6BBL0394 Kisspeptin 2016

6BBL0394 2015/16
Kisspeptin: hypothalamus and beta cell
James Bowe
Diabetes Research Group
Kings College London
james.bowe@kcl.ac.uk
Identified in 1996 in Hershey,

Pennsylvania and named after the
towns most famous export
Kisspeptins are a family of peptides encoded by the Kiss-1 gene.
Identified as the endogenous ligands for the orphan G-protein

coupled receptor, GPR54.
Originally identified in cancer research
Metastasis the spread of primary tumours to other sites in the body
Tumour cell lines transfected with normal chromosome 6 remained tumourigenic

but lost ability to metastasize

Kiss-1 gene on chromosome 6 identified as suppressor of metastasis

Kiss-1 gene on chromosome 6 identified as suppressor of metastasis
Kisspeptin research concentrates on a role in metastasis UNTIL
2 papers in late 2003 independently link kisspeptin and GPR54 to

hypogonadotrophic hypogonadism
Hypogonadotrophic hypogonadism - a condition in which there is impaired

pubertal maturation and subsequent lack of reproductive function

Different forms sometimes a hereditary genetic defect

In a family displaying the hereditary form of

hypogonadotrophic hypogonadism, all
affected children had a mutation of the
GPR54 gene
De Roux et al (2003) PNAS 100, 10972-10976

In a family displaying the hereditary form of

hypogonadotrophic hypogonadism, all
affected children had a mutation of the
GPR54 gene
GPR54 knockout mice did not go through

puberty and reproductive organs remained
undeveloped
De Roux et al (2003) PNAS 100, 10972-10976; Seminara et al (2003) NEJM 349, 1614-1627
The discovery that

hypogonadotrophic
hypogonadism may be caused
by a mutation of GPR54, led to
the discovery that kisspeptin
plays a crucial role in controlling
the onset of puberty.
Hypothalamic Kiss-1 and GPR54

mRNA:
during puberty
Roa et al (2006) Endocrinology 147,

2864-2878

mRNA:
during puberty
changes through
estrous cycle

2864-2878

mRNA:
during puberty
changes through
estrous cycle
also during pregnancy

2864-2878
Hypothalamic Kiss-1 mRNA:

in obese animals
Smith et al (2006) J. Neuroendo 18, 298-303

in obese animals
Fasting
Control
in fasted animals
Castellano et al (2005) Endocrinology

146, 3917-3925

in obese animals
in fasted animals
BUT GPR54 mRNA:
Fasting
Control
in fasted animals
Castellano et al (2005) Endocrinology

146, 3917-3925
Plasma kisspeptin levels are

normally very low.
EXCEPT during pregnancy when

they increase by several thousandfold.
Dhillo et al (2006) Am J Physiol

Endocrinol Metab. 291(5), E878-884
Why is this relevant to diabetes?

Kisspeptin
GPR54
Ohtaki et al (2001) Nature 411, 613-617

Kisspeptin
GPR54

Kisspeptin
GPR54

Kisspeptin in the hypothalamus
is involved in puberty, pregnancy
and obesity.
All states associated with
increased insulin release and
insulin resistance.
Kahn et al (2006) Nature, 444: 840-846
The pancreatic -cell is an important sensor and a regulator of

whole body fuel homeostasis.
Pancreatic kisspeptin / GPR54 may be expressed in islets of
Langerhans, and may play a local role in regulating islet
function.
How do we understand what kisspeptin does?
Questions...
Where in the pancreas is kisspeptin and its receptor located?
What effects does it have on islet function?
How might the effects be physiologically relevant?
Questions...
Where is kisspeptin in the pancreas?

