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Pluripotent Roles for Neuropeptides in Metabolic and Cardiovascular Regulation — Implications for Therapeutic Design
Alastair Ferguson
Department of Biomedical and Molecular Sciences
T
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CV Cent
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Cardiovascular Control Centers
Met Cent
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Metabolic Control Centers
Yeo and Heisler 2012
Met Peptid
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CV Peptides
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Levay
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The Hypothalamus
"People tend to stay away from the hypothalamus. Most brain scientists
prefer the sunny expanses of the cerebral cortex to the dark, claustrophobic
regions at the base of the brain. They think of the hypothalamus - though they
would never admit this to you - as haunted by animal spirits and the ghosts of
primal urges. They suspect that it houses, not the usual shiny hardware of
cognition, but some witches' brew of slimy, pulsating neurons adrift in a
broth of mind-altering chemicals.”
Simon LeVay Science 263:1994
Tracers
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The Paraventricular Nucleus
Org PVN
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Organization and peptidergic expression in the PVN
Posterior Pituitary
• Vasopressin
• Oxytocin
Brainstem/Spinal autonomic centers
• TRH • Oxytocin
3V
Median Eminence
• CRH • TRH
1. Magnocellular (MNC) 2. Parvocellular Preautonomic (PA) 3. Parvocellular Neuroendocrine (NE)
Cardiovascular and Energy Homeostasis
Ephys
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Electrophysiological fingerprinting in the PVN
3V
1. Magnocellular (MNC) 2. Parvocellular Pre-autonomic (LTS) 3. Parvocellular Neuroendocrine (NE)
PVN ATR
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Ang a Neurotransmitter in PVN
ANG immunoreactive projections
ANG receptors
Exogenous application
Endogenous release
Ephys MNC
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ANG depolarises magnocellular neurons
NE Cells
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ANG depolarises NE PVN neurons
NSCC
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ANG Influences NSCC and K+ Currents in NE Cells
25pA
-80 -60 -40 -20 0 20
Voltage (mV)
-80 -60 -40 -20 0
25pA
ANG
Control
ANG
Control
Recovery
Voltage (mV)
NSCC
25
0p
A
125mS
Control ANG
K+ Currents
LOS DIO
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Losartan abolishes body weight gain in DIO rats
Mort
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AT1 receptor knockout mice live longer
Benigni et al. JCI 2009
Summ
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Messenger VP OT NE PA Channel CV Metabolic
Angiotensin II + + + + NSCC Yes Yes
Orexin
Adiponectin
Nesfatin-1
Summary: ANG – Activation
ORX
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Orexins/Hypocretins
Two peptides from common 130 aa precursor
ORX-A - 33 aa
ORX-B - 28 aa
47% homology
Glp-Pro-Leu-Pro-Asp-Cys-Cys-Arg-Gln-
Lys-Thr-Cys-Ser-Cys- Arg-Leu-Tyr-Glu-
Leu-Leu-His-Gly-Ala-Gly-Asn-His-Ala-
Ala- Gly-Ile-Leu-Thr-Leu-NH2
From Yanagisawa
PVN BW
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PVN Orexin Decreases Body Weight
Novak and Levine, Obesity 2009
PVN MNC
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ORX-A Depolarizes MNCs
100 nM
500 nM
20 mV
60 s
M
em
bra
ne P
ote
nti
al
(mV
)
ORX (nM)
0 1.0 10 50 100 500
2.5
5.0
7.5
NE
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ORX-A Depolarizes NE neurons
100 nM
ORX-A
100 nM
ORX-B
500 nM
ORX-A
20 mV
60 s
M
em
bra
ne
Po
ten
tia
l (m
V)
0
5
10
15
OX-A (nM)
1 10 50 100 500
(6)
(10)
(29)
(10)
(4)
NSCC
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ORX-A activates NSCC
Vm (mV)
0.4
0.2
-0.2
Cu
rre
nt
(nA
)
0.6
0.8
Recovery
ORX-A
Control
1.0
0
-60 -20 0 -40 20
-80 -60 -20 0 20
50
25
-25
-50
0
Vm (mV)
Curr
ent (p
A)
Difference Current
PVN BP
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-30 0 30 60 90 120 150 180 210 240 270 300
-5
0
5
Time (s)No
rm B
P(m
mH
g)
Figure 2: Changes in blood pressure induced by microinjection
of 100nM ORX-A into the PVN (N=6) at time 0.
-30 0 30 60 90 120 150 180 210 240 270 300
-5
0
5
Time (s)No
rm B
P(m
mH
g)
Figure 2: Changes in blood pressure induced by microinjection
of 100nM ORX-A into the PVN (N=6) at time 0.
