L13-26: Cell signalling Flashcards
1
Q
How much of the genome accounts for signalling molecules?
A
10-15%
2
Q
What are the difficulties of targeting ABC transporters?
A
Physiological roles of ABC transporters
Ubiquitous expression
Transporter redundancy
Dose adjustment
Dose adjustment and monitoring when combining inhibitors with drugs with a narrow therapeutic window
3
Q
What are the basic features of cell communication?
A
Secreting cell
↓synthesis and release
Chemical signal
↓
Target cell
↓Reception
Secretion, metabolism, contraction, cell growth and excitability etc.
4
Q
What are the general signal processing pathway steps?
A
Chemical signal
↓
Receptor
↓
Transducer
↓
Amplifier
↓
2nd Messenger
↓
Effectors
↓
Response element
↓
Response
5
Q
What are the types of chemical signal in a processing pathway?
A
Pheromones, hormones, local hormones, neurotransmitters and cell surface molecules
6
Q
What are the different types of a receptors in processing pathways?
A
Ion channel-linked, G-protein linked, tyrosine kinase-linked
7
Q
What are the components of a transducer in a processing pathway?
A
G-proteins, non-receptor tyrosine kinases
8
Q
What are the components of an amplifier in a processing pathway?
A
Adenylyl cyclase, PLC
9
Q
What are the components of a 2nd messenger in a processing pathway?
A
Cyclic AMP, InsP3, IP3, Ca2+, DAG, proteins
10
Q
What are the types of effectors in a processing pathway?
A
Protein kinases, Ca2+-binding proteins,
11
Q
What are the types of response elements in processing pathways?
A
Enzymes, ion channels and TFs
12
Q
What are the types of a response in a processing pathway?
A
Metabolism, secretion, contraction, excitability, gene transcription and cell growth
13
Q
What are the basic principles of signal processing?
A
Amplification
Heterogeneity (diversity, multiple forms of components)
Information transducer (decoding)
Dynamics (temporal and spatial aspects)
14
Q
How does heterogeneity work?
A
Different G-proteins generate different responses
15
Q
What is an example of information transfer via conformational change?
A
Chemical signal to receptor
Receptor to G-protein
G-protein to amplifier
2nd messenger to protein kinase
16
Q
What is an example of covalent modification?
A
Phosphorylation (addition of terminal PO34- of ATP to OH group)
17
Q
Where are most common residues phosphorylated?
A
Serine, Threonine residues
18
Q
What does phosphorylation do to a compound?
A
Change activity or function
19
Q
How do cAMP dependent protein kinases work?
A
cAMP binds to PKA inducing conformational change
Causing release and activation of catalytic subunit
20
Q
What are the differences between type I and II PKAs?
A
Type I: Free in cytosol
Type II: forms stable interaction with AKAPs via R (not free in cytosol) catalytic subunits are not released after
21
Q
What are neurotransmitters?
A
Chemical messengers
Released from one neurone acting at a close site on another eliciting an effect determined by the specific nature of the receptor
22
Q
What are neurotransmitters mostly?
A
Amino acids, peptides and monoamines
23
Q
What are the most common neurotransmitters?
A
Acetylcholine - muscles
Glutamate - excitatory, memory and learning
Dopamine - motivation, pleasure (addiction and love)
Serotonin - emotions, wakefulness and temperature regulation
GABA - inhibitory
ATP - neuronal/glial communication, pain regulation
24
Q
What are the differences between ionotropic and metabotropic receptors?
A
Ionotropic: immediate, 1:1, ligand:channel, uses a single messenger, ionic itself
Meabotropic: Slower, 1:multiple channels, branched, can modulate ion channels, cascades to different functions
25
What is quantal release?
It is the release of neurotransmitter from 1 vesicle meaning they are smaller responses
Release from multiple vesicles means there is a larger response
26
What is the evidence of termination of an action potential from Ach?
Persistent Ach application produces persistent current, receptors can desensitise
Q10 = 2.8, not a diffusion dominated process
Voltage dependence of decay (faster at more negative holding potentials)
27
Who developed the patch clamp technique?
Sakmann and Neher
28
Why was patch clamp technique so important?
Opening and closing of membranes could be visualised
29
What are the properties of nicotinic ACh receptors?
At neuromuscular junction and pre and post synaptic cells in ANS
Permeable to Na+, K+ and Ca2+
5 subunits - 2α, 1β, 1σ and 1ε
α binds to ACh
30
What are the antagonists of nicotinic acetylcholine receptors?
α-bungarotoxin and curare
31
What is Myasthenia Gravis?
Autoimmune disease
Production of antibodies against nicotinic ACh receptors
EPP can't generate muscle stimulation
2nd subset is autoantibodies against muscle specific kinase
32
What type of receptors are GABAa, GABAc and glycine?
Ionotropic receptors
33
What are the GABAa, GABAc and glycine receptors permeable to?
Cl- and HCO3-
34
Why are the GABAa, GABAc and glycine receptors inhibitory?
As the Ecl is close to the Em and below action potential threshold
35
What structure do GABAa, GABAc and glycine receptors have?
Pentameric structures
36
What type of receptor is GABAb?
Metabotropic
37
How are the GABAa, GABAc and glycine receptors diverse?
Different combinations of their subunits
38
What does the subunit composition dictate?
Receptor properties
Cell surface distribution
Dynamic regulation
39
What are properties of δ subunits?
Sensitive, low desensitisation
Mediates tonic GABAergic currents
40
How are synaptic receptors modulated?
Bnzodiazepines
Mediate sedation
Bind at interface of α/γ2 subunits
41
How are extra synaptic receptors modulated?
Barbituates - increase affinity of GABA
Neurosteroids - both positive and negative allosteric modulation
Alcohol - enhances GABA action
42
What is the affect of barbiturates on GABA receptors?
It locks them in an open state (potentiates)
43
What is the affect of benzodiazepines on GABA receptors?
Eases the openings
44
What is the P2X receptor?
An ionotropic receptor using ATP as agonist
Na+, K+ and Ca2+ permeability
Widespread gila and neuronal expression
ATP released in synaptic vesicles
Neuronal-gila and gila-gila communication
45
What is glutamate?
An amino acid excitatory neurotransmitter in vertebrate nervous systems
46
What are the different types of glutamate receptors?
Ionotropic: transient opening allow net influx of cations, generate excitatory current
Metabotropic: Modulate synaptic transmission
47
What is glutamate most important for?
Learning, memory and various disorders
48
What are the 3 main glutamate receptors?
AMPA, NMDA and kainate (man made)
49
How are the main glutamate receptors activated?
Glutamate and kainate
50
Why are kainite receptors different to other glutamate receptors?
They are only activated by kainate
51
What are the properties of glutamate receptors?
Co-localised at synapses (mediate fast chemical synaptic transmission)
NMDARs, AMPARs and kainateRs both synaptic and extra synaptic
Pre and post-synaptic
52
Where are glutamate receptors localised?
At post synaptic sites mainly (found using fluorescent labelling)
53
What is the dominant molecule that travels through NMDARs?
