Cell Physiology Week 7-12

Normal Blood Glucose Levels

• Normal fasting blood glucose levels are between 3.9–5.4 mmol/L

• Normal blood glucose levels two hours after starting a meal are between 6-10 mmol/L

Hypo and Hyperglycaemia Blood Glucose Levels

• Hypoglycaemia – glucose levels < 4.0 mmol/L

• Hyperglycaemia – fasting glucose levels > 7.0 mmol/L

Insulin vs Other Hormones in Changing BGL

Multiple hormones can increase blood glucose levels but insulin in the only one that can decrease it.

Pancreatic Islets

• 1-2% of total pancreatic mass

• Islet receive ~10-15% pancreatic blood flow.

• ~ 0.5 to 14.8 million islets per adult human pancreas

• 1,000-3,000 endocrine cells per islets

• 50-75% beta cells, 25-35% alpha cells, 10% delta cells, <1% other cells

Formation and Transport of Insulin

1. Insulin starts as preproinsulin in the rough endoplasmic reticulum, where it is broken down into proinsulin.

2. Proinsulin is transported to the Golgi complex.

3. Within maturing secretory granules with a clathrin coat, enzymes remove the C-peptide, forming active insulin.

4. Insulin is stored in mature granules as a zinc-insulin hexamer and has a half life of up to 5 days.

Phases of Insulin Secretion

Secretion is bi-phasic and includes the triggering pathway and amplifying pathway.

Triggering Pathway for Insulin Release Process

1. GLUT2 mediated transport of glucose into the beta cell

2. G6P moves through the glycolysis pathway to form pyruvate, which enters the TCA cycle

3. In the mitochondria, oxidative respiration generates ATP, increasing the ATP/ADP ratio.

4. ATP binds and closes ATP-sensitive K+ channels

5. Depolarisation of plasma membrane, opening voltage-sensitive Ca2+ channels

6. Influx of Ca2+ triggers insulin granule exocytosis

Amplifying Pathway of Insulin Release Process

1. Glucose metabolism increases and additional coupling factors such as NADPH, GTP, cAMP, Glutamate, etc.

2. No further rise in Ca²⁺

3. Enhanced exocytosis of insulin granules

4. Results in sustained insulin release

Features of Triggering Pathway

Starts insulin secretion, raises ATP, closes K(ATP), Ca²⁺ influx, increases Ca²⁺, depolarizes the cell, and dominates the first phase.

Features of the Amplifying Pathway

Boosts insulin secretion already underway, makes Ca²⁺ more effective at releasing insulin, does not change Ca²⁺, no effect on membrane potential, and is a major contributor to the second phase.

Insulin Secretory Granules

• 10,000 ISGs per β-cell

• Only 1-5% of ISGs undergo exocytosis

3 theories of what determines which granules will be released

1. Proximity to the plasma membrane

2. Restless newcomer granules

3. Granule age

Proximity of Granules to the Plasma Membrane

• The closer to the membrane a granule is, the more likely it will be to released

• Readily releasable pool = granules that sit super close to the membrane and are ready to be released

• Once the readily releasable pool is drained then granules will come from the reserve pool

Restless Newcomer Insulin Granules

• Highly mobile, highly Ca2+ sensitive

• Contributes to the majority of secretory response

• A specialized class of insulin-containing granules within pancreatic beta cells that are newly recruited and immediately fuse to the plasma membrane upon stimulation, typically without a preceding docking phase. 

Insulin Granule Age

• When β-cells are stimulated by glucose there is preferential release of young insulin granules because they are more mobile and calcium sensitive, with older granules preferentially targeted for degradation.

• This preferential release of young insulin granules is dysregulated underconditions of metabolic stress and T2D

Insulin Secretion in Response to an Oral vs IV Glucose

Glucose given orally stimulates more insulin than intravenous or intraperitoneal glucose. Secretion of gastro-intestinal hormones, stimulate insulin secretion by beta-cells.

Dual Actions of Fatty Acids on Pancreatic Beta-cells

Fatty acids on their own don’t induce insulin secretion but with glucose they increase secretion. Fatty acids potentiate glucose-stimulated insulin secretion by activating GPR40. This activation triggers a signalling cascade to open calcium channels on the ER to increase calcium release. 