100nM
ORX-A
N=6
ORX-A in PVN increases BP
ICV ACTH
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0
50
100
150
200
250
300
350
Controls (prior to injection)
Saline vehicle controls
0.3 nmol Orexin A
1.0 nmol Orexin A
3.0 nmol Orexin A
0 15 30
Time (min) after i.c.v. administration
*
**
pg A
CT
H/m
l p
lasm
a (
Mea
n,
SE
M, n
=6
-10
ICV ORX-A increase plasma ACTH
T Summ
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Messenger VP OT NE PA Channel CV Metabolic
Angiotensin II + + + + NSCC Yes Yes
Orexin + + + + NSCC Yes
Yes
Adiponectin
Nesfatin-1
Summary: ORX - Activation
ADP
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Adiponectin
ICV BW
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ICV Adiponectin Decreases Body Weight
REC in PVN
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200 bp
500 bp
3v
AdipoR1
Magnocellular Parvocellular
ventral
dorsal
3v medial
AdipoR2
3v
FX
OC SCN SON
PVN
Adiponectin receptors in the PVN
PVN MNCS
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2
h h2hh2 adpon off h h2 h h3 h1 h1h
-53 mV
-57mV
10 nM ADP
10 nM ADP
Adiponectin Influences Magnocellular Neurons
SSRTPCR
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Single Cell rtPCR Magnocellular Neurons
(+) Control 200 bp
400 bp
(-) Control 200 bp
400 bp
suction
PCR sum
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Adiponectin Hyperpolarizes MNC OT Neurons
6/6
0/6
0/6
0/6
6/6
0/6
0/6
3/6
3/6
No Response
Hyperpolarization
Depolarization
Response to Adiponectin
Single cell
RT-PCR Identification
200 bp
400 bp
200 bp
400 bp
200 bp
400 bp
PA OT
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LTS OT-5
0
5
10
resp
on
se a
mp
litu
te (
mV
)
n = 15
Adiponectin Depolarizes PA OT neurons
-68 mV
10 mV
50 sec
NE CRH
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Neuroendocrine CRH neurons
ACTH
-68 mV
10 mV
50 sec
10 mV
100 sec
-73 mV
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Pla
sm
a A
CT
H L
evels
(pg/m
l, M
ean,
SE
M)
Plasma ACTH in response to ICV adiponectin
0
5
10
15
20
25
Vehicle (n = 12)
0.1 nmole ADP (12)
0.3 nmole ADP (7)
1.0 nmole ADP (5)
0 5 15 30
60
icv injections
* *
Time (minutes)
p=0.014
* p<0.05 versus vehicle (Betweeen Groups
ANOVA, Scheffe Multiple Comparisons)
p=0.007
PVN BP
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0 60 120 180 240 300
-10
-5
0
Time (sec)
Norm
BP
(m
mH
g)
Adiponectin in PVN Decreases Blood pressure
Summ
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Messenger VP OT NE PA Channel CV Metabolic
Angiotensin II + + + + NSCC Yes Yes
Orexin + + + + NSCC Yes
Yes
Adiponectin +/- - + (CRH)
+ (OT)
KATP Yes
Yes
Nesfatin-1
Summary: Adiponectin – differential regulation
NES
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Nesfatin-1 – non-specific actions in PVN
Product of the nucleobindin-2 gene
Described in 2006 by Oh et al.
Expressed in PVN
Profound inhibitor of food intake
Also profound inhibitor of drinking
NES PVN
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Nesfatin-1 Actions in PVN
Maejima et al. Cell Metab 2009
PVN Neur
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Nesfatin-1 Influences PVN Neurons
Ephys cell groups
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No Specific Effects on scRTPCR ID’d Cells
T BP PVN
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Nesfatin-1 Increases Blood Pressure
-30 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480
-5
0
5
10
PVN (n=6)
non PVN (n=7)
Time (sec)
no
rm m
ea
n B
P (
mm
Hg
)
Summ
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Messenger VP OT NE PA Channel CV Metabolic
Angiotensin II + + + + NSCC Yes Yes
Orexin + + + + NSCC Yes
Yes
Adiponectin +/- - + (CRH)
+ (OT)
KATP?? Yes
Yes
Nesfatin-1 +/- +/- +/- +/- KATP Yes
Yes
Summary – NES Apparently Disorganised
ABCs
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Signalling Molecules in PVN – ABC…..