Ca2+
54
What is the dominant molecule that travels through AMPARs?
Na+
55
What is the dominant molecule that travels through KARs?
Na+
56
What are the structural properties of glutamate receptors?
Multimeric protein complexes - 4 subunits (tetramer)
57
What are the structural properties of the glutamate receptor subunits?
3 transmembrane domains
A re-entrant loop
58
What are properties of the AMPAR subunits: GluA1-GluA4?
~900AAs, 68-73% identity
Two modifications (alternative splicing and RNA editing)
Homomeric and heteromeric channels can form
Heteromeric: in presence of edited GluA2 determines I-V curve and Ca2+ permeability
59
What is the I-V curve in AMPAR subunits?
It is inwardly rectifying - pass less outwards current than inward at equivalent distance from the reversal potential
60
What is the flip and flop of glutamate receptors?
It is a formation of the receptor using alternative splicing (weakens the strength)
61
How do the slice variants (flip/flow) affect the receptor kinetics?
Flow terminated by:
Deactivation - agonist unbinding so closing of channel, removal of transmitter
Desensitisation - channel closes while agonist is bound
62
How can rate of desensitisation of glutamate receptors be influenced?
By the subunit composition
63
How can the alternative splicing change receptor function?
They can differ between prenatal to adult form of flips or flops giving sustained or transient currents
64
How does subunit composition impact permeability?
The different arrangements can filter out ions they do not transport
For example
GluA1 - allows Ca2+ transport
GluA3 - allows Ca2+ transport
But GluA1 + GluA2 - filters Ca2+
65
How can editing determine permeability of AMPARs?
Editing Q/R site determines Ca2+ permeability
GluA2 receptors
Glutamine swapped for arginine by editing
Changes charge making it impermeable
66
Where are Ca2+ permeable AMPARs found?
Bergman glial cells, some hippocampus neurones and auditory neurone etc.
67
What can editing of AMPARs mean?
Can cause implications for plasticity and excitotoxicity
68
Where does glutamate bind?
Between S1 and 2 extracellular domains
69
What do auxiliary subunits modulate?
AMPAR trafficking and gating
70
Where are kainate receptors located?
Mostly around presynaptic area of neurone
71
Why are kainate receptors harder to study?
Because they have a lower conductance than other glutamate receptors
72
How could β-amyloid plaques build up in Alzheimers?
Using the NMDA receptors being blocked with Mg2+ so β-amyloid binds to the binding site forming plaques
73
What are the neuronal functions of Ca2+?
Transduces electrical into chemical signals
Axon and dendritic elaboration and retraction
Synaptic vesicle release
Synaptic plasticity
74
Why is Ca2+ used lots?
It is a common ion
East to construct proteins which bind to Ca2+ and change shape
75
What is the importance of Ca2+ transients?
They have different locations and shapes which get interpreted by presence and sensitivity using sensors
76
How can extra-synaptic NMDARs modulate synaptic NMDARs?
By causing Ca2+ entry once NMDARs are activated which activates CREB (TF) which expresses BDNF (Brain-derived neurotrophic factor) and release phosphorylating TrkB which forms a signalling cascade
All of this is turned off using extra-synaptic NMDA activation which (CREB inactivated) causes cell death pathways and loss of mitochondrial potential
77
What is bright-field imaging?
Limited ability to make out intracellular organelles
Impossible to identify individual proteins/processes
Make them stand out
78
What is luminescence?
It is the emission of light by a substance not resulting from heat
79
What are the types of photoluminescence?
Bio: luciferase - firefly tails, aequorin- jellyfish
Chemi: glow-sticks
80
What is phosphorescence?
Slow emission of light that has been previously absorbed by a substance is slow
Light emission after illumination
81
What is fluorescence?
Emission of light by a substance that has absorbed light fast
Light emission only during illumination
82
What is auto-fluorescence?
It is fluorescence that is naturally occurring
83
What is fluorescein?
It is a universal dye used in engine coolant and opticians eye drops
84
What is the Jablonski energy diagram?
Excitation of electrons to higher energy level, the fluorescence emission occurs when the energy levels drop to ground state
85
What are the components of an Epi-Fluorescence microscope?
Eyepiece, observation tubes tube lens, fluorescence filter cube turret, objective, stage, stage, stage translation control, base, field diaphragm, condenser aperture diaphragm, mercury arc lamp lamp house, frame and focus knob
86
What is a dichroic mirror?
It reflects below a certain wavelength
Transmits above it
87
What is the dichroic filter block?
Excitation filter
Dichroic mirror
Emission filter
88
How does a confocal microscope work?
Focal plane has an out/in focus light then through the lens which has a laser pointing towards the pinhole through the dichroic mirror which is then detected
89
How can the smaller image be seen?
Dependent on the the chemical used and the lateral resolving power (d) the wavelength and numerical aperture of the lens can be used
90
What are the 3 major ways to overcome the resolution limit?
Structured illumination (SIM)
Stimulated emission depletion (STED)
Localisation (STOM & PALM)
91
What are the properties of STED super resolution imaging?
Up to 60nm X-Y resolution
Up to 130nm Z resolution
Fixed samples
92
What are the problems with fluorescent microscopy?
Get the probe on target
Only label the target
Overcome any sample autofluorescence
Phototoxicity - live-cell consideration
Photobleaching - dye resistance
93
How can dyes target?
Small-molecule probes:
dye chemistry and antibodies
94
How can fluorescent proteins be used to help overcome problems?
They can be genetically manipulated to target protein to express a fluorescent tag
95
How does dye chemistry help targeting?
Live-cell imaging applications
Gets through the cell membrane
Only become fluorescent in certain environments
Accumulate in certain organelles
Very easy to use and visualise
96
What are the problems of dye chemistry?
Limited retention time in the cell/organelle
Limited targets
Specificity
Toxicity
97
What is used for fixation of label?
formaldehyde
98
What is used for permeabilisation for the label?
Mild detergent
99
What is used for blocking the label?
Excess non-specific protein
100
What is the secondary label of microscopy?
Fluorescent
101
What are the problems with sample labelling?
Small-molecule chemical probes - cannot be fixed sometimes, few specifically target individual proteins
Immunofluorescence techniques - difficult with live cells and required permeabilisation
102
What is the main solution to the fluorescence problems?
Fluorescent proteins
103
How are fluorescent proteins used?
They are genetically manipulated into the DNA which expresses the GFP
104
Why do fluorescent proteins work well?
Because they cover most of the visible spectrum
Monitor events in live cells
105
How are fluorescent proteins visualised?
Using confocal microscopy
106
What are the problems with fluorescent protein microscopy?
Fusion constructs - not native, strong promotors ethane signal, transient transfection (expression level), may perturb protein function
Over-expression artifacts - protein found in unexpected areas
107
What are the problems with live organelle/ protein tracking?
Short exposure times
Bleaching issues
Toxicity
Not known if they are active
108
What is interaction colocalisation?
Used to identify cells/organellse that co-express certain protein and also identify the location of proteins
109
What is FRET imaging?
