Formation and degeneration of pancreatic beta cells Flashcards
Islets of the endocrine pancreas
- islets are clusters of ~1000 endocrine cells
- ~1 million islets are in human pancreas
- islets make up ~1-2% of pancreatic volume: remainder mostly exocrine pancreas (acini and ducts) which secrete digestive enzymes
Islet endocrine cell types
- alpha cells (38% human, scattered) secrete glucagon
- beta cells (55% human, scattered) secrete insulin
- delta cells secrete somatostatin (<5%)
- PP cells secrete pancreatic polypeptide (~1%)
- epsilon cells secrete Ghrelin
- RANDOM ASSORTMENT IN HUMAN COMPARED TO ORGANISED IN MOUSE
Maintenance of normoglycaemia (5-7mmol)
- fast/exercise: hypoglycaemia and hypolipidaemia; glucagon increase, insulin decrease; return glucose production to normal (catabolism, lipolysis)
- feeding: hyperglycemia and hyperlipidaemia; insulin increase, glucagon decrease; return glucose production to normal (anabolism, lipid storage)
- NOT JUST ABOUT GLUCOSE!! Lipid metabolism is also important
The role of the beta cell in glucose homeostasis
insulin release suppresses pancreatic output and stimulates glucose uptake into muscle and fat
Biogenesis of insulin
- active secretory pathway
- insulin is expressed from pre-pro-insulin gene, which is translated to proinsulin in the rough ER
- trafficks to golgi, packaged to vesicles where it is cleaved to mature insulin and c-peptide
- dense core vesicles are released upon glucose stimulation
- when beta cells are stressed they can begin to degranulate and loose insulin vesicles
Essential molecular components of B-cell glucose sensing
Facilitates dose-dependent stimulation of insulin secretion by glucose
- GLUT1/2
- GCK
- oxidative metabolism
- K(ATP) channels
- VDCC
- exocytotic machinery
Pathway of glucose sensing in beta cells
1) glucose taken up by glucose transporters
2) glucokinase creates a wave of carbohydrates through glycolysis into the mitochondria
3) wave of ATP generation; closes ATP sensitive K channels, depolarising the membrane
4) depolarisation stimulates opening of VDCC, triggering Ca influx and the fusion and secretion of insulin-containing vesicles
Principles of tissue development
gradients of growth factors and transcription factor activation guide cell lineages and tissue development
Structure of the endocrine pancreas
- ‘TIP TRUNK STRUCTURE’
- commitment to tip phenotype = acinar cell (produce digestive enzymes, e.g. amylase)
- commitment to trunk structures = ductal cells (drain digestive enzymes into gut, endocrine cells of islets) or endocrine progenitors via transient activation of Neurog3
Describe the process of beta cell neogenesis during development (key TFs)
Key TFs:
- PDX1: master regulator; initates pancreatic formation and plays a role in insulin expression of beta cells in mature pancreas
- Neurog3: commits cell to endocrine lineage
- MAFA
- NKX6.1: beta cell marker (and PDX1)
- PAX6
Discuss the role of beta cell proliferation in the expansion of beta cells mass during adulthood
- neogenesis is greatly reduced in adult organ, but can be activated by tissue injury
- forming partial duct ligation in mouse pancreas, within 7 days there is mass neogenesis in positive ductal cells and increase in beta cell mass
- reactivation of neogenesis as a way to regenerate beta cells during diabetes
Concept for transdifferentiation
- under experimental conditions of extreme beta cell loss, lineage tracing has shown alpha and delta cells can transdifferentiate into insulin-expressing cells in mice
- evidence for transdifferentiation is limited in humans: difficult to test experimentally as lineage tracing is not possible
Evidence for the transdifferentiation of pancreatic endocrine cell types (hint: mice)
Herrera lab and Geneva
- mice do not express the human Diptheria toxin receptor (DTR) and tissues are resistant to Dipetheria toxin (DT)
- expression of the DTR in beta cells enables selective ablation of beta cells by DT
- expressing DTR in mouse beta cells using RIP (rat insulin promoter) driven constructs
- giving DT to this mouse, we oblate 99% of beta cell mass (extreme model) - can study what happens in organ as a response
Glucagon promoter
- activate a Cree in alpha cells; snips STOP cassette, YFP is expressed
- important as it is genetic labelling of cell and cell lineage