Kisspeptin
GPR54

Kisspeptin
Kisspeptin
GPR54
319bp
Islet
MIN6
Islet
MIN6
175bp
GPR54
Hauge-Evans et al (2006) Diabetologia 49,

2131-2135

Kisspeptin
Kisspeptin
GPR54
319bp
Islet
MIN6
Islet
MIN6
175bp
GPR54
Hauge-Evans et al (2006) Diabetologia 49,

2131-2135
Questions...
Localised to the islets.
Questions...
Effects of kisspeptin in vivo
kisspeptin iv
Kisspeptin stimulates insulin

release in vivo when
administered intravenously.
3.5
Insulin (ng)
Insulin (ng)
3
2.5
2
1.5
1
0.5
0
0
30
60
90
120
150
Time (min)
180
210
240
270
kisspeptin iv

3.5
Insulin (ng)
Insulin (ng)
3
2.5
2
1.5
1
0.5
0
0
30
60
90
120
150
180
210
240
270
Time (min)
1.6
kisspeptin icv
(ng)
Insulin
Insulin (ng)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
30
60
90
Tim e (m in)
120
150
Kisspeptin has no effect on

insulin release when
administered icv.

7
6.5
6
4
5.5
5
3.5
4.5
Insulin (ng)
Insulin (ng)
2.5
3.5
2
1.5
3
0
30
60
90
120
150
180
210
240

270
1
0.5
0
0
30
60
90
120
150
180
210
240
270
Time (min)
1.6
kisspeptin icv
1.4
(ng)
Insulin
Insulin (ng)
Plasma Glucose (mM)
kisspeptin iv

administered icv.
1.2
Plasma glucose levels do not

rise prior to the increase in
insulin.
1
0.8
0.6
0.4
0.2
0
0
30
60
90
Tim e (m in)
120
150

7
6.5
6
4
5.5
5
3.5
4.5
Insulin (ng)
Insulin (ng)
2.5
3.5
2
1.5
3
0
30
60
90
120
150
180
210
240

270
1
0.5
0
0
30
60
90
120
150
180
210
240
270
Time (min)
1.6
kisspeptin icv
1.4
(ng)
Insulin
Insulin (ng)
Plasma Glucose (mM)
kisspeptin iv

administered icv.
1.2
Plasma glucose levels do not

rise prior to the increase in
insulin.
1
0.8
0.6
0.4
0.2
0
0
30
60
90
120
150
Tim e (m in)
Suggests a direct effect of kisspeptin on the pancreas.
Effects of kisspeptin on insulin secretion
How quickly does kisspeptin act and is it reversible?
Are the effects of kisspeptin glucose dependant?
What dose of kisspeptin is required to cause insulin release?
What intracellular signalling pathways are involved?

All in vitro experiments used isolated mouse islets
Mouse pancreas was injected with collagenase to

digest exocrine tissue
Endocrine islets of langerhans were then

separated from digested exocrine tissue using a
density gradient
Isolated islets were then cultured at least overnight

before experiments
Perifusion
40 mouse islets placed in a chamber
Physiological salt solution containing treatments is pumped through them

and collected every 2 minutes for insulin measurement
Useful for testing the dynamics of insulin response
Perifusion
40 mouse islets placed in a chamber
Physiological salt solution containing treatments is pumped through them

and collected every 2 minutes for insulin measurement
Useful for testing the dynamics of insulin response
Static Incubation
3 mouse islets placed in a tube with physiological salt solution and
treatment
After 1 hour salt solution is collected for insulin measurement
Useful for testing a wide range of different treatments
14
20mM Glucose
12
Insulin (pg/islet/min)
Control
10
8
6
4
2
0
0
10
20
30
40
50
Time (min)
60
70
80
90

