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

M.Lohse
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