Forster Resonance Energy Transfer
Measures the interaction and location of interaction of two proteins
Typically one structure is labelled with a donor fluorophore the other an accept fluorophore
110
What are typical FRET dye pairs?
CFP (donor) YFP (acceptor)
Fluorescein (donor) Rhodamine (acceptor)
111
What is the problem with ion imaging?
Photobleaching
Difficult to accurately measure Ca2+ concentration
112
What is ion imaging?
Single excitation - single emission
Only fluoresces when bound to Ca2+
Large increase in fluorescence when bound to Ca2+
113
What is Fura-2 ion imaging?
Dual excitation
Only fluoresces when bound
Large increase in fluorescence when bound
Ratiometric - easy to correct
Can be used to accurately measure Ca2+
114
What is GCaMP -genetic Ca2+ indicator ion imaging?
Based on GFP, calmodulin, M13
Single excitation single emission
Only fluoresces when bound
Large increase when bound
115
What is the main name of the Ca2+ signalling pathway?
The phosphoinositide pathway
116
What are the main molecules that triggers Ca2+ response?
Hormones
Growth factors
Neurotransmitters
117
What are the different responses from intracellular Ca2+ signalling?
Ion permeability
Secretion
Contraction
Metabolism
Fertilisation
DNA synthesis
Development
118
Why is Ca2+ kept low in cells?
Can cause cell death if too high
119
How is intracellular Ca2+ kept low in cells?
Ca-ATPase pumps (Plasma membrane Ca2+ ATPase and Sarco-endoplasmic reticulum Ca2+ ATPase)
Na/Ca exchanger
Mitochondria + other organelles
Proteins (+lipid)
120
What is the approximate concentration of Ca2+ in and outside of cells?
Inside ~100nM
Outside ~ 2-3mM
121
How do Ca2+ OFF mechanisms work?
They operate over a range of concentrations
Needed for constant high concentrations
122
What is the Ca2+ signalling toolkit?
Intracellular Ca2+ increases occur when Inositol 1,4,5-triphosphate acts on InsP3 receptors or Ryanodine receptors
Intracellular Ca2+ decreases when OFF mechansims are stimulated like buffering proteins or sensors allow transport out of the cell using SERCA/PMCA
123
What are the 3 types of Ca2+ signal?
Elementary events
Global Ca2+ wave (intracellular)
Global Ca2+ wave (intercellular)
124
What are the steps of the phosphoinositide pathway?
1) Channel signal induces conformational change at GPCR (7 spanning TMDs)
2) 3 cytoplasmic loops of receptor activates G-protein
3) Stimulates an amplifier PLC
4) PLC acts on membrane phospholipid phosphatidyl inositol 4,5-bisphosphate (PIP2) gives DAG and IP3 both are second messengers
5) DAG acts within lipid bilayer stimulates PKC
6) IP3 diffuses into cytosol acts on ion channels releasing Ca2+
7) Ca2+ signal augmented by Ca2+ induced Ca2+ release (CICR)
8) Ca2+ act through protein kinase
9) Ca2+ use specific binding protein to induce muscle contraction
10) Ca2+ act directly on ion channels to influence excitability
125
What is phospholipase C-β?
It is an amplifier
Causes the formation of DAG and IP3 from PIP2(in the membrane)
126
Why is IP3 soluble?
To find receptors in the cell (inositol)
127
How do Ca2+ spikes increase?
With hormone frequency
128
What is the visualisation of Ca2+?
It is in the form of a series of spikes
129
What are Ca2+ waves?
They are formed from Ca2+ promoting more Ca2+ release (CICR) which does not diffuse but aim to promote more release of Ca2+ causing a Ca2+ wave
130
What are the 2 components of Ca2+ signals?
Temporal (spike)
Spatial (wave)
131
How do the waves and spikes of Ca2+ signals impact the cell?
They are both the equivalent of each other:
Wave is spatial correlate of the spike
Spike is the temporal correlate of the wave
132
What would happen if there was no CICR?
Too little Ca2+ release:
Sensor proteins stimulate buffer proteins which bind to 98-99% of them
So the rise is buffered meaning there are no spikes and waves
133
How is too little Ca2+ overcome?
Regenerative Ca2+ signal is used to saturate the buffers and generate waveband spikes
134
How does Ca2+ stop being released?
The CICR halts as it would use too much energy to be constantly pumped out (ATPase pumps)
135
What happens once OFF mechanisms relax?
Another spike is produced
136
How can Ca2+ waves be propagated?
Using InsP3 and Ryanidine receptors
137
What stimulus is used from the membrane ryanidine receptors?
Unknown
138
What is produced stimulate ryanidine receptors directly?
Cyclic adenosine diphosphate ribose
139
Are ryanidine receptors sensitive the IP3?
NO
140
Where are most InsP3 receptors?
Hepatocytes
141
Where are most ryanidine receptors?
Cardiac myocytes
142
How are InsP3 and ryanidine receptors regulated?
In a biphasic manner
High cytosolic Ca2+ inhibits Ca2+ release after CICR
143
What is used to stimulate InsP3 and Ryanidine receptors?
InsP3R: IP3 and Ca2+
RyR: Ca2+ only
Both: ATP binding (NO HYDROLYSIS)
144
How is the Ca2+ spike and wave generated?
Initiation through an agonist producing IP3 releasing Ca2+
Amplification using CICR
145
How is the Ca2+ spike and wave terminated?
Inhibition of CICR stopping Ca2+ release turning OFF mechanisms on
146
What are elementary Ca2+ release events?
Comes and goes without generating a wave which is reduced in space
Called a Ca2+ puff: InsP3R
Also called a Ca2+ spark: RyR
147
What are the spatial and temporal aspects of puff and sparks?
Spatial ~3um
Temporal ~200ms
148
What are the hierarchical organisations of intracellular Ca2+ signalling?
Fundamental event - blip/quark
Elementary event - puff/spark (abortive waves)
Global event - lots of CICR
149
How are elementary release events physiological signals?
Ca2+ release leads to global Ca2+ stimulating contraction in muscle cells
BUT
K+ leaves the cell using Ca2+ activated K+ channel leading to hyper polarisation which relaxes the cell
150
What is the global Ca2+ wave (intercellular)?
It is the spread of a Ca2+ wave through a monolayer of cells
Can occur in endothelial cells lining the windpipe
151
How does the wave take place over the monolayer?
Using gap junctions which allow the movement of IP3 and Ca2+ so the wave can take place
152
What is the phosphorylation cascade?
Ca2+ rises in the cell
↓
Ca2+ calmodulin
↓
Cam kinase II
↓ Phosphorylation
Phosphorylase kinase - P
↓Phosphorylation
Phosphorylase - P
153
How was the phosphorylation cascade found?
Enzyme was attached to beads - chromatography
Activating solution of Ca2+, CaM and ATP
poured down column
Deactivating solution of EGTA pouted down which stops activation of glycogen to glucose
Complex falls apart
154
How can the frequency of Ca2+ spikes be decoded?