1M Kisspeptin
14
20mM Glucose
12
Control
+ Kisspeptin
10
8
6
4
2
0
0
10
20
30
40
50
Time (min)
60
70
80
90
1M KP
20 mM glucose
30
Mouse
Islets
25
20
15
10
5
0
0
10
20
30
40
Time (min)
50
60
70
1M KP
1M KP
20 mM glucose
30
Mouse
Islets
Rat Islets
20
25
20 mM glucose
25
20
15
10
15
10
5
0
0
0
10
20
30
40
Time (min)
50
60
70
10
20
30
40
Time (min)
50
60
70
1M KP
1M KP
20 mM glucose
30
Mouse
Islets
Rat Islets
20
25
20
15
10
15
10
5
0
0
0
10
20
30
40
Time (min)
50
60
70
1M KP
20 mM glucose
8
7
20 mM glucose
25
Pig Islets
6
5
4
3
2
1
0
0
10
20
30
40
Time (min)
50
60
70
10
20
30
40
Time (min)
50
60
70
1M KP
1M KP
20 mM glucose
30
Mouse
Islets
Rat Islets
20
25
20 mM glucose
25
20
15
10
15
10
5
0
0
0
10
20
30
40
Time (min)
50
60
70
10
20
60
70
20 mM glucose
Pig Islets
Human Islets
50
1M KP
1M KP
20 mM glucose
30
40
Time (min)
5
4
3
2
1
0
0
0
10
20
30
40
Time (min)
50
60
70
10
20
30
40
Time (min)
50
60
70
1. Stimulation of insulin release by kisspeptin was rapid in onset

and readily reversible.
3
Control
2.5
Insulin (ng)
1.5
0.5
0
2mM Glucose 5mM Glucose
7.5mM
Glucose
11mM
Glucose
15mM
Glucose
20mM
Glucose
3
Control
2.5
+ 100nM Kisspeptin
Insulin (ng)
1.5
0.5
2mM Glucose 5mM Glucose
7.5mM
Glucose
11mM
Glucose
15mM
Glucose
20mM
Glucose

2. Stimulatory glucose levels are required for a significant effect on
insulin release in islets.
6
2mM Glucose
20mM Glucose
Insulin (ng)
0
0
100pM
1nM
10nM
62.5nM
100nM
125nM
Kisspeptin concentration
250nM
500nM
1M

2. Stimulatory glucose levels are required for a significant effect on
insulin release in islets.
3. A kisspeptin concentration of at least 62.5nM is required for a

significant stimulation of insulin release from islets.
Intracellular signalling
Different G-proteins tend to be associated with different intracellular

signalling pathways.
Gs proteins tend to signal through PKC and increased cAMP
Gq proteins signal through PLC and increased intracellular Ca2+
GPR54 is a Gq protein coupled receptor
Ca2+
K+
Ca2+
Insulin
Ca2+
Insulin
Insulin
Insulin
Glucose
Ca2+
ATP
Glucose
Insulin
Insulin release from cells usually requires an increase in intracellular Ca2+
Calcium microfluorimetry
Islets are dispersed into separate cells and then treated with FURA-2AM
Islet cells will then fluoresce when intracellular calcium levels increase
Cells can be treated and changing calcium levels measured in real time
Useful for measuring changes in intracellular calcium in response to
treatments
1M Kisspeptin
100M
Tolbutamide
1M Kisspeptin
0.8
2mM Glucose
20mM Glucose
2mM Glucose
0.7
0.6
0.5
0.4
0.3
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Time (min)
Kisspeptin increases intracellular Ca2+ in islet beta cells
25
Also PLC inhibitors block the effects of kisspeptin on insulin release,
but PKC inhibitors do not.
PKC
Ca2+
DAG
IP3
Ca2+-sensitive
signalling elements
Gq
GPR54
KP
PLC
PIP2
Insulin release
Also PLC inhibitors block the effects of kisspeptin on insulin release,
but PKC inhibitors do not.
PKC
Ca2+
DAG
IP3
Ca2+-sensitive
signalling elements
Gq
GPR54
KP
PLC
PIP2
Insulin release
Kisspeptin works through PLC signalling and increased intracellular

