GLP-1 secretion as a prerequisite for the
antidiabetic activity of DPP-4 inhibitors:
Can it be modified?
Fiona M Gribble
Cambridge Institute for Medical Research, UK
Underlying basis for the incretin effect
appetite
insulin
Vagal
GIP Hormonal 90%
Ingested food inactivated
DPP4
GLP-1
L-cell peptides
Ingested food
insulin release
(incretin effect)
GLP-1
L-cell peptides PYY appetite
Oxyntomodulin
GLP-2
Epithelial regeneration
Food ingestion
K-cells GLP-1
(GIP) secretion
Other GI
hormones
• CCK L-cells
• 5-HT
• neurotensin
• ghrelin
• somatostatin
• secretin
How are L cells stimulated physiologically ?
Sugars
Peptides
Fats
Bile acids
Hormonal
/ paracrine?
Receptors?
Uptake? GLP-1
release
Metabolism?
Neural?
Studying L-cell function in vivo
• Plasma GLP-1, GLP-2 or PYY levels after nutrient ingestion in humans /
animal models
• Isolated perfused intestine preparations
Studying L-cell function in vitro
Cell lines e.g. GLUTag Primary intestinal cultures
Venus-fluorescent L-cells
L-cells are electrically active and nutrient responsive
40
V (mV)
0
-40
60 s
glucose
40
V (mV)
-40
500 ms
Activity dependent GLP-1 release
Ca2+
GLP-1
secretion
Ca2+
Luminal
Na+
signal
How do L-cells sense?
Nutrient Na+, H+
Electrogenic GPCR
uptake
Metabolism IP3 cAMP
ATP
Ca2+
KATP
channel
SGLT1 as a glucose sensor in primary L-cells
GLP-1small
Upper secretion
intestine
Small intestinal villus
secretion
2.5 ** **
19 12
release
2.0
GLP-1
1.5
GLP-1
1.0
Relative
Venus
SGLT1
0.5
Con Gluc αMG
SGLT1 Expression
Relative mRNA expression
2.2
GLP-1 release
0.4 FACS
0.3 L+ 1.8
0.2 L- 1.4
0.1 1.0 EC50 0.2 mM
0 0.6
α β δ/pp
GLUTag
L+ L- L+ L- 0.1 1 10 100
SI colon [αMG] (mM)
A working model for luminal nutrient detection
Ca2+
Amino acid GLP-1
transporters secretion
PEPT Ca2+
Na+
Glucose
H+
Na+
SGLT1
Na+
Expression of KATP channel subunits and glucokinase
Kir6.2 3 SUR1
0.4
0.3 2
0.2
Expression relative to β-actin by qRT-PCR
1
0.1
0 0
α β δ/pp
GLUTag
α β δ/pp
GLUTag
L+ L- L+ L- L+ L- L+ L-
SI colon SI colon
Glucokinase
0.12
0.08
0.04
0
β
GLUTag
L+ L- L+ L-
SI colon
What is the role of L-cell KATP channels?
Neurotransmitters?
Plasma glucose?
KATP channels
+
K +
K
Luminal
glucokinase
nutrients
Na++
Na
Na+
+
Na
H+
How might you target L-cells therapeutically?
Ca2+
GLP-1
secretion
Ca2+
Na+
cAMP
GLP-1 secretion from primary colonic L-cells – importance of cAMP
†††
12 6
***
Relative GLP-1 secretion 10
8 9
***
6
†††
7
4 *** 13
14 ***
*** ***
29 3
2
**
0
Con Fsk/IBMX PMA Bombesin SS
glucose - + - + - - + -
Monitoring dynamic cAMP changes in GLUTag cells using FRET sensors
Modified cAMP
binding protein
cAMP
520nm
Cyan Yellow
[Link]
430nm
FRET: excite cyan, detect yellow
100 µ M IBMX
1 µ M Forskolin
10 µ M Forskolin
1.20
CFP/YFP
1.15
1.10
1.05
1.00 1 min.
Gαs-coupled receptors under therapeutic evaluation
0.2
GPR119
0.16
0.12
Expression relative to β-actin by qRT-PCR
0.08
0.04
0
L- α β
GLUTag
K+ K- L+ L- L+
SI SI colon
GPBAR
0.02
(TGR5 / GPR131)
0.01
0
L- α β
GLUTag
K+ K- L+ L- L+
SI SI colon
Adaptation of enteroendocrine cells in vivo?
Nutritional Surgical
Genetic
??
Inflammatory
∆ L-cell number/function
Therapeutic
Metabolic
With thanks to:
Abdella Habib
Gwen Tolhurst
Helen Parker
Gareth Rogers
Asan Ramzan
Ronn Friedlander
Frank Reimann
Ria Diakogiannaki
Katie Moss
Daisuke Fukuoka
Giles Yeo Dermot Cooper Anna Petrunkina
Ian MacFarlane Martin Lohse
The Lister Institute of
Preventive Medicine