Acute vs Chronic Fatty Acid Stimulation of Beta Cells

Potentiation of insulin release only works for acute stimulation: 

• If you give beta cells fatty acids for 1 hour with high glucose then there will be a doubling in insulin secretion

• If you give beta cells fatty acids for 24 hours with high glucose then the beta cells become dysfunctional

Beta Cell Adaptability and Plasticity during Pregnancy

Pregnancy causes beta cell mass expansion, increased insulin synthesis and secretion, increased glucose sensing, and increased anti-oxidative function.

Bioactive Lipids

Function as either extracellular or intracellular signals. Function in Inflammation, Development, Neurogenesis, Cognition, Motor Control, Feeding, Pain, Proliferation, Migration, and Apoptosis.Dysregulation implicated in many pathological conditions.

Major Classes of Bioactive Lipids

Eicosanoids, Endocannabinoids (eCBs), phospholipids/sphingolipids, and steroids.

What are the eicosanoids?

Leukotriene, prostaglandin, thromboxane, and prostacyclin

What are the endocannabinoids?

Anandamide, arachidonoylglycerol, and palmitoylethanolamine.

What are the phospholipids/sphingolipids?

Phosphatidylserine, phosphatidylinositol, lysophosphatidic acid, and sphingosine-1-phosphate.

Steroids

• Glucocorticoids, mineralocorticoids, androgens, estrogens, progesterones

• Most act through nuclear receptors to regulate gene expression

Lipid Receptors

G-Protein Coupled Receptors

• Lysophosphatidic acid (LPA) - LPA1, LPA2, LPA3 Receptors

• Sphingosine-1-phosphate (S1P) - S1P1-5 Receptors

• Platelet activating factor (PAF) - PAF Receptor

• Endocannabinoids - CB1 and CB2 receptors

• Prostaglandins - 9 different Eicosanoid Receptors

• Retinol derivatives - Rhodopsin Receptor

Production of Lipid Signalling Molecules

• Made by phospholipases that cleave the lipids off 

• Depends on what tail and head groups you have what it will produce

• Phospholipase A₁ produces free fatty acids

• Phospholipase A₂ produces arachidonic acid

• Phospholipase C produces inositol-phosphates

• Phospholipase D produces anandamide

Arachidonic Acid

• A precursor for many bioactive lipids such as COX-1, COX-2, and prostaglandin

• A precursor for the biosynthesis of eicosanoid

• Obtained from the diet (meat or eggs) or synthesized from linoleic acid

• Classified as an omega-6 fatty acid

Endocannabinoids

• Derivatives of arachidonic acid

• Anandamide and 2-Arachidonylglycerol are the predominant ones

• Regulate synapse formation and neurogenesis

• Can influence cognition, motor control, feeding behaviour, pain

• CB1-R are the most abundant GPCRs in the brain

• CB₁-R and CB₂-R are G_i/o coupled receptors

• Brain functions are mediated predominantly by CB₁-Rs

Biosynthesis of Anandamide

Phosphatidulethanolamine → N-arachidonoyl-PE → anandamide

Biosynthesis of 2-arachidonyl glycerol (2-AG)

Phosphatidylinositol → Lyso-Pl or 1,2-DAG → 2-AG

Anandamide Transport

• Endocannabinoids (ECs) are soluble in both aqueous and membrane environments

• Various proteins have been suggested as being EC transporters.

• EC Transporters may be involved in both release and re-uptake of ECs

• Cholesterol appears to regulate EC transport

• Not clear if this is due to a direct interaction between EC and cholesterol or due to cholesterol regulating the activity of the transporter

Metabolism of Endocannabinoids

• Fatty Acid Amide Hydrolase (FAAH) breakdown of anandamide

• Monoacylglycerol lipase (MAGL) breakdown of 2-AG

• Both break down into arachidonic acid

Possible Mechanism of Action of Acetaminophen (Paracetamol/Panadol)

• Not actually quite sure how it works

1. Acetaminophen is first converted to p-aminophenol and then conjugated with arachidonic acid (possibly by FAAH) to produce AM404

2. AM404 is a TRPV1 agonist

3. AM404 also inhibits cyclooxygenase (COX)-1 and COX-2 and prostaglandin synthesis

4. AM404 also inhibits anandamide transport Combination of effects could partially explain its analgesic, anti- pyretic and weak anti-inflammatory activities

Locations of enzymes involved in biosynthesis and breakdown

• Phospholipase C, Diacylglycerol lipase (2-AG biosynthesis) - located postsynaptically

• N-acyltransferase, N-acylphosphatidyl-ethanolamine phospholipase D (AEA biosynthesis) - located postsynaptically

• Transporter (EMT) - both pre- and post-synaptic

• Breakdown

  • Fatty acid amide hydrolase – postsynaptic 

  • Monoacylglycerol lipase - presynaptic

Cannabinoids Receptors

• Endocannabinoids were identified being the endogenous ligand for CB1-R

• A lot of searching eventually led to the realisation that it bound the active ingredient of cannabis – ΔTHC

What does cannabinoid receptor activation cause?

