3. Metabolic Disease 1: Animal Models Flashcards
How does T1D lead to hyperglycaemia?
- Pancreas makes little/no insulin due to immune system attacking B cells
- Little insulin in bloodstream means glucose cannot enter cells and therefore accumulates in bloodstream
- Leads to hyperglycaemia
How does T2D lead to hyperglycaemia?
- Pancreas make insulin which enters bloodstream
- Due to insulin resistance of peripheral cells glucose cannot enter and therefore accumulates in bloodstream
- Leads to hyperglycaemia
How can T1D and T2D be similar?
Both T1D and late-stage T2D show B cell depletion despite early mechanistic differences
Describe the natural history of T2D.
- Before metabolic defects that lead to T2D, fasting and post-meal glucose levels are similar and constant
- As T2D develops, insulin resistance increases over time, leading to compensatory increases in insulin secretion t prevent blood glucose levels rising
- Eventually insulin resistance peaks and stabilises whilst insulin secretion continues to increase
- At crisis point, B cells become depleted/dysfunctional and so insulin secretion cannot match insulin resistance leading to hyperglycaemia
What is thought to be a major risk for T2D?
There is a strong correlation between obesity and T2D
Why is it unclear how obesity leads to T2D?
So many tissues are involved in glucose homeostasis and they all interact with one another therefore hard to understand cause vs consequence
What is the relationship between obesity, insulin resistance and dyslipidemia?
- Central obesity is associated with an increase in free fatty acids (FFA) and insulin resistance (unclear whether increased FFA causes insulin resistance or vice versa)
- These 2 effects stimulate increased hepatic apolipoprotein B and hepatic lipase activity (catalyse removal of lipids from HDLs)
- This leads to hypertriglyceridemia, increased small, dense LDLs and decreased HDLs
- These abnormal lipid patterns increase risk of developing plaques in the CV system and therefore increases the risk of co-morbidity
What is co-morbidity?
Having more than 1 recognised disease
What is the cause of hyperglycaemia in T2D?
3 major body metabolic defects:
- Increased hepatic glucose production
- Decreased pancreatic insulin secretion
- Increased peripheral insulin resistance
How have animal models been used to study how obesity can trigger T2D?
What is the problem with this study?
- Obesity characterised by accumulation of excess fat in ectopic sites (liver and skeletal muscle) instead of accumulation in adipocytes - this is associated with insulin resistance and T2D
- Hypothesis: Adipocytes are critical to prevent T2D
- Genetically-engineered mice lacking adipocytes show hyperphagia, insulin resistance and T2D
- However, due to lack of adipocytes these mice are leptin-deficient - leptin’s primary effects are on the brain therefore primary trigger may be brain dysfunction
How can transgenic reporter lines be used in the study of T2D?
Allow us to analyse specific tissues involved in glucose homeostasis in real time and space:
- In healthy animals
- In animals with genetic mutation (to study impact of human genetic variation)
- In animals subjected to different environmental conditions (e.g. high fat diet)
What is a future idea for a study to understand triggers in T2D?
- Cross 3 reporter lines to make fish that reports pancreas, liver and adipose tissue
- Feed fish high fat diet and observe which tissue is affected first
What is a reporter line used to specifically visualise B cells?
Tg(ins:Kaede)
Describe a technique for analysing proliferation.
EDU labelling
- EDU is an analog of Thymidine that has been modified to allow visualisation of proliferation
- Incubate cells with EDU
- EDU is incorporated into DNA during active DNA synthesis that occurs prior to cell division
- Incorporated EDU in DNA can then be detected with click chemistry procedure which makes nucleus fluoresce
What is the current treatment for diabetes?
What is wrong with this?
What is a potential new treatment?
What must be known for this to be possible?
- Insulin injections
- Does not increase life expectancy or decrease morbidity
- Restore B cell numbers
- Need to know what controls B cell number in development and regeneration
What 2 things shape development of an embryo?
- Gene/Chemical signals (e.g. Wnt, Shh)
- Environmental cues (e.g. signals from nearby bacteria)
What did the Hill et al (2016) paper on B cell development show?
Certain gut bacteria (specifically a protein they secrete) are necessary for the pancreas to populate itself with a robust number of B cells
What did rearing fish conventionally and in microbe-free environments show?
- In conventionally-reared fish, number of B cells increases steadily between 3-6dpf
- In microbe-free-reared fish, number of B cells remains the same during this period
What effect did the lower number of B cells in fish reared in microbe-free environments have?
Lower number of B cells means less insulin secretion resulting in increased blood glucose levels
What could restore the normal number of B cells in fish reared in microbe-free environments?
Exposing fish to specific bacteria (aeromonas)
What did the bacteria that restored the normal number of B cells in fish reared in microbe-free environments have in common?
How does this increase B cell numbers?
- They all secrete a protein - BefA which is sufficient to restore normal number of B cells
- Using EDU labelling they showed BefA increases B cell numbers by inducing proliferation
- BefA has homologs produced by bacteria in humans which were also sufficient to restore normal number of B cells
What future studies did Hill et al (2016) suggest?
Investigate the mechanism by which proteins affect B cell development and if they have the same effect in humans
What shows that the pancreas has the capacity to regenerate?
Experimental ablation of B cells by chemical treatment/partial pancreatectomy in rodents is followed by significant recovery of B cell mass
What is the stumbling block for using regeneration of B cells to treat diabetes?
Intrinsic transcriptional cascade that regulates B cell formation is well known, but the extrinsic signals regulating B cell regeneration in unclear
What transgenic fish line was used by Andersson et al (2012) in their screen to identify enhancers of B cell regeneration?
Double transgenic fish:
- Tg(ins:Kaede); Tg(ins:CFP-NTR)
Explain how conditional genetic ablation works.
- Identify cell-specific promoter for cell to be ablated and clone coding sequence of Nitroreductase (NTR) downstream
- NTR catalyses the conversion of MTZ into cytotoxic product
- NTR will only be expressed in the cells expressing the cell-specific promoter
- When MTZ is added it will be only be converted to cytotoxic product in cells that express NTR resulting in their ablation
How was the screen performed by Andersson et al (2012)?
- Fish were treated with MTZ and then transferred to 96-well plates
- Each well contained 1 of 7000 small molecules and fish were left to recover for 2 days
- Looked if any compound enhanced B cell regeneration by measuring fluorescence
What were the results of the high-throughput screen by Andersson et al (2012)?
- Hit compounds that enhanced B cell regeneration converged on the Adenosine signalling pathway, including exogenous agonists and compounds that inhibit degradation of endogenous adenosine
- Most potent enhancer of B cell regeneration was NECA (adenosine agonist) which increased B cell proliferation and accelerated restoration of normoglycemia
What did treatment of diabetic mice with NECA show?
- B cell regeneration and glucose-lowering effects were seen
- Suggests an evolutionary conserved role for adenosine signalling in B cell regeneration
