues.
Adipokines - defintion, functions
• synthesised and secreted in/by adipose tissue
• many can act auto-, para- and/or endocrine
• many are involved in glucose and/or lipid metabolism in the periphery
• some possess central nervous actions, e.g. in energy homeostasis
Adipokines with known central nervous actions
- Visfatin, FGF21, Leptin, Adiponektin, NUCB2/Nesfatin
What are the central nervous actions?
- food and water intake
- energy expenditure
- blood pressure
Visfatin - CNS and BBB
- mainly synthesized by visceral adipose tissue
• can (most likely) pass the BBB
• also synthesised in brain (dogs)
Visfatin - blood glucose homeostasis
• i.v. administration reduces plasma glucose independently from insulin secretion (also in mice with type 2 diabetes)
• chronic ‘administration’ (visfatin adenovirus) slightly lowered plasma glucose and insulin
• insulin-sensitiser Rosiglitazone (thiazolidindione) (a PPAR𝛾 agonist) increases visfatin mRNA expression (in obese OLETF rats)
• stimulates glucose uptake in adipocyte- and muscle cell culture
• suppresses glucose release from cultured hepatocystes; mediation via insulin receptor (IR)
• activates IR signal transduction, however, does not compete with insulin
Visfatin - secretion
• detected in plasma (humans, mice), although primary amino acid sequence does not comprise a signal sequence
• correlates with fat mass
• also evidenced in nucleus and cytoplasm
• explanation: visfatin is secreted by adipocytes via a highly-regulated non-classical secretory pathway
Visfatin - core body temperature
• visfatin increases the expression of proinflammatory cytokines and enzymes of prostaglandin synthesis (TNFalpha, IL1b, Cox2, mPGES1) in hypothalamus
• indomethazin inhibits Cox (prostaglandin-synthesis enzyme)
-> thermogenesis is mediated via prostaglandins
• reduction of food intake is independent of prostaglandins
FGF 21 - expression / localization
• highly expressed in liver, pancreas and white adipose tissue (WAT)
• not expressed in the CNS
FGF 21 - expression / induction
• induced by PPAR alpha activation in liver
• PPAR alpha is activated by fasting, fatty acids, pharmacological ligands (fibrates)
• induction by cold stress (most likely independent of PPAR alpha)
• overexpression prevents diet induced increase in body weight
• also expressed in WAT and brown adipose tissue (BAT); in WAT, expression is induced by PPAR gamma
FGF 21 - receptor - FGFR1c
• most important receptor: FGFR1c, expressed in many tissues
• co-receptor betaKlotho is needed for binding at the FGFR1c
• betaKlotho expression only in distinct tissues (amongst others in liver, WAT, pancreas, testes) -> tissue specific action
• both FGFR1c and betaKlotho are expressed in hypothalamus
FGF 21 - effects
• effects in CNS and periphery
• CNS effects are hardly investigated
• can pass the BBB
• no expression in CNS
FGF 21 - lipid and glucose metabolism
• induces lipolysis (unequivocal findings), hepatic fatty acid oxidation, ketogenesis
• blocks growth hormone signal transduction
• improves insulin sensitivity and reduces plasma insulin, glucose, triglycerids
FGF 21 - food intake and thermogenesis
• induces PPAR gamma receptor co-activator PGC-1alpha in the liver
PGC1alpha is a transcriptional regulator of metabolism
PGC1alpha participates in TRANSDIFFERENCIATION OF CELLS IN THE WAT INTO BAT(LIKE)
• s.c. application INCREASES THERMOGENESIS (UCP1 upregulation in WAT and BAT), fatty acid oxidation is increased (reduction of respiratory quotient (RQ))
• s.c. administration REDUCES BODYWEIGHT, adipose tissue in particular
• s.c. administration does not affect food intake
• i.c.v. administration INCREASES THERMOGENESIS and FOOD INTAKE, bodyweight and body composition unaffected
• s.c. application INCREASES AGRP AND NPY EXPRESSIONin hypothalamus – is discussed as compensation of increased thermogenesis
• FGF21 SENSITIES FOR TORPOR and reduces physical activity (hungry/underweight animals?)
FGF 21 - a complicated story
• data on direct effects of FGF 21 on WAT lipolysis and BAT thermogenesis are unequivocal
• distinct functions of FGF 21 in obesity and fasting?
• prolonged fasting: FGF 21 -> thermogenesis down regulation
• well nourished condition or obesity: FGF 21 -> thermogenesis upregulation
• pharmacological activation of PPAR (which is physiologically activated e.g. by fasting):
-> induction of hepatic FGF 21
-> body temperature downregulation
Nesfatin 1
• precursor: NUCB2/nesfatin
• synthesized in white adipose tissue (and elsewhere)
• can cross the blood-brain-barrier (BBB)
Nesfatin 1 - CNS - expression
expressed (among other regions) in
• hypothalamus (ARC, PVN, SON, LH)
• limbic system(NAc, amygdala, VTA)
• brainstem (NTS, dorsal motor nucleus of the vagus)
Nesfatin 1 - intracellular localisation
• located in secretory vesicles in perikarya, but not in axon-terminals → dendritic release (autocrine or paracrine action?)