Activation of CB1/2 stimulates

1. Gi/o heteromeric proteins inhibits Adenylate cyclase (AC) – which reduces PKA activity

2. Mitogen activated protein kinase

• Both lead to changes in gene expression

Stimulation of Gi/o by CB1 receptors

1. Inhibits voltage dependent Ca2+ channels and

2. Stimulation of K+ channels

• which inhibits neurotransmitter release

3 Types of Endocannabinoid Signalling at the Synapse

Retrograde, non-retrograde, and enuron-astrocyte signalling.

Retrograde eCB Signalling

eCBs are mobilized from postsynaptic neurons and target presynaptic cannabinoid type 1 receptors (CB1Rs) to suppress neurotransmitter release.

Non-Retrograde eCB Signalling

eCBs produced in postsynaptic neurons activate postsynaptic CB1Rs or transient receptor potential vanilloid type 1 (TRPV1) channels.

Neuron-Astrocyte eCB Signalling

eCBs released from postsynaptic neurons stimulate astrocytic CB1Rs, thereby triggering gliotransmission. Glu, glutamate.

3 molecular mechanisms underlying endocannabinoid-mediated short and long-term synaptic signalling

Short-term plasticity, homosynaptic excitatory long-term depression, and heterosynaptic inhibitory long-term depression.

Short-Term Plasticity

• Release of 2-AG acts on CB₁R to cause inhibition of neurotransmitter release

• This temporary reduction in transmitter release is a form of short-term plasticity, because it transiently changes synaptic strength without altering synaptic structure.

Homosynaptic Excitatory Long-Term Depression

• 2-AG acts on the same neuron that the glutamate was released from to cause downregulation of the release mechanisms and inhibition of glutamate release

• CB1 receptor activation triggers persistent reductions in presynaptic neurotransmitter release. Unlike the short-term forms, this can last hours, days, or longer. It often involves changes in presynaptic signaling pathways, such as

Heterosynaptics Inhibitory Long-Term Depression

2-AG acts on cannabinoid receptors on GABAergic neurons which suppresses GABA release over the long term.

Current Bioactive Lipid Research in Vandenberg Lab

• Glycine neurotransmission in the spinal cord

• Glycinergic interneurons play a gate keeper role in regulating pain signals sent to the brain

• In neuropathic pain there is a reduction in glycinergic neurotransmission

• Glycine transport inhibitors will elevate [glycine] and restore normal pain processing

N-Arachidonyl Glycine is an endogenous analgesic

• Structurally related to the endocannabinoid anandamide

• An endogenous lipid found in highest concentrations in the spinal cord

• Reduces mechanical allodynia and thermal hyperalgesia in rat models of neuropathic pain

• No activity on Cannabinoid receptors, but inhibits Glycine Transporters

Problems with N-Arachidonyl Glycine

• Not very potent

• Readily metabolised

• Acts of a range of targets

How was N-arachidonyl glycine improved?

Modify the head and tail groups to produce a more potent Glycine Transporter 2 inhibitor. Made Oleoyl-D-Lysine which has a D-lysine attached to a Oleoyl tail. Found that there was no activity on off target sites after screening so it is selective.

Prediabetes T2

Blood glucose levels are higher than normal but not yet high enough for a diabetes diagnosis. During this phase, pancreatic beta cells begin working overtime to compensate for resistance.

Type 2 Diabetes Diagnosis

When the pancreas can no longer produce enough insulin to overcome resistance, blood sugar levels rise to a diagnostic range. At diagnosis, it is estimated that 50-70% of insulin-producing pancreatic cells may have been lost.