Ca2+ - the expected pathway for a Gq-protein coupled receptor.
BUT
Silvestre et al (2001) J. Endocrinol. 196, 283-290
BUT
Silvestre et al (2001) J. Endocrinol. 196, 283-290
In perfused pancreas kisspeptin

appears to inhibit insulin secretion
7
6.5
6
4
5.5
5
3.5
4.5
Insulin (ng)
Insulin (ng)
2.5
3.5
2
1.5
3
0
30
60
90
120
150
180
210
240
270
30
60
90
120
150
180
210
240
270
1
0.5
0
Time (min)
Perfused Pancreas
In vivo
The effect seen in vivo is most likely to be reliable.
Plasma Glucose (mM)
kisspeptin iv
7
6.5
6
4
5.5
5
3.5
4.5
Insulin (ng)
Insulin (ng)
2.5
3.5
2
1.5
3
0
30
60
90
120
150
180
210
240
270
30
60
90
120
150
180
210
240
270
1
0.5
0
Time (min)
Perfused Pancreas
In vivo
The effect seen in vivo is most likely to be reliable.
Is an unknown factor causing kisspeptin to have a different effect

in perfused pancreas?
Plasma Glucose (mM)
kisspeptin iv

Questions...
Potentiates glucose-induced insulin secretion in vivo and in
vitro. No significant effect on other islet hormones or
apoptosis.

Questions...
apoptosis.
Physiological relevance
Kisspeptin in the hypothalamus is

involved in puberty, pregnancy and
obesity.
All states associated with increased
insulin release and insulin resistance
Kisspeptin in pregnancy
Kisspeptin
GPR54

Endocrinol Metab 291, 878-884
Kisspeptin in pregnancy
The effects of the large quantities
of kisspeptin released from the
placenta during pregnancy are
poorly understood.
Role in trophoblast invasion?
Possible role in fetal
development?
Islet function?

Endocrinol Metab 291, 878-884
The islet during pregnancy

During pregnancy there is a progressive increase in maternal
insulin resistance.
In a healthy pregnancy this insulin resistance is countered by
both functional and structural changes to the islets of
Langerhans.
Increased insulin secretion

Increased -cell proliferation
Hypertrophy (increased -cell size)

Under normal physiological
conditions -cells have an
extremely low proliferative
rate.
Dhawan et al (2007) Curr Opin Cell Biol.

19(6), 634-45

Under normal physiological
conditions -cells have an
extremely low proliferative
rate.
Pregnancy is one of the few

physiological states in which
-cells rapidly proliferate.
What is the purpose of kisspeptin in islets?

Does kisspeptin play a role in the adaptation of islets to
pregnancy?

pregnancy?
What happens to the islets in kisspeptin or GPR54 knockout mice?

pregnancy?

undeveloped

pregnancy?

undeveloped
What effect does administering chronic kisspeptin in non-pregnant mice

have on islet function?
What effect does blocking the endogenous kisspeptin during pregnancy

have on islet function?
Day 16: IPGTT
Day 1: Plug
date
Day 8: Implant
Osmotic
minipumps
Day 10: Start

BrdU treatment
Pregnant mice were implanted with osmotic minipumps containing kisspeptin

antagonist (200nmol/day) on day 2 of pregnancy.
Control mice were implanted with osmotic minipumps containing kisspeptin
(50nmol/day)
Day 18: IPITT
Day 18: Collect

pancreas for
histology
Day 16: IPGTT
Day 1: Plug
date
Day 8: Implant
Osmotic
minipumps
Day 10: Start

BrdU treatment
Day 18: IPITT
Day 18: Collect

pancreas for
histology

(50nmol/day)
1mg/ml BrdU administered in the drinking water for 10 days starting from day 10
of pregnancy until day 18.
Day 16: IPGTT
Day 1: Plug
date
Day 8: Implant
Osmotic
minipumps
Day 10: Start