Nesfatin 1 - food intake
• involved in the regulation of food and water intake and also energy expenditure:
• CNS application reduces food intake
• application of nesfatin-1 antibody (CNS) increases food intake
Nucleobindin 2 (NUCB2) - first description
• precursor of nesfatin-1
• first discovered in human acute lymphoblastic leukemia cell line (KM3) and, because of its characteristic features, originally named DNS binding/EF-hand/acidic amino rich region (NEFA)
• NUCB2’s EF hand domains can bind Ca++ and thus, as a golgi resident protein, is implied in Ca++ homeostasis
• ‘rediscovered’ in hypothalamus of rodents
Nesfatin 1 - hypothalamus and more
• some neurons in PVN and ARC respond to nesfatin-1
• increases c-Fos expression in PVN, ARC, SON, locus coereuleus, raphe pallidus, NTS, ventrolateral medulla
Nesfatin 1 - receptor
• nesfatin-1 induces Ca2+ influx – can be blocked by pertussis toxin → G-protein coupled receptor, not identified yet
Nesfatin 1 and the melanocortin system
• in hypothalamus: interaction of NUCB2/nesfatin and melanocortin (MC)-system
- melanocortin receptor (MC) 3/4 blockade abolishes nesfatin-1 induced anorexia and its effects on blood pressure
- i.c.v. administration of α-MSH increases NUCB2/nesfatin expression in PVN
• however: anorexigenic effect of nesfatin-1 is not dependent on direct agonistic activity at the MC-receptors
• MC systems of hypothalamus and brainstem are involved in thermogenesis
Nesfatin 1 and oxytocin
• nesfatin-1 and oxytocin are often/mostly co-localized
• oxytocin reduces food intake
• nesfatin-1 depolarises oxytocinergic neurons
• nesfatin-1 stimulates oxytocin-release from magnocellular and parvocellular PVN neurons, these project to the NTS
• blockade of oxytocin-receptors inhibits nesfatin-1 effects on food intake and blood pressure
• α-MSH induces dendritic release of oxytocin (via a Ca2+-dependent mechanism)
How does nesfatin-1 increase energy expenditure?
Nesfatin-1 recruits the melanocortin system to increase energy expenditure
- Nesfatin 1 increases thermogenesis in brown adipose tissue
- Nesfatin 1 Ab administration into the PVN increases food intake and decreases energy expenditure
Leptin
• identified in 1994 by Zhang et al.
• synthesized in white adipose tissue (and elsewhere)
• can cross the blood-brain-barrier (BBB)
• feedback signal from adipose tissue to brain
• crucially involved in (longterm) bodyweight regulation
- increases energy expenditure
Leptin - animal models
• ob/ob mouse -> mutation in leptin-gene
• db/db mouse, S1138 mouse -> mutation in leptin-receptor gene
• fa/fa (Zucker) rat -> mutation in leptin-receptor gene
ob-gene deficiency in mice (ob/ob)
• characterized by Coleman before mutation was identified
- down regulation body temperature
- down regulation energy expenditure
- upregulation food intake
- downregulation immune system
- downregulation fertility
• Coleman performed parabiosis experiment
Leptin - receptors
• members of class I cytokine receptors
• different isoforms
- Ob-Rb
o longest isoform
o fully functional signaltransduction (all pathways)
o expressed in hypothalamus (e.g. ARC)
- other isoforms (Ob-Ra, c, d)
o differ in length of intracellular domain
o expressed in hypothalamus, chorioid plexus, various peripheral organs
o blood-brain-barrier (BBB) transport protein, but longer forms possess also certain signal transduction capabilities (at least theoretical)
- soluble isoform (Ob-Re)
o shortest isoform, modulates circulating leptin levels
Leptin - central nervous actions
• central nervous administration of leptin reduces body weight
• i.c.v. administration is more potent than peripheral application
Leptin - transport into CNS
• participation of ObR
• however: also involvement of another transport system
• saturable
• temperature dependent
• specific
• serum-leptin is proportional to adipose tissue
• significant correlation between CSF- and serum-leptin
- in lean
- but not in obese humans
o in obese, CSF/serum quotient decrease
• saturation of BBB transport at ~ 25 ng/ml
• higher serum levels do not lead to proportional increase in CSF
Leptin-resistance at the blood-brain-barrier (BBB)
• develops with increasing adipose tissue
-> reduction of leptin transport across the BBB
-> peripheral application of leptin becomes ineffective
• central nervous application remains active
• additionally, centralnervous leptin resistance is discussed
Arcuate nucleus (ARC) -> target area
• central nervous leptin target area
white fat can turn brown