Changes During Development of T2 Diabetes

1. Insulin resistance increases to a point where it plateaus

2. Insulin secretion increases before decreasing

3. Beta cell mass and function increase before decreasing

4. Beta cell workload increases not quite exponentially

Insulin Resistance Lead to Beta-Cell Compensation

• Increase in beta cell mass and individual beta cells also secrete more insulin

• This is often sufficient to keep the glucose tolerance “normal” because most people have the genetic predisposition to be able to do this

• When people have a genetic predisposition for type 2 diabetes this compensatory mechanism fails and leads to a decrease in beta cell mass and insulin secretion

Inability to Compensate for Insulin Resistance is Genetically Driven

• > 80% of human genetic variants associated with T2 diabetes are involved in β-cell function (from genome-wide association studies)

• T2 diabetes occurs at the intersection between genetics and environment

• Environment = obesity, diet, smoking…

3 Key Mechanisms of Beta-Cell Failure in T2D

1. Inflammation

2. Oxidative stress and mitochondrial dysfunction

3. ER stress

Which all cause dysfunction, apoptosis, and/or dedifferentiation

Role of Inflammation in Beta-Cell Failure

• Chronic metabolic stress recruits and activates macrophages in islets, and their cytokines directly impair beta-cell function and survival

• Islets normally contain resident macrophages for homeostasis and remodelling.

• In T2D, macrophage numbers increase, driven by metabolic stress

Macrophage Polarization

• M1-like macrophages → pro-inflammatory, secrete pro-inflammatory cytokines

• M2-like macrophages → anti-inflammatory, tissue-repairing

• Obesity and nutrient excess push macrophages toward an M1-dominant state, amplifying inflammation

Oxidative Stress and Mitochondrial Dysfunction in Beta-Cell Failure

•  β-cells are uniquely vulnerable to oxidative stress because they have low antioxidant capacity.

• When exposed to chronic hyperglycaemia and elevated fatty acids the mitochondria work harder to produce ATP and they generate excessive reactive oxygen species, which damage mitochondrial electron transport chain components.

• Ros damages DNA, proteins, and lipids contributing to beta-cell dysfunction and apoptosis.

ER Stress in Beta-Cell Failure

• β-cells are highly secretory cells that depend on a tightly regulated ER to fold proinsulin.

• Chronic metabolic stress (obesity, insulin resistance) overwhelms this system, activating the unfolded protein response (UPR) and eventually driving β-cell failure

Key ER Stressors

• Increased insulin demand → more proinsulin synthesis → folding burden

• Hyperglycaemia and lipotoxicity → misfolded proteins accumulate

• Genetic susceptibility → impaired ER proteostasis

Unfolded Protein Response (UPR)

• There are UPR sensors that are always found in the membrane of the ER. These include ATF6, PERK, and IRE1 which are bound to BIP to keep them in an inactive state.

• When there is misfolding of insulin within the lumen of the ER BIP will dissociate and the sensors will be activated.

ATF6 (UPR)

• Upregulates genes encoding ER chaperons, folding enzymes, and ER-degrading components

• Expands the ER’s capacity to fold proteins and clear misfolded ones

• Key function: Increase ER folding machinery and degradation components

PERK (UPR)

• Reduces global protein translation, lowering the influx of new protein into the ER

• PERK at the same time activates ATF4 transcription

• If ER stress persists, ATF4 induces CHOP, a pro-apoptotic transcription factor.

• Key function: Reduce ER workload; trigger apoptosis if stress is chronic

IRE1 (UPR)

• Increases ER folding capacity, ER-associated degradation and lipid synthesis

• Degradation is to reduce ER load

• Key function: Increase ER folding and degradation capacity; fine-tune UPR adaptiveness

Effect of Unfolded Protein Response in Chronic Metabolic Stress

• Adaptive UPR becomes maladaptive

• CHOP and other pro-apoptotic pathways are activated

• ER Ca²⁺ handling becomes disrupted

• Beta-cells lose their ability to maintain insulin production

Signal Transduction Key Steps

• Extracellular ligand binding (hormones, neurotransmitters, cytokines)

• Activation of transmembrane receptors (conformational change / enzymatic activation)

• Intracellular signalling cascades (e.g. kinase-mediated phosphorylation)

• Second messenger generation (Ca²⁺, cAMP, DAG, IP₃ – rapid and transient)

• Amplification and integration of signals

• Altered cellular responses (metabolism, gene expression, transport, motility)

Types of Receptors

GPCRs, enzyme-linked receptors (RTKs), channel-linked receptors, and intracellular receptors.