It can help show the different amounts of enzyme (Ca.CaMKII) activity
155
What affect do the different isoforms of Ca2+ signalling receptors have?
Different affinities for their activators
156
What is it an example of when forms from the different isoforms are being expressed?
Heterogeneity so different isoforms of different receptors all form different pathways which have different affinities for each molecule
157
What is the cardiac specific calcium signal some?
Et-1R
PLCβ1(form PIP2 and IP2)
L-type: RyR2
SERCA2a (pump)
PV (parvalbumins)
CaM, TnC (troponinC)
158
What is the T-cell specific calcium signal some?
TCR; IL-2R
PLCγ1; PI 3-K
Orai1; IP3R1
SERCA2b
CR (calreticulin)
CAM
159
What are the different steps in the Ca2+ signalling toolkit?
Receptors
Transducers
Channels
Pumps
Buffers
Sensors
160
How can the singalsomes be remodelled?
Phenotypically and genotypically
161
What is phenotypic remodelling of signalsomes?
It is a change from the outside/ external event
e.g. phosphorylation changing activity of a component
162
What is genotypic remodelling?
Mutations that cause activity of components (e.g. CF)
163
When does phenotypic remodelling occur in cells under normal conditions?
Heart - increased force of contraction with exercise (cAMP phosphorylation of Ca2+ signalling components)
Liver - calcium signalling altered during regeneration
164
What are the main diseases from phenotypic and genotypic remodelling?
Phenotypic - Alzheimers disease
Genotypic - Brody's disease
165
How does phenotypic remodelling occur leading to Alzheimers disease?
Extracellular plaque deposits of the β-amyloid peptide disrupt synaptic transmission
β-amyloid oligomers increase Ca2+ entry via the NMDA receptor
Amyloid precursor protein intracellular domain increased Ca2+ release from stores
Abnormal amyloid metabolism results in an up regulation of neuronal Ca2+ signalling to induce an initial decline in memory
166
How does amyloid metabolism lead to more phenotypic remodelling?
AICD (precursor protein transcription factor) used to increase RyR and decrease Calbindin (binds to Ca2+ acting as a buffer) producing more Ca2+ for more processing of prion protein so more amyloid oligomers
167
What is the end result of Alzheimers?
It is an amyloid-dependent up-regulation of Ca2+ signalling
Causes constant erasure of memory stores (LTD) during the day and night as Ca2+ levels are constantly high
168
What are the potential therapies for Alzheimers disease?
Vitamin D3 (Ca2+ off mechanism)
169
How does positive feedback impact Alzheimers?
Bidirectional relationship between Ca2+ signalling and amyloidogenic pathway
Amyloid's increase Ca2+, stimulation of amyloid precursor protein
170
What is Brody disease?
A skeletal muscle genetic disorder
Characterised by stiffness and cramp by prolonged Ca2+ elevation and slowing of relaxation
Mutation is SERCA1
SR unable to refill Ca2+; cytosolic Ca2+ remains elevated
171
How can cancer cells cause remodelling in Ca2+ signalling?
Alter SERCA pump activty
Alter Ca2+ release through InsP3Rs
Alter resting levels Ca2+
172
What is the structure of InsP3R?
It is a tetramer (310kDa subunits)
Half pore complex
N-terminal binding domain
Isoforms from 4 alternatively spliced forms of subunits
173
What is the structure of RyR?
Plant alkaloid
Tetramer (560kDa subunits)
Big protein
4 TMDs line the pore
N-terminal cytosolic domain
174
What are the 3 genes for RyR?
RyR1 - skeletal muscle
RyR2 - cardiac muscle
RyR3 - non-muscle cells
175
What is the cAMP signal pathway?
G-protein coupled receptor
↓
Adenylyl cyclase
↓
cAMP
↓
Protein Kinase A
↓
Response from cell
176
How many types of adenylyl cyclase are there?
2
177
What are the 2 types of adenylyl cyclase?
Type 1: Found in plasma membrane
9 isoforms - activated by Gαs
Activated by plant alkaloid forskolin (by-passes GPCR)
Regulated by cytosolic Ca2+ (airways)
Type 2: Cytosolic enzyme activated increases HCO3- and Ca2+
178
What is the pharmacological agent that activates type 1 adenylyl cyclase?
Forskolin
179
Where are type 2 adenylyl cyclase found?
In sperm and epithelia
180
What is the structure of adenylyl cyclase?
12 TMDs 2NBDs
181
What are the different OFF mechanisms of cAMP?
Inhibit cAMP production
Breakdown cAMP
Remove cAMP from cell
182
How does inhibition of cAMP take place?
Active the inhibitors G-protein, Gαi, reduces adenylyl cyclase activity, opposing stimulation by Gαs lowering cAMP
183
How does breakdown of cAMP take place?
Using phosphodiesterases
184
What are phosphodiesterases?
They are enzymes used to break down cAMP as an OFF mechanism
185
What are the properties of phosphodiesterases?
11 isoforms
8 breakdown cAMP, other cGMP or both
Expression is tissue specific
Important in 'shaping' local cAMP signal (duration, amplification and spatial localisation)
186
How are phosphodiesterases inhibited?
By caffeine and PDE inhibitors used to treat disease symptoms
187
What are the clinical uses of PDE3 inhibitors?
Cilastazol - peripheral vascular disease (+cAMP - causes relaxation of blood vessels which increases blood flow)
Milrinone - used for failing hearts
(+cAMP - increase HR and inotrophy)
188
What are the clinical uses of PDE4 inhibitor?
Roflumilast - used for COPD
(+cAMP - relaxes airway smooth muscle reducing airway obstruction)
189
How is cAMP removed from cells?
ABC transporters actively pump cAMP out of the cell
MRPs also affect duration and aptitude of cAMP signal, plus spatial aspects
190
What is the problem with a linear on and off mechanism of cAMP?
Different agonists increase cAMP levels but produce distinct responses in same cells
Some physiological agonists produce cAMP dependent responses but do not appear to change global cAMP cells
191
What findings arise from cAMP activation and termination not being linear?
cAMP must be highly localised (compartmentalised) to spatially distinct areas inside cells (microdomains), changes in cAMP are agonist specific
192
How is cAMP compartmentalised?
GPCRs localised to different regions of the cell
Restrict diffusion of cAMP from plasma membrane (adenylyl cyclase) to cytosol
Target PKA to distinct sites and substrates in cells
193
What are examples of cAMP signalling being compartmentalised?
CFTR activity in epithelial cells
Isolated heart cells response to different cAMP agonists
194
What do phosphodiesterases restrict from PM to cytosol?
Diffusion
195
What were the phosphodiesterase restricting diffusion CFTR experiments?
CFTR activated by GCPR agonist (adenosine) in polarised airway cells
Cell-attached patch used
Experiment 1 + ADO = lots of activity
Experiment 1 + ADO + 8-STP(antagonist) = less activity
Experiment 2 + ADO to bath = little/no activity
Experiment 2 + ADO + Forskolin = lots of activity
196
What is the effect of inhibiting PDE4?
