Glucose Homeostasis Flashcards
Hyperinsulinaemic Clamp / Glucose Clamping
- A glucose clamp set up is a method used to study how the body processes glucose (which is a type of sugar), it helps to measure how sensitive person is to insulin a hormone that controls blood sugar levels.
- infuse insulin to reduce blood glucose
- difference in GIR and hormones indicate whether a drug improves or lessen glucose homeostasis
- Used for assessing counterregulatory response to hypothermia.
Setup in humans:
- Measurement of baseline blood sugar levels
- Insulin infusion- a person is given a steady amount of insulin through IV, this insulin infusion is meant to keep the insulin level in the blood constant.
- Glucose (dextrose) infusion - at the same , glucose is also infused through another IV, the amount of glucose given is adjusted to keep the persons blood sugar level at the target range —-> 20% glucose (weight/volume)
- Adjusting glucose - researcher then constantly monitor the blood sugar level and adjust glucose fusion rate to maintain the target blood sugar level. If the person body is sensitive to insulin, less glucose will be needed to keep the blood sugar . If a person is less sensitive to insulin (insulin resistant) more glucose will be needed.
- Blood sampling/ measurement - is taken every 5-10 minutes using glucose meter - the amount of glucose required to maintain the blood sugar level is recorded which tells researchers how sensitive a person body is to insulin- the more glucose needed the less sensitive (more resistant) the person is to insulin.
The clamp usually last 90-180 minutes
More insulin = more resistant (Insulin resistance)
Less insulin = more sensitive (Insulin sensitivity)
Glucose clamp in rodent:
- not easy as blood sampling is challenging
- stays functional for up to 7 days and catheters needs to be flushed with saline solution containing small amount of heparin to prevent blood cots.
Euglycaemic clapms
- typical clamps, utilised in many studies
- it assess whole body insulin sensitivity/ insulin resistance
- if using tracer, hepatic insulin sensitivity
- skeletal muscle/ adipose/ brain glucose usage
- steady state of the clamp is a crucial aspect - this is when glucose levels between the two experimental groups are matched.
Limitation of glucose clamps
- glucose clamping requires a constant infusion i.e. Prolonged high insulin levels - it does not occur post-prandially and requires fasting before the clamp - depletes glycogen stores.
- In rodent require surgery and post surgical recovery (5-7 days)
- In mice donor blood is required to prevent anaemia
- Expensive, particularly with rodents and time-consuming with humans
- Frequent blood drawers required and operator skill in deciding what glucose infusion is required - potential for biases.
What are the main glucose sensing centres in the hypothalamus?
Hypotonus main sensing centres include VN, ARC and LH.
How do hypothalamic neurons sense changes in glucose?
- All neurons utilises glucose as major fuel source and chronic removal of glucose will inhibit neural activity globally
- Glucose sensing neurons, alter membrane, potential action, potential frequency and transmitter/peptide release to acute physiological changes in glucose.
- Studies have identified two main types of glucose sensing neurons:
1. Glucose-excited (GE) neurons — as glucose level decreases , the activity/firing rate of glucose excited neurons decreases.
2. Glucose-inhibited (GI) neurons — as glucose level drop glucose inhibited neurons become more active. These neurons are inhibited by glucose so when glucose level fall inhibition is reduce leading to an increase in the firing rate.
Classic glucose sensor
- Pancreatic beta cells are known as the classical glucose sensor within the body
- glucose is transported across the membrane by glucose transporter 2 (GLUT 2)
- glucokianse (GK) performs the rate limiting step to phosphorylate glucose to produce glucose 6 phosphate
- subsequent metabolism by glycolysis and oxidative phosphorylation increases ATP levels within the cell - which inhibits the ATP sensitive potassium channels
- this leads to membrane depolarisation - this allows activation of voltage dependent calcium channels - which leads to increase in intracellular calcium levels = which then act as a signal for insulin vesicle movement = infusion and secretion of insulin granules.
Within the brain glucose neurons uses the same glucose sensing mechanism- GLUT 1/3 may also be involved in glucose transport- these glucose excited neurons do not release insulin but rather release neurotransmitters- either GABA or Glutamate.
Evidence suggest that proportion of glucose neurons within the hypothalamus are likely to be GABAergic
How do hypotonic neurons changes in glucose? Key enzymes:
GLUT2 - is a high capacity glucose transporter - high Km so rate of transport is proportional to ambient glucose
GK - has low affinity hexokinase- Km in Mm range - considered to be rate limiting and the beta cell primary glucose sensor
K-ATP channel- partly open at ambient glucose -responsible for change in cell membrane potential- drives excitability changes.