First and Second Messengers

First messengers are extracellular ligands (such as hormones or neurotransmitters) that bind to cell-surface receptors to initiate signaling. Because they do not enter the cell, they activate second messengers, which are intracellular molecules that amplify and relay the signal to target proteins within the cytosol

Second Messengers Overview

• Small molecules and ions (Ca²⁺, cAMP, DAG, IP₃ – rapid and transient)

• Present at low or inactivated states in resting cells

• Activation leads to amplification and integration of signals

3 Classic Second Messenger Pathways

1. Activation of adenylyl cyclase by GPCRs to generate cAMP

2. The receptor is an enzyme that stimulates the production of PI3K by growth factors to generate the lipid second messenger PIP3

3. Activation of PLC by GPCRs to generate two second messengers: the membrane bound DAG and water soluble IP3

• IP3 activates IP₃R to cause the release of calcium from the ER

• DAG activates PKC

GPCRs Overview

• Receptors have 7 transmembrane domains and have various extra and intracellular loops

• Ligands bind at various different sites on the receptors

• Largest family of cell-surface receptors in many organisms.

• Largest and among the most efficacious class of therapeutic targets

How do GPCRs work?

• When GPCRs are inactive the alpha subunit of the g-protein heterotrimeric complex is bound to GDP.

• When a ligand binds to the extracellular domain of the GPCR, GPCRs act as guanine nucleotide exchange factor (GEFs) and exchange the GDP with GTP on the alpha subunit.

• The activated GTP bound alpha subunit will dissociate from the rest of the complex and interacts with downstream effect proteins which are mostly enzymes or ion channels

What do the different alpha subunits act on?

• α_i - activates ion channels, inhibits cAMP, acts on phospholipases and phosphodiesterases

• α_s - increases cAMP

• α_q - actviates phospholipase C

• α₁₂ - Rho GEFs

Activation-Inactivation Cycle of Heterotrimeric G-proteins

• G_α proteins are GTPases that catalyse the hydrolysis of GTP to GDP.

• Once downstream proteins are activated the alpha subunit deactivates itself as it is a GTPase itself that catalyses the hydrolysis of GTP to GDP.

• The GDP bound alpha subunit will now bind to the beta-gamma dimmer and form the inactive heterotrimeric complex

Signalling Through G_αi Protein

• Inhibitory G protein

• Inhibits adenylyl cyclase

• Decreases cytosolic cAMP levels

Signaling through G_αs Protein

• Stimulatory G protein

• Activates adenylyl cyclase

• Increases cytosolic cAMP levels

Activation of Adenyl Cyclase to Generate cAMP

• cAMP is synthesised from ATP via the action of adenyl cyclase and is inactivated by hydrolysis by cyclic nucleotide phosphodiesterase (PDE).

• The principal task of cAMP is to stimulate protein phosphorylation by cAMP dependent protein kinase PKA.

• cAMP dependent PKA mediates most of the effects of cAMP

• cAMP activates PKA and then will be hydrolysed by the cyclic nucleotide phosphodiesterases to downregulate the signal

Action of cAMP on PKA

Inactive PKA has a regulatory subunit and inactive catalytic subunit. cAMP binds to the regulatory subunit to cause the catalytic subunits to dissociate and become active. These subunits travel to the nucleus to activate CRED which binds to CRE on target genes.

Regulation of cAMP/PKA Activity

• Spatial and temporal organisation of signal transduction.

• AKAP: A-Kinase Anchoring Protein - one of the main targets of PKA

• AKAPS also interact with PDEs which maintain the local pool of cAMP

Signaling through G_αq Protein

• Activates phospholipase C

• Generates inositol 1, 4, 5-triphosphate and diacylglycerol

• IP3 mobilises Ca2+ from ER

• DAG activates PKC

• PKC phosphorylate target proteins

Inactivation or Desensitization of GPCRs

Activated GPCR stimulates GRK to phosphorylate the GPCR on multiple sites. Arrestin binds to the phosphorylated GPCR.

Enzyme-Linked Receptors

• Catalytic receptors

• Cytosolic domain has intrinsic enzyme activity

• Single pass transmembrane proteins - only have a single transmembrane domain

• Involved in cell division, programmed cell death or cell differentiation.

• Most common example : receptor tyrosine kinase

Activation of Enzyme-Linked Receptors

1. Ligand binding stimulates kinase activity

2. Autophosphorylation phosphorylates the tyrosine causing intracellular signalling proteins to bind to them

Receptor Tyrosine Kinase Activation of PIP3

1. RTK is activated by ligand binding

2. The phosphotyrosines recruit PI3K

3. Activated PI3K phosphorylates the membrane lipid PIP2

4. This generates PIP3 (phosphatidylinositol 3,4,5-trisphosphate).

Receptor Tyrosine Kinases in Disease

• Cancer (EGFR, HER2, PDGFR mutations)

• Insulin resistance & metabolic disease

• Therapeutic targeting (RTK inhibitors)