It eliminates compartmentalised cAMP signalling in human airways
197
What are the AKAP?
A Kinase Anchoring Proteins
198
What is the structures of AKAPs?
Targeting domain
Interaction sites
2 C subunits
PKA holoenzyme
2RII
Docking domain on enzyme
199
How do PKA and AKAP interact?
PKA binds to AKAP via docking domain on RII
Then is targeted to plasma membrane, organelles and sub cellular structures via targeting domain, PKA close to substrates
AKAPs help assemble signalling complexes forming signalling hubs or signalsomes
200
What is the role of AKAPs in activation of CFTR by cAMP?
If PKA is present via AKAP it activates CFTR by cAMP alone (+ATP) and block activation by cAMP by preventing PKAII binding to AKAP
201
What is required for Ezrin (AKAP) to target PKA to CFTR?
NHERF1 as it contains PDZ1 binding domains CFTR binds and ERM domain that binds Ezrin
202
How is cAMP dynamic measured in living cells?
Using genetically encoded fluorescent sensors to study spatiotemporal cAMP dynamics
Use cAMP binding domains from PKA/EPAC attached to fluorescent proteins
Expressed using plasmids
203
What are the 2 experimental approaches to measure cAMP?
FRET - cAMP binding domain, 2 fluorescent proteins undergoing FRET, FRET decreases when cAMP rises
Intesity - cAMP binding domain + fluorescent protein tag, rise in cAMP increases fluorescent intensity
204
Why do 2 different GPCR cAMP agonists cause different functional effects in heart cells?
Noradrenaline causes increased contractility
Prostaglandin(PGE1) causes no effect on contractility
They have different spatiotemporal changes in cAMP
205
How was the effect of the same receptor but different response investigated?
Activation of -adrenergic receptor elevated cAMP to T-tubules and SR
Phosphorylation of key proteins excitation-contraction coupling - LTCCs, phospholamban (SERCA) and RyR2
Activation of prostanoid receptor = phosphorylation of enzymes in metabolism and TFs
Inhibiting PDEs abolished dependent cAMP signalling + PKA phosphorylation
Key roles illustrated
206
How does increased contraction lead to PKA-dependent phosphorylation of Ca2+ signalling components?
Components involved: L-type Ca2+ channels (LTCCs)
Phospholamban
Ryanodine type 2 receptors
Enables:
Larger Ca2+ signal generated, + trigger Ca2+ using LTCCs and Ca2+ store via RyR (increased inotropy)
Removes Ca2+ quicker into stores during diastole enable relaxation (lustropy)
Reload Ca2+ stores better help increase inotropy
207
How does PKA increase LTCC activity?
PKA phosphorylation increases open state probability of individual LTCCs in sarcolemma
PKA also recruits more LTCCs to sarcolemma
208
How does phenotypic remodelling by cAMP/PKA pathway affect the SERCA pump?
PKA phosphorylation and increase Ca2+ causes dissociation of PLB from SERCA
Increased ATP consumption
Increased chronotropy and inotropy
209
What conclusions can be inferred from β-adrenergic stimulation?
Leads to enhanced muscle contraction because cAMP/PKA signalling is restricted to sites and substrates directly involved in excitation-contraction coupling
cAMP and Ca2+ signalling interact to maximise contractile response from cardiac cells
210
How is spatial cAMP signalling disrupted in CF airways?
FRET studies used to find a decrease in Cl- influx in CF so less interaction with proteins in cAMP signalling meaning it is not as localised
211
What are the protein kinases activated by increased Ca2+?
PKC - Ca2+ and/or lipids
Ca2+/Calmodulin-dependent PKs
212
What is the cGMP signalling pathway?
GCPRs NOT INVOLVED
generated guanylyl cyclase (GC)
Soluble and plasma membrane GCs
Binds to protein kinase G to activate (2 isoforms)
PKG phosphorylates serine/threonine residues
cGMP broken down by cGMP-dependent PDEs
213
How is cGMP generated?
Guanylyl cyclase
214
What are the different types of guanylyl cyclases'?
Soluble form - activated by NO
Plasma membrane bound (pGC) - activated by some peptide agonists
215
What are the different treatments using cGMP?
Nitric oxide - activates cGMP in VSM leading to vasodilation and fall in BP - drugs often used to treat angina
Viagra - type 5 cGMP PDE inhibitor; rise in cGMP relaxes smooth muscle in some tissues - treat erection problems and pulmonary hypertension
Heat stable enterotoxin - E.coli in intestine - activates cGMP.PKG phosphorylates and activates CFTR - secretory diarrhoea
LPS (endotoxin) - from gram negative bacteria - increase expression iNOS, causing excessive NO production
216
What are the 3 groups of protein kinase C?
Conventional (cPKC) - α, βI, βII, γ
Novel (nPKC) - δ, ε, θ, η
Atypical (aPKC) - ζ, ι/λ
217
What is the structure of PKC?
Single polypeptide with regulatory and catalytic domains
11 different isoforms, divide into 3 groups (C, N and A)
218
What is the function of PKC?
Require phospholipid binding to be active and Ca2+
R domain has pseudo substrate motif - keeps kinase inactivate by occupy substrate binding site
219
What is the activation cycle for PKC?
Agonist binds
Ca2+ binds C2 domain; PKC translocates to PM and binds DAG and C1 domain
PS motif disengages from C4 domain, substrates bind and phosphorylate
All forms artificially activated by phorbol esters directly bind to PKC
220
What do the 2 main types of Ca2+/Calmodulin-dependent protein kinases do?
Show narrow substrate specificities and those with broad substrate specificities
221
What are the functions of CaMKII?
Regulate activity of NMDA receptors phosphorylate sites on NR2A and NR2B
Enhances InsP3 formation by inhibiting inositol polyphosphate 5-phosphatase
Frequency decoding of Ca2+ signals and act as molecular switch ing learning and memory
Phosphorylate PLB to control SERCA2 pump, work in synergy with PKA to inhibit PLB effect
222
What are protein phosphatases?
Remove phosphate from phosphorylated proteins
4 major classes Ser/Thr:
1, 2A and 2C broad and overlapping substrate specificity
2B (calcineurin) more restricted specificity - requires Ca2+/CaM
Regulated by inhibitory proteins (type I&II)
One major class of tyrosine PPs - cytosolic and membrane bound
223
What are the chemical inhibitors of protein phosphatases?
Okadaic acid (OA) - blocks PP1 and PP2A
Cyclosporin A - specific for calcineurin, clinically for immunosuppresion
224
What is Okadaic acid?
Toxin produced by dinoflagellates accumulate in marine sponges & shellfish
Causes diarrhetic shellfish poisoning
Symptoms: severe, acte diarrhoea, vomiting and nausea
Caused by:
Increase paracellular permeability
More active CFTR, fluid loss
Inhibits PP1
PP2A completely inhibited
225
What is the role of phosphatase inhibitory proteins?
PKA phosphorylates key enzymes leading to breakdown of glycogen
PKA activates PP inhibitory proteins bind and inhibit glycogen breakdown
226
How do different signalling pathways interact?