Location within hypothalamus :
GLUT mainly expressed in expendymal cells of 3V and some ARC neurons
GK reported to be present in many, but not all glucose sensing neurons or brain areas
K-ATP present in nearly all ARC and many VMH neurons but not all neurons exhibit glucose sensing
Glucose inhibited (GI) neurons
- GI neurons are metabolic sensors
- stimulation of a+/K+ ATPase activity (not is now considered unlikely)
- opening if chloride channels (CFTR proposed as mediator) - glucose range - 0.1/0.5 to 2.5/5.0 mM- GI type
Both of the above will inhibit neuron activity by hyperpolarisation
- closure of K-ATP channels (like pancreatic alpha cells) - results in cell depolarisation — inhibits action potential generation and same effect is observed on the release of neurotransmitters- inhibition of neurotransmitter release
AMP- activated protein kinase AMP-K
AMP-K is described as a master regulator of cellular energy homeostasis.
- AMP- K is an energy sensing kinase which is activated by an increased intracellular AMP level - which typically indicates low cellular energy
- AMPK can also be activated by increase in ADP levels or other upstream kinases.
- it is composed of a heterotrimer complex with 3 subunits: 1 alpha, 1 beta and 1 gamma subunits
The alpha sub unit contains catalytic domain and information within this domain is required for kinase activity
There are 2 isoforms of alpha subunit - A1 and A2
- the function of kinase is to restore cellular energy balance
- when activated AMK promotes AT production by enhancing catabolic pathways (e.g. Glucose uptake /fatty fatty acid oxidation) ——> inhibit ATP consuming anabolic processes (like fatty acid synthesis/protein synthesis)
- In this way AMP K acts as a fuel gauge tanning on energy producing pathways and shutting down energy consuming pathways —> this insures that the cell maintains its energy homeostasis
MPK plays a significant role in glucose homeostasis by promoting glucose uptake (especially in muscle cells) and by inhibiting pathways that consume glucose or produce glucose unnecessarily it also influences insulin sensitivity.
** AMP is a molecule that activates AMK by binding to gamma subunit ——> is heterotrimer as it consists of three subunits (a,b and y) ——> AMPK is activated by changes in the AMP or activated by increase in ADP levels**
AMPK : mechanism to monitor cell energy availability fluctuation in (glucose) detected via AMP/ATP ratio.
AMPK phosphorylation is high at low glucose concentration and low at elevated glucose level
Key points
Hypothalamus shows expression of AMP neurons
Decrease in glucose increases AMPK activity
Fasting AMP activity and increased AMP = increased food intake .
Does a MPK activity play a role in GE glucose sensing?
Yes, AMP K activity does play a role in glucose excited (GE) glucose sensing but the relationship is somewhat complex and contents dependent.
AMPK act as a crucial energy sensor in cells including neurons . in GE neurons activity inversely correlates with glucose levels- when glucose is high, AMP is less active = leading to increased neural firing. Conversely, when glucose level drop, AMPK becomes more active which contribute to the suppression of GE neuron activity.
Therefore suggest that AMPK ability plays an essential role in glucose sensing mechanism S of gene neurons by linking changes in cellular energy status to neuron activity.
Some ARC, POMC and AgRP neurons exhibit properties of GE neurons- and loss of AMPKa2 subunit makes ARC neurons unresponsive to glucose.
AMPK activity also plays a role in beta cell glucose sensing - by generated mice lacking the AMPKa2 subunit in beta cell causes defect in glucose sensing and insulin secretion.
AMPKa2 knockout mice = AMPKa2KO beta cell exhibit defects in glucose sensing and impaired insulin secretion = which result in alteration/ abnormality of glucose homeostasis
Insulin
Insulin is a peptide hormone secreted by pancreatic beta cells in response to high blood glucose levels, it binds to the extracellular alpha subunit of the insulin receptor.
——> Integrated stress response (ISR) regulates downstream signalling of insulin receptor and insulin like growth factor one receptor (IR/IGF-1R)
- Insulin activates insulin receptors
- Insulin receptor is a heterotetrameric protein- composed of 2 alpha and 2 beta subunits
- there are 4 types of insulin receptors —-> IRS1-4
- Insulin receptors causes phosphorylation with 3- kinase protein (PI-3K)
- 2 main subunit p85 and p110
- recruitment of PI3K allows PI3K to convert PIP2 = PIP3
- PIP 3 activates PDK1 - PDK1 phosphor and activates Akt ———> which is a key target which regulates variety of other pathways.