in humans too: brown-adipose- tissue activity as assessed by PET–CT with 18F-FDG
Intranasal application
Intranasal application can circumvent the blood brain barrier
Leptin intranasal application (mouse)
- Leptin i.n. reduces body weight and food intake in lean and diet induced obesity (DIO) animals
-> Leptin treatment doesn’t work in “normal” obesity -> useful to treat lipodystrophy
Melanocortin receptors
• 5 known types of melanocortin receptors
• MC3-5 are expressed in CNS
• MC4 is widely distributed within the CNS (e.g. cortex, thalamus, brainstem, spinal cord, amygdala, hypothalamus)
• within hypothalamus: ARC, PVN, DMN, VMN, MPO……
• distribution indicates an involvement of MC4 in the control of food intake
• MC4-/- -mice develop hyperphagia, hyperinsulinemia, hyperglycemia and obesity
• MC4- and POMC-mutations are associated with obesity in humans
• importance of MC3 for the regulation of food intake and bodyweight is less clear
Melanocortin receptor and POMC gene
Ligands for melanocortin-receptors are (amongst others) products of the proopiomelanocortin (POMC) gene
POMC processing products alpha-MSH and ACTH
• alpha-MSH is (a) physiological ligand of MC3 and MC4
- anorexigenic action
• centralnervous application of ACTH reduces food intake
• ACTH binds to MC4 with similar affinity as alpha-MSH
Agouti-protein (Agp)
• normally only expressed in skin
• acts in skin as MC1-receptor antagonist
-> blocks alpha-MSH-action
• in Ay, Avy, Aiy, Asy –mice, ubiquitous expression, incl. CNS
• acts in CNS at MC3- and MC4-receptors
- expression in hypothalamus (PVN, VMH, DMN and nuclei in medial hypothalamus)
-> blocks alpha-MSH-action
• induces
- obesity
- hyperglycemia
- hyperinsulinemia
- increases longitudinal growth
• expressed physiologically in the ARC of the hypothalamus
• acts as an antagonist for alpha-MSH at MC3- and MC4-receptors
• downregulated in well fed animals
• increased expression as consequence of low leptin
Neuropeptide Y (NPY)
• belongs to the pancreatic polypeptide (PP) family
• potently stimulates food intake and decreases energy expenditure via interaction with the noradrenergic system
• important for food intake:
- NPY Y1 receptor
- NPY Y5 receptor
• NPY-synthesizing neurons are located e.g. in
- brainstem (e.g. LC)
- hypothalamus (ARC, DMN)
- in the ARC, NPY is colocalized with AgRP
• hypothalamic NPY-neurons are involved in the regulation of food intake
- NPY inhibits BAT thermogenesis via reduced SNS outflow
- NPY increases adipogenesis and decreases lipolysis via reduced sympathetic outflow
Neuropeptide Y administration
- Acute i.c.v. administration of NPY increases food intake
- Chronic PVN administration of NPY increases food intake and promotes body weight gain and adiposity
- NPY in the Arc of WT and NPY-/- mice affects TH hydroxylase expression in PVN and LC
NPY and Nile tilapia
- Food intake suppresses NPYa mRNA expression of the hypothalamus in Nile tilapia
Ghrelin
• hungersignal
• increases pre-prandial (= before a meal)
• synthesized and released in gastrointestinal system
• circulating ghrelin is reduced in obesity
• exists in two forms: acyl- and desacyl ghrelin
• both act via ghrelin receptor 1a
Central nervous application of corticotropin releasing factor (CRF)…
a) possesses antidepressant actions
b) increases blood pressure
c) induces milk ejection
d) induces hypothermia
e) increases food intake
b) increases blood pressure
Acromegaly is caused by:
a) Overproduction of ACTH during childhood
b) Overproduction of growth hormone during childhood
c) Adrenal tumors during adulthood
d) Overproduction of growth hormone during adulthood
e) Adrenal tumors during childhood
d) Overproduction of growth hormone during adulthood
Which of the following statements is correct?
a) ob/ob mice only become obese on feeding with high-fat diet
b) obesity in db/db mice can be treated by leptin administration
c) fa/fa (Zucker) rats have been developed by genetic manipulation
d) db/db mice have been developed by genetic manipulation
e) obesity in ob/ob mice can be treated by leptin administration
e) obesity in ob/ob mice can be treated by leptin administration
Which statement on melatonin is WRONG?
a) Melatonin secretion is independent of the activity pattern
b) The basal secretion of melatonin is age-dependent
c) In shift workers, melatonin secretion is often suppressed
d) The duration of nocturnal melatonin secretion is age-independent
e) Melatonin has anti-oxidative action
b) The basal secretion of melatonin is age-dependent