Synergy between cAMP and Ca2+ in skeletal muscle, key role of phosphorylase kinase
227
How does phosphorylase kinase become active?
Requires PKA phosphorylation and Ca2+ binding in skeletal muscle
δ subunit = calmodulin, γ = catalytic
PKA phosphorylation of α and β subunits increases Ca2+ sensitivity
228
How is calcium sensitivity increased in PKA?
When phosphorylated
229
What are the principles of blood glucose homeostasis?
Blood glucose concentration ~5 mM
Total blood volume ~5 L
Total glucose in blood <5g
Daily intake of glucose ~300g
Glucose concentration rises moderately after meal ~5-7 mM
In disease state can rise to 60 mM
230
What are the 2 regulatory hormones of blood glucose?
Insulin and glucagon
231
What would blood profiles of insulin and glucagon be like after 75g glucose consumed?
Insulin - high so stimulates insulin secretion
Glucagon - initially high but lowers as it is inhibited
232
What is the role of insulin?
Secreted when blood glucose is high and causes it to decrease
233
What is the role of glucagon?
Secreted when blood glucose is low and causes it to increase
234
When insulin increases what processes are initiated?
Muscle
An increase in glucose uptake
Liver
Increase glycogenesis
Decrease gluconeogenesis
235
When glucagon increases what processes are initiated?
Liver
Increased glycogenolysis
Increased gluconeogenesis
236
What are the pancreatic islets?
They are scattered throughout the pancreas
Make up 1-2% of pancreatic tissue
α, β and δ cells secrete hormones
<0.5 mm diameter
237
What are the contents of α, β and δ cells?
α - Glucagon
β - Insulin
δ - Somatostatin
238
What is regulated in insulin release?
Major: Increase glucose
Minor: Increase AAs
Increase neural input
Increase gut hormones
Decrease adrenaline
Decrease somatostatin
239
What is regulated in glucagon release?
Major: Decrease glucose
Decrease insulin
Increase AAs
Minor: Increase neural input
Increase adrenaline
Decrease gut hormones
Increase cortisol
240
What is the sequence of events of insulin production?
1 - Nucleus to ER: insulin gene 3 exons, signal peptide, B-chain, C-peptide and A-chain
2 - ER: Preproinsulin - primary peptide
3 - Immature secretory granules: proinsulin signal peptide cleaved and disulphide bridges formed
4 - Mature secretory granules: Mature insulin C-peptide cleaved
241
What is proinsulin?
A single chain of 86 AAs
242
What happens in insulin production after formation of disulphide bonds?
Residues 31-65 are cleaved forming C-peptide and insulin (51 AAs)
243
How is insulin stored?
As a hexatrimeric form complexed with zinc and released by β cells
244
Why does insulin have a short half life?
To be able to release more
245
What can be a diagnostic value of type 1 diabetes?
C-peptide as it is inactive but stable in the blood stream
246
What is the glucose sensor in insulin secretion?
Glucokinase
247
What happens when little glucose is transported?
Glucokinase is stimulated causing glycolysis and TCA cycle producing little ATP which allows the KATP channel to stay open so hyperpolarised
Ca2+ channel closed
Low insulin secretion
248
What happens when lots of glucose is transported?
Glucokinase stimulated
Glycolysis and TCA
More ATP
K+ stops leaving as channel closes
Membrane depolarised
Ca2+ channel opes so high Ca2+
High insulin secretion
249
What is a key regulator of insulin?
Extracellular glucose concentration
250
What other compounds can regulate insulin secretion?
Increased by gut hormones (incretins) and amino acids
Decreased by adrenaline
251
Which monogenes cause diabetes?
Inactivating glucokinase MODY(young)/PNDM(neonatal)
Activating KCNJ11 PDNM(neonatal)
Activating ABCC8 - SUR1 PDNM(neonatal)
252
Which conversions are most important posprandial(after food)?
Liver: conversion glucose to glycogen and triglyceride
Adipose: glucose to triglyceride
253
What is glycogen?
It is a branched polymer of glucose
Formed by 1,4 and 1,6 glycosidic bonds
Converted in the liver
Made using glycogen synthase (1,4) and branching enzyme (1,6)
Degraded using debranching enzyme (1,6) and glycogen phosphorylase (1,4)
254
What happens to dietary glucose when stores are full?
Forms VLDL (very low density lipoproteins)
255
How are ketones formed?
In a fasting state
Used to provide energy for the brain
256
What is the insulin action on the liver?
Increased glycogen synthesis
Increased fatty acid synthesis
Increased protein synthesis
Decreased glycogen degradation
Decreased gluconeogenesis
257
What is the insulin action on the muscle?
Increased glucose transport (GLUT4)
Increased glycogen synthesis: glycogen synthase(dephosphorylation)
Increased glucose oxidation: pyruvate dehydrogenase (dephosphorylation)
Increased protein synthesis
258
What is the insulin action on adipose tissue?
Increased glucose transport (GLUT4) vesicle to PM
Increased triacylglycerol synthesis (+ acetyl-CoA carboxylase and fatty acid synthase)
Decreased triacylglycerol breakdown release of fatty acids (decrease hormone sensitive lipase (HSL) TAG not broken-down)
259
What is TAG role in adipose tissue when insulin is present?
TAG uptake is stimulated when very low density lipoprotein and chylomicrons are provided creating TAG and lipoprotein lipase digests into fatty acid and monoacylglycerol which is transported making TAG and being stored in adipose
260
Which enzymes are activated or repressed in liver during covalent modification?
Activated: glycogen synthase and Acetyl-CoA carboxylase
Repressed: Phosphorylase
261
Which enzymes are activated or repressed in liver during gene transcription?
Activated: Glucokinase, acetyl-CoA carboxylase and fatty acid synthase
Repressed: Glucose-6-phosphase and PEPCK (phosphoenolpyruvate carboxykinase)
262
How is glycogen synthase stimulated?
Insulin and glucose/G6P produces regulatory protein phospohtases
Dephosphorylated glycogen synthase causes activation
Insulin also represses glycogen synthase kinase
263
How is glycogen synthase repressed?
PKA, PhK, PKC and CAMKK and glycogen synthase kinase which causes ADP to ATP
Phosphorylated glycogen synthase causes repression
264
How is glycogen phosphorylase stimulated?
Ph-kiase is phosphorylated using glucagon (cAMP to PKA) which allows ATP to ADP
Phosphorylates glycogen phosphorylase causing stimulation
265
How is glycogen phosphorylate repressed?
Protein phosphotases produced by insulin and increase in G6P/glucose
Dephosphorylation of glycogen phosphorylase causes repression
266
What are the different protein domains involved in insulin signalling?
PH - Plekstrin homology
PTB - phosphotyrosine binding domain
SH2 - Src Homology 2
SH3 - Src Homology domain
267
What is the PH domain?
Pleckstrin homology domain binds to phosphorylated inositol phospholipid in plasma membrane
268
What is the PTB domain?
