Formation and degeneration of pancreatic beta cells

Question 1

Islets of the endocrine pancreas

Answer 1

• 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

Question 2

Islet endocrine cell types

Answer 2

• 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

Question 3

Maintenance of normoglycaemia (5-7mmol)

Answer 3

• 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

Question 4

The role of the beta cell in glucose homeostasis

Answer 4

insulin release suppresses pancreatic output and stimulates glucose uptake into muscle and fat

Question 5

Biogenesis of insulin

Answer 5

• 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

Question 6

Essential molecular components of B-cell glucose sensing

Answer 6

Facilitates dose-dependent stimulation of insulin secretion by glucose

• GLUT1/2
• GCK
• oxidative metabolism
• K(ATP) channels
• VDCC
• exocytotic machinery

Question 7

Pathway of glucose sensing in beta cells

Answer 7

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

Question 8

Principles of tissue development

Answer 8

gradients of growth factors and transcription factor activation guide cell lineages and tissue development

Question 9

Structure of the endocrine pancreas

Answer 9

• ‘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

Question 10

Describe the process of beta cell neogenesis during development (key TFs)

Answer 10

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

Question 11

Discuss the role of beta cell proliferation in the expansion of beta cells mass during adulthood

Answer 11

• 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

Question 12

Concept for transdifferentiation

Answer 12

• 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

Question 13

Evidence for the transdifferentiation of pancreatic endocrine cell types (hint: mice)

Answer 13

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

Question 14

Rates of beta cell proliferation

Answer 14

• 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)

Question 15

Measuring beta cell proliferation

Answer 15

• 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

Question 16

Describe the mitogenic effect of GLP1 on beta cells

Answer 16

• 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

Question 17

Define diabetes and describe the aetiology of type 1 and type 2 diabetes

Answer 17

• 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)

Question 18

Risk factors for T2DM

Answer 18

• 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

Question 19

Discuss the contributions of beta cell apoptosis and de-differentiation to beta cell failure in T2D

Answer 19

• 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

Question 20

Beta cell apoptosis or dedifferentiation (Butler et al., 2003)

Answer 20

• 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)

Question 21

Beta cell apoptosis or dedifferentiation (Marselli et al., 2014)

Answer 21

• 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

Question 22

Summarise the interactions between the beta cell and immune cells during autoimmune destruction of beta cells in T1D

Answer 22

• 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

Question 23

Summarise and evaluate the evidence for transdifferentiation in the endocrine pancreas

Question 24

Discuss potential strategies to regenerate or replace functional beta cells during diabetes

Answer 24

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