BrdU treatment
Day 18: IPITT
Day 18: Collect

pancreas for
histology

(50nmol/day)
Intraperitoneal glucose tolerance test at day 16 and intraperitoneal insulin
tolerance test at day 18.
Day 16: IPGTT
Day 1: Plug
date
Day 8: Implant
Osmotic
minipumps
Day 10: Start

BrdU treatment
Day 18: IPITT
Day 18: Collect

pancreas for
histology

(50nmol/day)
Intraperitoneal glucose tolerance test at day 16 and intraperitoneal insulin
tolerance test at day 18.
Whole pancreas was removed and fixed for histology.
Glucose tolerance test

Intraperitoneal glucose injection
25
Plasma Glucose (mM)
20
15
10
0
0
30
60
Time (min)
90
120

25
Plasma Glucose (mM)
Control
Pregnant
20
15
10
0
0
30
60
Time (min)
90
120

25
25
Control
Pregnant
20
15
10
Plasma Glucose (mM)
Plasma Glucose (mM)
Control
Control + KP
20
15
10
0
0
30
60
Time (min)
90
Kisspeptin improves glucose

tolerance in non-pregnant mice.
120
30
60
Time (min)
90
120

25
25
Control
15
10
Plasma Glucose (mM)
Pregnant
20
Control + KP
20
15
10
0
0
30
60
Time (min)
90
120
30
60
Time (min)
90
120
25
Pregnant
Kisspeptin improves glucose

tolerance in non-pregnant mice.
Kisspeptin antagonist impairs
glucose tolerance in pregnant mice.
Plasma Glucose (mM)
Plasma Glucose (mM)
Control
Pregnant + KP antagonist
20
15
10
0
0
30
60
Time (min)
90
120

Changes in glucose tolerance can be due to either changes in insulin
resistance or due to changes in insulin release
Insulin release
3
0 min
30 min
Plasma Insulin (ug/L)
2.5
1.5
0.5
0
control
control & kp
In non-pregnant mice kisspeptin

causes an increased insulin
response to glucose.
Insulin release
3
0 min
30 min
Plasma Insulin (ug/L)
2.5
In non-pregnant mice kisspeptin

causes an increased insulin
response to glucose.
In pregnant mice blocking
endogenous kisspeptin causes a
reduced insulin response to
glucose.
1.5
0.5
control
control & kp
pregnant
pregnant & kp
antagonist

Improved insulin release in response to glucose
Insulin tolerance test

Intraperitoneal insulin injection
9
8
Plasma Glucose (mM)
6
5
4
3
2
1
0
0
15
30
Time (min)
45
60

9
Plasma Glucose (mM)
8
7
6
5
4
3
2
Control
1
Pregnant
0
0
15
30
Time (min)
45
60
Plasma Glucose (mM)
Plasma Glucose (mM)
6
5
4
3
2
Control
1
6
5
4
3
2
Control
Pregnant
Control + KP
0
0
15
30
Time (min)
45
Kisspeptin has no effect on insulin

sensitivity in non-pregnant mice
60
15
30
Time (min)
45
60
Plasma Glucose (mM)
7
6
5
4
3
2
Control
1
6
5
4
3
2
Control
Pregnant
Control + KP
0
0
15
30
Time (min)
45
60
15
30
Time (min)
45
60
45
60
Kisspeptin has no effect on insulin

sensitivity in non-pregnant mice
Or pregnant mice.
Plasma Glucose (mM)
Plasma Glucose (mM)
7
6
5
4
3
2
Pregnant
Pregnant + KP antagonist
0
0
15
30
Time (min)

No change in insulin resistance
Endogenous kisspeptin improves glucose tolerance during pregnancy

through increasing insulin release


Is kisspeptin involved in -cell proliferation during pregnancy?
-cell proliferation
KP
25
20
% BrdU positive -cells
Non-pregnant
Control
Control
KP /antagonist
KP antagonist
15
10
0
Non-pregnant
Pregnant
-cell proliferation
KP
Non-pregnant
Control
25
Pregnant
% BrdU positive -cells
20
Control
KP /antagonist
KP antagonist
15
10
0
Non-pregnant
Control
KP antagonist
Pregnant