Ablating beta cell mass, using DT, we can see the appearance of insulin (+) cells, some of which also express YFP, meaning that they must have been alpha cells at the point when the tetracyclin pulse lineage was used - shows that alpha cells can differentiate into beta cells using linear tracing approach
Rates of beta cell proliferation
- proliferation in adult pancreas is low: <0.5% of beta cells actively proliferate in rodents and humans
- rodent beta cells show stronger mitogenic response to Harmine (via Dryk1a inhibition)
Measuring beta cell proliferation
- incorporation of Bromodeoxyuridine (BrdU), a thymidine analogue, into newly synthesised DNA during replication; daughter cells are labelled
- Harmine is Dyrk1a inhibitor - induces cell proliferation
- presence of the Ki67 protein (typically by immunoflorescence) indicates that cells in active cell cycle are labelled
Describe the mitogenic effect of GLP1 on beta cells
- GLP-1 can drive proliferation in both human and rodent beta cells
- produced by endocrine cells in gut (L-cells; ileum)
- release GLP-1 in response to food intake, travels via bloodstream and activates receptor on target tissues (beta cell)
- initiates GPCR signalling to promote cell proliferation via a cAMP and PKA dependent process
- drugs derived from exenatide and DPP-4 inhibitors target GLP-1 receptor
Define diabetes and describe the aetiology of type 1 and type 2 diabetes
- beta cell dysfunction occurs in both T1DM and T2DM
T2DM:
- insulin secretion defects observed early (pre-diabetes); become more profound and get defects in both phases
- isolating islets from people, see inherent defect in islets of people with T2Dm (Deng et al., 2004)
Risk factors for T2DM
- genetic factors (polygenic): >200 genes are identified by GWAS; most are B-cell genes
- environmental factors (e.g. obesity, age, pregnancy, calorie-dense diet, sedentary lifestyle) increase demand for insulin
- epigenetic factors: maternal and paternal metabolic health influences offspring T2DM risk
Discuss the contributions of beta cell apoptosis and de-differentiation to beta cell failure in T2D
- excessive workload, high levels of glucose, and oxidative stress can result in ER stress; triggering unfolded protein response and beta cell apoptosis
- defects in protein degradation pathways (aggregation of IIPP), oxidative stress (low antioxidant defence mechanism) and inflammation
- beta cell stress, dysfunction, and apoptosis
Beta cell apoptosis or dedifferentiation (Butler et al., 2003)
- loss of insulin B-cell mass during T2DM
- immunohistochemistry of pancreatic sections using anti-insulin antibody
- quantify 2D insulin(+) area and extrapolate to three dimensions
- should normalise to weight of pancreas
- other groups report a more modest change in insulin(+) area
- beta cell apoptosis increased during T2DM (TUNEL stained, insulin(+) cells)
Beta cell apoptosis or dedifferentiation (Marselli et al., 2014)
- Beta cells degranulate but persist during T2D
- IHC of pancreatic sections using anti-insulin and anti-chromogranin antibody
- pancreatic insulin(+) area and mature insulin granules reduced in T2D
- chromogranin A was unaltered
- suggests degranulation or dedifferentiation of beta cells during T2D
Summarise the interactions between the beta cell and immune cells during autoimmune destruction of beta cells in T1D
- autoimmunity
- activation of innate, adaptive immune system
- presence of autoantibodies recognising beta cell antigens
- increased level of apoptosis
- reduction in beta cell mass; hypoglycaemia and T1D diagnosis
- want to protect beta cell mass! also need to find ways to restore beta cell funciton/mass in individuals with T1D
- therapeutic options: replacement or regeneration
Summarise and evaluate the evidence for transdifferentiation in the endocrine pancreas
Discuss potential strategies to regenerate or replace functional beta cells during diabetes
Primary human islets:
- islets given by donor are graphted into liver of T1D recipient
- donor supply is limited and require strong immuno-suppression
- ectopic graft (may limit function); often function poorly and recipients rarely maintain long-term insulin independence
Stem cell (hESC or iPSC) derived beta-like cells generated in vitro
- currently in experimental stage
- allogeneic graft requires immunosuppression or shielding from immune system
- ectopic graft may limit function
- long term functionality and durability of cells is unknown