RTKs Overview

• 1 TM

• Ligands: growth factors

• Activation: dimerisation

• Speed: slow, long-term

• Signalling: kinase cascades

• Outcomes: growth, metabolism

GPCRs Overview

• 7 TM

• Ligands: hormones, neurotransmitters

• Activation: G-protein activation

• Speed: fast, transient

• Signalling: second messengers

• Outcomes: rapid physiological responses

Points of Crosstalk Between RTKs and GPCRs

• GPCRs can activate MAPK

• RTKs can influence cAMP levels

• Integration allows fine-tuned cellular responses

Fasted vs Fed Insulin Metabolism

• Fasted: liver provides glucose to the brain and adipose tissue provides fat to the muscles

• Fed: pancreas secretes insulin due to high blood glucose and stops the liver from producing glucose and increases uptake of glucose by adipose tissue and muscle.

How does insulin increase glucose uptake into muscle and adipose tissue?

• Insulin increases the abundance of glucose transporters on the surface of muscle and fat cells 

• Muscle and fat cells have a unique glucose transporter called GLUT4

• Insulin binds to its receptor which activates a signaling cascade which moves GLUT4 to the cell surface

Why is GLUT4 regulated?

• To prevent hypoglycemia - otherwise muscle would just continually be taking the glucose out of the blood

• Prevents muscle stealing the brain’s glucose

Why do muscle, adipose and liver cells respond to insulin?

• Because they have the insulin receptor but not all cells do

• Note: same receptor, different cell, different outcome

Ozempic and the Glp1 receptor as an example

• Type 2 diabetes medication that targets GLP1R on beta-cells to cause insulin secretion

• Caused weight loss because the GLP1R receptor also happens to be found in the hypothalamus and attenuates hunger

• This wasn’t the original target but is actually a bit of a side effect

• Same receptor, different cell, different physiological outcome

Insulin Signal Transduction

• Insulin activates a network of kinases

• Kinases phosphorylate lipids, metabolites and proteins

• Alter protein activity, localization, and interactions

Protein Kinases

• Serine and Threonine kinases are the most common

• 64% of tyrosine kinases are hormone receptors (only 3% for S/T kinases).

• The insulin receptor is an example of an RTK

Requirements for a protein kinase to phosphorylate a serine, threonine or tyrosine:

1. The phosphorylation site needs to be exposed

2. The target protein needs to be near the kinase

3. The surrounding amino acids needs to match the kinases targeting motif.

• Ex. Akt motif: RXRXXS (don’t need to remember)

Insulin Signalling Process

1. Insulin binds to the insulin receptor on the outside of the cell. This changes the shape of the insulin receptor and activates tyrosine kinase activity.

2. The insulin receptor phosphorylates itself and IRS-1 (insulin receptor substrate-1).

3. Phosphorylation of tyrosine on IRS-1 attracts a lipid kinase called phosphoinositide 3-kinase (PI3K).

4. PI3K phosphorylates a lipid in the inner leaflet of the plasma membrane called PIP2, converting it to PIP3.

5. PIP3 attracts proteins which contain a PH-domain (pleckstrin homology).

6. PDK1, Akt and mTORC2 all contain PH-domains. By coming together at PIP3 on the plasma membrane they come into proximity. Both PDK1 and mTORC2 phosphorylate Akt, thereby activating it.

Overall Phosphorylation Caused by Akt

• Phosphorylates the protein AS160 which sits on the outside of vesicles that contain GLUT4. This then allows them to be transported and fuse with the cell membrane

• Phosphorylates PFKFB2 which increases glycolysis - metabolised for fuel

• Phosphorylates GSK3 which then activates glycogen synthase

• Phosphorylates FOXO (not in diagram)

Insulin-mediated GLUT4 translocation: AS160

• Myosin and Rab proteins work together to move GLUT4

• AS160 inhibits Rab proteins. This means it keeps GLUT4 inside the cell

• Akt inhibits AS160 by phosphorylation of 4 different sites

• Exercise through AMPK also triggers this process via muscle contraction not just insulin

FOXO and Regulation of Transcription by Insulin

• FOXO is a transcription factor normally found in the nucleus

• When it is phosphorylated by Akt it is removed from the nucleus because of the negative charge of the phosphate

• In low energy conditions (starvation) FOXO activates genes involved in survival

• When fuel is abundant FOXO is switched off because these genes are no longer needed

• Insulin is one signal which communicates fuel (glucose) availability