Yes, endogenous kisspeptin is involved in increasing -cell proliferation

during pregnancy
kisspeptin
GPR54


Yes, endogenous kisspeptin is involved in increasing -cell proliferation

during pregnancy
Is this relevant in humans?
Links to gestational diabetes
Cetkovic et al (2012) Endocrine Research, 37(2): 78-88

Questions...
apoptosis.
Possible physiological role of kisspeptin in gestational
diabetes?
Take-home message
Potentially kisspeptin is an important regulator of

insulin release in altered reproductive and
nutritional states, particularly pregnancy.
HOWEVER, much more work is required to
understand its physiological role.

6BBL0394 Kisspeptin 2016

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6BBL0394 2015/16

Kisspeptin: hypothalamus and beta cell

Identified in 1996 in Hershey,

Kisspeptins are a family of peptides encoded by the Kiss-1 gene.

Identified as the endogenous ligands for the orphan G-protein

Originally identified in cancer research

Metastasis the spread of primary tumours to other sites in the body

Tumour cell lines transfected with normal chromosome 6 remained tumourigenic

Tumour cell lines transfected with normal chromosome 6 remained tumourigenic

Kiss-1 gene on chromosome 6 identified as suppressor of metastasis

Tumour cell lines transfected with normal chromosome 6 remained tumourigenic

Kiss-1 gene on chromosome 6 identified as suppressor of metastasis

Kisspeptin research concentrates on a role in metastasis UNTIL

2 papers in late 2003 independently link kisspeptin and GPR54 to

Hypogonadotrophic hypogonadism - a condition in which there is impaired

Hypogonadotrophic hypogonadism - a condition in which there is impaired

Hypogonadotrophic hypogonadism - a condition in which there is impaired

In a family displaying the hereditary form of

De Roux et al (2003) PNAS 100, 10972-10976

Hypogonadotrophic hypogonadism - a condition in which there is impaired

In a family displaying the hereditary form of

GPR54 knockout mice did not go through

The discovery that

Hypothalamic Kiss-1 and GPR54

Roa et al (2006) Endocrinology 147,

Hypothalamic Kiss-1 and GPR54

Roa et al (2006) Endocrinology 147,

Hypothalamic Kiss-1 and GPR54

Roa et al (2006) Endocrinology 147,

Hypothalamic Kiss-1 mRNA:

Smith et al (2006) J. Neuroendo 18, 298-303

Hypothalamic Kiss-1 mRNA:

Castellano et al (2005) Endocrinology

Hypothalamic Kiss-1 mRNA:

BUT GPR54 mRNA:

Castellano et al (2005) Endocrinology

Plasma kisspeptin levels are

EXCEPT during pregnancy when

Dhillo et al (2006) Am J Physiol

Why is this relevant to diabetes?

Why is this relevant to diabetes?

Ohtaki et al (2001) Nature 411, 613-617

Why is this relevant to diabetes?

Ohtaki et al (2001) Nature 411, 613-617

Why is this relevant to diabetes?

Ohtaki et al (2001) Nature 411, 613-617

Why is this relevant to diabetes?

Kahn et al (2006) Nature, 444: 840-846

Why is this relevant to diabetes?

The pancreatic -cell is an important sensor and a regulator of

How do we understand what kisspeptin does?

What effects does it have on islet function?

How might the effects be physiologically relevant?

How do we understand what kisspeptin does?

What effects does it have on islet function?

How might the effects be physiologically relevant?

Where is kisspeptin in the pancreas?

Ohtaki et al (2001) Nature 411, 613-617

Where is kisspeptin in the pancreas?

Hauge-Evans et al (2006) Diabetologia 49,

Where is kisspeptin in the pancreas?

Hauge-Evans et al (2006) Diabetologia 49,

How do we understand what kisspeptin does?