Phosphotyrosine binding domain binds P-Y residues
269
What is the SH2 domain?
Src homology domain 2 binds phosphotyrosine surrounded by unique protein sequences
270
What is the SH3 domain?
Src homology domain 3 binds specifically proline rich regions
271
What are the properties of the insulin receptor?
It is part of the RTK superfamily
Heterotetramer
190kDa glycoprotein
ααββ subunits held by disulphide bonds
α subunit - insulin binding
β subunit - transmembrane; tyrosine kinase (cytoplasmic tail) PTB and SH2 domains
272
How does insulin bind to the α subunits?
transphosphorylation of β units
Activation of tyrosine kinase activity
Phosphorylation of IR generates binding sites for proteins with PTB and SH2 domains
273
What is the function of the kinase activation loop on insulin receptor?
Activation of tyrosine kinase
Phosphorylation leads to binding site of SH2 domain
274
What is the function of the juxtamembrane domain on insulin receptor?
Phosphorylation creates binding site for PTB domain
275
What is the function of the C-terminal domain on insulin receptor?
Regulates IR kinase activity and kinase-adaptor interactions
276
What is the function of the Set/Thr residues on insulin receptor?
Inhibits receptor kinase activity
277
What is the Shc-SH2 containing protein?
From 3 genes A,B and C
Shc comprises: PTB, CH1 and SH2 domains
Shc binds insulin receptor through PTB and SH2
CH1 has tyrosine residues and becomes phosphorylated which binds proteins with SH2 domains
Grb-Sos signals through MAP kinase
278
What is the insulin receptor substrate?
Comprises 4 genes: IRS1-4
IRS1 and 2 are essential for insulin biological action in liver, muscle and adipose
IRS contains PH and PTB domains enable binding to phosphorylated insulin receptor
279
What binding domains does IRS contain?
PH and PTB domains
280
What happens to the IR and IRS once binding takes place?
It becomes phosphorylated on tyrosine by insulin receptor tyrosine kinase activity
281
What is the Grb2 protein?
Growth factor receptor bound protein-2
282
What does the Grb2 protein do?
Binds phosphorylated IRS/Shc through SH2 the activates and uncovers the SH3 domains which binds SOS (son of seven less) via SH3 domain
283
What is SOS?
Son of Sevenless
Acts as GDP/DTP exchange factor (GEF)
284
When is Ras active/inactive?
Inactive: Bound to GDP
Active: Exchanged for GTP (using SOS)
285
What happens when Ras-GTP forms?
Activates Raf which activates MAPKK (MEK)
MEK activates MAP-kinase (ERK)
286
What is ERK?
Drives growth, differentiation and proliferation
287
What factor of IRS makes it useful?
IRS1 has 21 potential Y-phosphorylation sites which makes it a docking protein with SH2 domains
288
How many SH2 domains does PI3K have?
2
289
How many SH2 domains does Grb2 have?
1
290
How many S/T-phosphorylation sites does IRS1 have?
30
291
What are the properties of the S/T-phosphorylation sites on IRS1?
They are phosphorylated by either components of insulin signalling or stress kinases which are activated by stress/inflammation
292
How does PI3Kinase pathway lead to AKT activation?
Insulin binds IR α subunit → activates IR tyrosine kinase → autophosphorylation of tyrosine
Recruitment of IRS to IR → phosphorylation of IRS on tyrosines
Recruitment of proteins to IRS via SH2 domains → formation of PIP3
Recruitment of AKT/PTB and PDK1 by PIP3 to PM
Dual phosphorylation of AKT
Phosphorylation of AKT substrates
293
What are the properties of PI3K?
Phosphatidylinositol 3-kinase
2 subunit enzyme, 85 and 110 kDa
p110 - catalytic subunit
p85 - regulatory subunit, 2 SH2 domains and one SH3
294
What is the function of PI3K?
Adds a phosphate group to 3 position of inositol ring in phasphatidylinositol
295
What happens once PI3K add a phosphate group to phosphatidylinositol?
PIP3 forms which remains membrane bound
296
What is PIP3 function in insulin signalling?
Acts as a binding site for PH-domain containing proteins
297
What is a factor about PIP3 during insulin signalling?
It is undetectable in unstimulated cells and produced transiently to high levels during insulin stimulation
298
What is PIP3 pleiotropic functions?
Insulin signalling
Cell proliferation, apoptosis, motility and immune activation
299
How is PIP3 switched off?
Using PTEN (phosphatase and tensin homologue)
Mutations in PTEN associated with cancer and other diseases
300
What is AKT?
Protein kinase B (PKB)
3 genes α, β and γ
301
What are the 3 domains of AKT?
N-terminal PH domains
Central kinase domain (Thr308)
C-terminal hydrophobic regulatory domain
302
How is AKT activated?
By phosphorylation on Thr308 (PDK1) and Ser473 (mTORC2)
303
How does PIP3 activate PDK1 and AKT?
As they both have PH domains
Locates proteins to cytoplasmic side of the membrane
304
Where do PDK1 and mTORC2 phosphorylate AKT?
PDK1 Thr308
mTORC2 Ser473
305
Which substrates does AKT phosphorylate?
AS160 (Rab-GAP) - GLUT4 translocation, glucose transport
TSC2/TSC1 > GAP - cell growth
FOXO1, PGC1α and SIK2 - inhibition of transcription of gluconeogenic enzymes
Glycogen synthase (using GSK-3) - glycogen storage
306
How is insulin stimulation of glut4 using AS160?
Glut4 - main glucose transporter in skeletal muscle and adipose
Absence of insulin means glut4 is present intracellularly in storage vesicles
Phosphorylation of AS160 by AKT causes inactivation sp glut4 translocation to PM
307
What is AS160?
It is a GTPase that negatively regulates translocation by Rab proteins
308
How does AKT cause protein synthesis?
mTORC1 regulates protein synthesis
Regulated by TSC1/2
AKT phosphorylate TSC2 leads to inhibition
Rheb-GTP activates mTORC1
mTORC1 activates eIF4E-BP1(eukaryotic translation initiation factor) and S6K1 leading to translation initiation and ribosome biogenesis
309
What does TSC1/2 complex do?
Inhibits rheb by converting form GTP to GDP bound state
310
What is Rheb?
Ras homologue enriched in brain
311
What is FOXO1?
A transcription factor
Induces G6PC and PEPCK (gluconeogenesis)
Recpreses GCK (glucokinase)
312
What effect does AKT have on FOXO1?
It increases degradation
313
How does AKT degrade FOXO1?
Insulin → phosphorylates FOXO1 → translocation to cytoplasms → degradation by proteasome → no induction of G6PC/PEPCK expression → relieve inhibition of GCK gene → increased GCK expression
314
What is GSK3?
Belongs to CMCG family of proline directed kinases
Downstream of PI3K/AKT
Negatively regulated by Der-phosphorylation
GSK-3 substrates - enzymes, TFs, eIF2B, cytoskeletal proteins and IRS1/2 etc
315
What is AKT effect on GSK3?