- Mammalian target of rapamycin (mTOR)
Insulin modulation of AgRP neuronal firing
- insulin inhibits AgRP neuronal firing
- Mouse with genetically engineered loss of insulin receptors on AgRP neurons do not respond
- pharmacological blockage of K-ATP channels reverse the effect of insulin
Insulin modulation of HGP via the brain
HPG = hepatic glucose production
- Insulin suppresses HPG (to lower blood glucose levels)
- Loss of insulin receptors only on AgRP neurons- diminished insulin action
- Insulin receptors AgRP neurons are required for full regulation of blood glucose by insulin
Loss of insulin receptors on AgRP neurons diminishes insulin which contributes to the suppression HGP axis.
Insulin modulation of POMC neuron firing
- both increased and decreased firing
- Inhibition caused by activation of ATP sensitive potassium channels- K-ATP
Leptin regulation of glucose homeostasis
- Leptin is adipose tissue derived hormone
- acts via the leptin receptors (Ob) and there are 6 variants - ObRb in the main signalling isoform
- main signalling mechanism uses:
- JAK2
- STAT3
ObRb is expressed in skeletal muscle, pancreas, brown adipose tissue and the brain.
Leptin and neuronal activity
- Similar to insulin, Leptin also hyperpolarises some hypotonic neurons
- it activate the ATP- sensitive K+ channels —— K-ATP
- tolbutamide is K-ATP blocker (it also blocks pancreatic beta cell K-ATP channels)
- Leptin can also depolarised other neurons like POMC
- activation of calcium channels - and genetic loss of CaV2.3 abolishes sustained leptin-induced depolarisation
CaV2.3 knockdown - decreased hepatic insulin sensitivity
Hypothermic Leptin and hepatic insulin sensitivity
- hypothalamic leptin signalling increases liver insulin sensitivity
Evidence:
- kolesky rats - this is an obese rat around 700g =
- leptin receptors deficient - doesn’t have leptin induced inhibition of feeding
- measured hepatic glucose production using the glucose clamp technique - allows detail measurement of glucose production freely moving animals
- Insulin induced signalling events are enhanced by restoration of hypothalamic leptin receptor expression
- Ad-leprechaun have increased insulin receptor substrate 1 and Akt phosphorylation
- Improved hepatic insulin sensitivity
- skeletal muscle insulin sensitivity not altered.
Hypothalamic leptin and skeletal muscles AMP-K activation
- leptin directly and indirectly increases skeletal muscle glucose uptake
- AMP-K
- which is a mater cellular energy sensor
- activated by energy stress and some hormones
- increases in GLUT4 translocation in muscle to enhanced glucose transport into muscle
- cutting sympathetic nerves to the muscles abolishes leptin-induced AMPK activation
- leptin induced phosphorylation is also abolished
Hypothalamic leptin and peripheral glucose uptake
- VMH injection of Lapin increases glucose uptake onto:
Brown adipose tissue
Skeletal and cardiac muscle
Spleen
Summary of leptin
Leptin activities, sympathetic nervous system to increase sensitivity of liver to insulin and to increase glucose disposal into a number of peripheral tissues (Skeletal muscles, heart- cardiac muscles, brown adipose tissue and the spleen) and it increases the insulin sensitivity of the liver
Taken together these actions decrease blood glucose levels suggesting that Lipton resistance will contribute to obesity induced hyperaemia and possibly the development of type two diabetes . 
Hypoglycaemia and diabetes
- hypoglycaemia = low blood glucose level
- still prevalent despite better treatment and technology
- those with diabetes especially those taking insulin are at higher risk hypoglycaemia- excessive insulin administration can cause blood glucose level to drop too low
Hypoglycaemia symptoms are characterised as neurogenic and neuroglycopenic:
**Neurogenic symptoms **:
- Shakiness or tremors
- palpitations
- Anxiety or nervousness
- Sweating
- Hunger
- Nausea
Neuroglycopenic symptoms:
- Confusion or difficulty concentrating
- Dizziness or lightheadedness
- Blurred vision
- Headache or fatigue
- slurred speech
- irritability or more changes
-Seizures in severe cases
And loss of consciousness of coma in very severe cases
Physiological response to hyperglycaemia in humans
Decreased insulin production can lead to increased glycogen breakdown and elevated adrenaline secretion. This cascades of event results in heightened stress responses and symptoms which can ultimately impair cognitive brain function.
Pathophysiological response to hyperglycaemia and type one diabetes
Type one diabetes result in impaired insulin production leading to ongoing hyperglycaemia/the liver continues to release glucose due to the lack of insulin whether the adrenal gland secret adrenaline in response to the stress of hyperaemia this casket of events leads to a various symptoms that can negatively impact cognitive function.
