Differentiation of Enteroendocrine Cells

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New Roles for Gastrointestinal Hormones In
Development, Health, and Disease Andrew
Leiter M.D., Ph.D.
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Multipotential Pancreatic Endocrine Tumors-An
Example

• 1982 35 yo patient with secretory diarrhea,
  metastatic islet cell tumor stains for VIP,
  pancreatic polypeptide resected
• 1984 Diarrhea (1-2 L/day) responded to
  Octreotide
• 1985 Stool volume 3-5 L/day blood- VIP
  855pm/L (nllt30), PP 3226pm/L (nllt300)
  partial response to octreotide
• 1986 Diarrhea, dehydration, hyperglycemia-
  Stool volume 10-20 L/day refractory to
  treatment. Patient expired during surgery.

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The Beginning c. 1822
Alexis St. Martin
William Beaumont, MD
The Wound
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Proliferation of Gut Hormones
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Gastrointestinal Hormones

• 1902 - concept of blood-borne messenger
  controlling digestive organs- secretin
• Messengers originated from scattered mucosal
  cells
• Ended Pavlovian era of exclusive CNS control of
  gut
• Next 50 years, new hormones discovered by
  reexamination of CNS-controlled functions
• 1905 gastrin
• 1928 cholecystokinin (CCK)
• 1960s - Discovery of many new gut hormones
• 1980s-present New peptides continue to be
  identified by molecular biologic approaches
• The GI tract is the largest endocrine organ in
  the body!
• The function of gut peptides extends beyond
  control of digestive organs and includes the
  brain, cancer growth control,etc.

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Insulinotrophic Actions of GLP-1 and GIPProduce
the Incretin Effect
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Pancreatic Polypeptide

• One of the four pancreatic islet hormones
• Produced only in pancreatic PP cells
• Inhibitor of vagally stimulated pancreatic
  exocrine secretion
• Problem
• PP Receptors (NPY4) not present on pancreatic
  acinar cells
• Only present in the brain-?how to access
  receptors on other side of blood brain barrier

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Pancreatic Polypeptide is a FeedbackModulator of
Vagal Afferents to the Pancreas
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Loss of Meal-related Oscillations of Ghrelin
Levels Following Gastric Bypass Surgery
Cummings, D. E. et al. N Engl J Med
20023461623-1630
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PYY3-36 Inhibits Food Intake in Rodents and
Humans
Rat
Human
Batterham RL et al, Nature 2002 418650-4
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Sustained Effects of PYY3-36 on Food Intake by
Humans
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Peptide YY An Anorectic Hormone?
From Nature 2002
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How Cells Become Specialized-Differentiation

• Genes controlling differentiation are often
  transcription factors, proteins that bind to DNA
  to activate expression of specific genes.
• Differentiation results from the sequential
  activation of specific genes at the right time
  and and place.
• Differentiated cells usually stop dividing.
• Loss of cell proliferation controls in cancer may
  block cellular differentiation.

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bHLH Proteins and Enteroendocrine Differentiation
Secretin
CCK
GLP-1/PYY
Endocrine Progenitor
BETA2
GIP
Serotonin
Crypt Stem Cell
Substance P
Neurogenin 3
Somatostatin
MATH1
Gastrin
Secretory Progenitor
Goblet Cells
Paneth Cells
Enterocytes
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Neurogenin 3 (NGN3) and Endocrine
Differentiation in the Intestine and Pancreas

• NGN3 is a basic helix loop helix (bHLH)
  transcription factor related to the atonal
  protein in flies.
• Transiently expressed in scattered epithelial
  cells in intestinal crypts and the fetal
  pancreas.
• NGN3 is no longer expressed in hormone producing
  cells.
• NGN3-/- mice lack endocrine cells in their
  pancreas and intestine. Some ghrelin, serotonin,
  and ECL are present in the stomach.

Do all gut endocrine cells arise from Ngn3
cells? Does Ngn3 commit cells to an endocrine
cell fate?
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Recombination Based Lineage Tracing of NGN3 Cells
Indicator Mouse - ROSA26
NGN3-Cre Recombinase Mouse
X
Rosa26
Stop
ß-gal
ß-gal OFF
Rosa26
ß-gal
ß-Gal ON
?-gal activity marks all cells and all
descendants of cells that express Ngn3 during
differentiation. This includes cells that only
transiently express Ngn3.
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?-galactosidase activity marks intestinaldescenda
nts of NGN3 progenitors
Proximal small intestine
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Ngn3 is Expressed in a heterogeneouspopulation
of precursor cells
Pluripotent Crypt Cells
Precursor Cells
Ngn3
Math1
Ngn3/Math1
Enterocytes
Goblet Cells
Paneth Cells
Enteroendocrine Cells
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The Stomach and Intestine Produce Similar Hormones
Stomach
Intestine
CCK Secretin GIP Neurotensin Substance
P Gastrin Glucagon/Peptide YY Somatostatin ghrelin
Serotonin
Gastrin Somatostatin Serotonin Ghrelin ECL
Are gastric and intestinal enteroendocrine cells
specified by the same pathway?
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Stomach Anatomy
Body-acid producing
Antrum- hormones
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Most Endocrine Cells in the Antral Stomach Arise
from NGN3 and BETA2 Cells
The same is true for descendants of BETA2
cells.
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Most Endocrine Cells in the Stomach BodyArise
Independently from NGN3
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Most Serotonin Cells in the Body of the Stomach
Are Not From Ngn3or BETA2 Cells
NGN3-Cre
BETA2-Cre
Endocrine Cells Expressing ßgal
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Ngn3 Independent Differentiation of Serotonin
Cells in the Small Intestine
45 of serotonin cells arise from Ngn3
progenitors
55 of serotonin cells arise independently of Ngn3
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The Future

• Can we isolate serotonin cells for genetic
  profiling?
• What cells do carcinoids arise from? Ngn3 or
  Ngn3- or ?
• Most importantly, there has been a resurgence of
  interest in research that will impact our
  understanding of carcinoid tumors.
• In the future, this new information may drive the
  development of novel therapies.

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