Causes inactivation at position 21 (Ser)
316
What is type 2 diabetes?
A polygenic disorder
Defects in insulin action
Defects in glucose-induced insulin secretion
317
Which genes characterise monogenic diabetes?
HNF4-α , HNF1-α and PDX1
318
What are the major diabetic complications?
Retinopathy
Nephropathy (kidney)
Peripheral neuropathy (feeling in feet)
Autonomic neuropathy (CV, gut, urinary)
Macrovascular (heart attack, stroke)
319
What can happen after major diabetic complications?
Decreased life expectancy
Quality of life compromised
320
What was the timeline for discovery of insulin?
1921: inject pancreatectomised dog with pancreatic extracts
1922: First injection of a person with insulin
1923: Commercial production
1923: Nobel prize to Fred Banting & C McLeod (supervisor)
321
How does obesity increase diabetes risk?
Can cause insulin resistance and enlargements of islets
Genetic background still important factor for compensation
Good β-cell capacity compensate
Type 2 develops once β-cells no longer compensate
322
How does obesity drive type 2 diabetes?
Causes insulin resistance
Can lead to robust β-cell or susceptible cell
Either increased insulin secretion or β-cell dysfunction and failure leading to hyperglycaemia
323
What lifestyle changes can people with T2D make?
diet and increased exercise
324
What drugs can be used to treat T2D?
Monotherapy or combination
First: metformin (decrease hepatic glucose production)
325
How can carb absorption be targeted in type 2?
Using α-glucosidase inhibitors
Inhibition conversion of oligosaccharides to glucose (competition)
326
What are the different α-glucosidase inhibitors?
1st gen: Acarbose tetrasaccharide (not absorbed)
2nd gen: Iminosugar (absorbed not metabolised)
Miglitol analogue of 1-deoxynojirimycin
327
What are the benefits and adverse effects of AGIs?
Benefits:
Dec. intestinal glucose absorption
Dec. glycemic index of food
Dec. post-prandial BG
Dec. post-prandial TLGs
No risk hypoglycaemia
Adverse:
Abdominal discomfort, mimics malabsorption
Fermentation of undigested carbs
328
How is renal glucose increased using excretion?
Inhibiting SGLT2 in kidney
Symport glucose and sodium using gradient so stop glucose reabsorption
Allow excretion of glucose in urine
329
Which drugs are used to increase glucose excretion in urine?
Phlorizin (non-selective SGLT1/2)
Lowers BG
Sergiflozin and Dapagiflozin (SGLT2 only)
40-60% inhibition
Lowers BG
Weightloss
330
What are the adverse effects of SGLT2 inhibitors?
Increased urine volume
Risk of UTIs
Risk of genital fungal infections
331
What are sulphonylureas?
Bind SUR1 (receptor)
K+ATP channel
Low glucose = channel open = no insulin secreted
High glucose =channel closed = increased insulin secretion (action of these drugs)
332
What type of drugs target insulin secretion?
Sulphonylureas
GLP-1R agonists
Dipeptidyl peptidase-4 inhibitors
333
What are incretins?
Intestinal peptide produced in response to food that stimulates insulin secretion
334
What is GIP?
Glucose-dependent insulinotropic peptide
Located in K cells (proximal) in gut
335
What is GLP-1?
Glucagon-like peptide-1
Located in L cells (distal) in small intestine
336
What are the different GLP-1 agonists?
Exenatide
Liraglutode
Semaglutide
337
What are the GIP agonists?
Tirzepatide (dual agonist)
338
What is the function of GLP-1 in diabetes?
Less gastric emptying
Less chylomicron production
Less caloric intake
More insulin secretion
Less glucagon secretion
More glucose uptake
Less hepatic glucose production
Less intrahepatic fat
339
What is the function of GIP in diabetes?
Less caloric intake
More increase secretion
More glucagon secretion
More glucose and TG uptake
More TG storage
More glucose uptake
Less hepatic glucose production
340
What is the mechanism of action on GLP1 in β-cells?
More cAMP
More PKA
More cAMP guanine nucleotide exchange factor
More Ca2+
Less KATP channel
More insulin secretion
More PI3K to AKT
Increase gene transcription
Decrease apoptosis
Stimulate cell growth
341
How are incretins degraded?
Using DPP-4 (dipeptidyl peptidase-4)
342
What is Dipeptidyl peptidase-4?
Degrades incretins
2 forms:
Membrane anchored extracellular enzyme
Soluble form retaining catalytic activity
DPP4 inhibitors: gliptins (increase GLP-1/GIP and decrease BG
343
What are the benefits and adverse effects of sulphonylureas, GLP-1R agonists and DPP4 inhibitors?
Sulphonylureas
Benefits:
Dec. BG
Inc. Insulin secretion
Adverse effects:
Inc. Risk hypoglycaemia
Inc. Weight gain
Inc. CV events
GLP-1R agonists and DPP4 inhibitors
Benefits:
Moderate Dec. BG
Inc. insulin secretion
Dec. Glucagon secretion
No risk hypoglycaemia
Dec. food intake and body weight
Adverse effects: Pancretitis/ cancer (disproved)
Gastric discomfort
344
How is adipose tissue expanded in obesity?
Hypertrophy of cells (increase in volume)
Hyperplasia of cells (increase cell number)
345
What is the function of PPARγ's?
They favour adipocyte proliferation and storage which prevents damage by lipids in other organs
346
Which drugs are used to target adipose tissue in T2D?
Thiazolidinediones
347
What is the function of Thiazolidinediones?
Dec. BG
Dec. Blood insulin
Dec. Blood TGs
Inc. insulin sensitivity
348
What are PPARγ's?
Perosisome proliferator activated receptor gamma - nuclear receptor TF promote adipocyte proliferation and differentiation
349
What is the process of PPARγ activation?
Ligand binds
Heterodimer with retinoid acid receptor
Recruitment to PPRE on DNA promotor
Recruitment of coactivator
P300 = histone acetylase
Acetylation of histones exposes chromatin
Increased TXN of PPARγ target genes
350
What are the benefits and adverse effects of Thiazolidinediones?
Benefits:
Chronically lower BG and insulin
Increase insulin sensitivity
Increase body weight
Adverse effects:
Troglitazone withdrawn - liver damage
Rosiglitazone prescribing issues - heart attacks
Pioglitazone only in clinical use
351
What are the biological effects of metformin?
Dec. hepatic glucose prodution
Inc. FA oxidation
Inc. Insulin sensitivity
Inc. Glucose utilisation
352
What is the mechanism of metformin?
Enters cells via OCT1
Accumulates in energised mitochondria
Inhibits complex I (Dec. ATP/ADP, Inc. AMP, AMPK activation)
353
What happens during complex I inhibition using metformin?
Dec. ATP/ADP - decreases energy for gluconeogenesis
Inc. AMP - inhibits FBPase activity, inactivates AC, prevents glucagon signalling
Dec. gluconeogenesis
Inc. AMPK - decreased acetyl-CoA carboxylase
Inc. FA oxidation
Dec. FA synthesis