Adiponectin
Adrenomedullin
ANP
Apelin
BDNF
BMP
CRH
Endothelin
Estradiol
Gastrin Releasing Peptide
Ghrelin
Glucocorticoids
GLP-1
Hydrogen Sulfide
Insulin
Interleukin
Leptin
MIF
MSH
Nesfatin
Neuronostatin
Nitric Oxide
PVN Levay
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Conclusions
All of these messengers exert both metabolic and cardiovascular actions by modulating the excitability of PVN neurons
Two potential explanations:
Very specialised subsets of PVN neurons control CV vs Metabolic Function
OR
The same neurons control both functions
and are thus involved in regulating and “integrated autonomic state”
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Thanks
Saint Louis University
Rick Samson
Queen’s University
Trainees
Jaideep Bains
Christie DeVille
Matt Follwell
Ted Hoyda
Kevin Latchford
Chris Price
Pauline Smith
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"People tend to stay away from the hypothalamus. Most brain scientists
prefer the sunny expanses of the cerebral cortex to the dark, claustrophobic
regions at the base of the brain. They think of the hypothalamus - though they
would never admit this to you - as haunted by animal spirits and the ghosts of
primal urges. They suspect that it houses, not the usual shiny hardware of
cognition, but some witches' brew of slimy, pulsating neurons adrift in a
broth of mind-altering chemicals.”
The Hypothalamus PVN
Simon LeVay Science 263:1994
Ack
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Methodology
Sterile Technique used for all procedures Materials
Microelectrode Glass: heat over-night at 300°C Clean DNase/RNase free filtered Intracellular Recording solution:
keep at -80°C in RNase free sterile 0.5 ml PCR tubes Clean syringe to expel cytoplasm from micropipette glass Dedicated latex examination gloves
Reagents: Dithiothreitol (DTT), dNTPs, MgCl - RNase inhibitor, random
primers Superscript II reverse transcriptase Qiagen Multiplex PCR Kit (for first round PCR) Outside/Nested Primers for genes of interest Platinum Taq DNA polymerase Kit
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RTPCR
Perform Reverse Transcriptase reaction to make cDNA (sooner is better).
Remove from incubator and freeze at -80°C
Perform first round Multiplex reaction using Qiagen kit and outside primer sets for genes of interest
Second round PCR using multiplex PCR products as template, nested primer sets and Platinum Taq DNA polymerase kit
Run second round products out on 2% agarose gel visualized with ethidium bromide
Confirm Sequence of Products
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Extracellular in vitro
PVN
0 50 100 150 200 250 300 3050 3100
Time (s)
0
5
10
Sp
ikes
/s
75 m
ANG LOS
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tota
l CRH
NE C
RH
LTS C
RH
-8
-6
-4
-2
0
2
4
6
8
10
12
resp
on
se a
mp
litu
te (
mV
)
13 10 3
-68 mV
Neuroendocrine CRH neurons
10 mV
50 sec
10 mV
100 sec
-73 mV
ACTH
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Pla
sm
a A
CT
H L
evels
(pg/m
l, M
ean,
SE
M)
Plasma ACTH in response to ICV adiponectin
0
5
10
15
20
25
Vehicle (n = 12)
0.1 nmole ADP (12)
0.3 nmole ADP (7)
1.0 nmole ADP (5)
0 5 15 30
60
icv injections
* *
Time (minutes)
p=0.014
* p<0.05 versus vehicle (Betweeen Groups
ANOVA, Scheffe Multiple Comparisons)
p=0.007
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Receptor expression in MNC OT neurons
scRT-PCR
OT 6/7
Parvo OT
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Losartan Blocks effects of SFO Stim. On MNCs
0 100 200 300 400
Time (ms)
0
15
Sp
ikes
/Bin
0 100 200 300 400
Time (ms)
0
15
Sp
ikes
/Bin
Control Losartan SFO
PVN
Pituitary T
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SFO efferents excite PVN Cells
SFO
PVN
Pituitary Time (ms)
Spik
es/b
in
+
Median
Eminence
IML
Cycling
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Mean ORX-A evoked NSCC
-80 -60 -40 -20 0 20
-100
100
-50
50
0
Vm (mV)
Cu
rre
nt
(pA
)
Model
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Adiponectin Effects Magnocellular Neurons
-15
-10
-5
0
5
10
15
23/55 17/55 15/55
Change in M
em
bra
ne P
ote
ntial (m
V)
*** ***
RTPCR
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Adiponectin and the oxytocin system
Adiponectin hyperpolarizes magnocellular oxytocin
neurons These neurons express mRNA for both AdipoR1 and AdipoR2
Pre-autonomic oxytocin neurons depolarize in the
presence of adiponectin
Adiponectin has opposite effects on functionally distinct OT expressing neurons in the PVN
T
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Intracerebroventricular Injection of Nesfatin-1 InhibitsOvernight Water Restriction-Induced Drinking in Male Rats
Time (min) after replacementof water bottles
Cum
ula
tive w
ate
r in
take
(ml/100 g
body w
eig
ht, M
ean, S
EM
)
0
1
2
3
4
5
6
7
Saline vehicle (n=27)
180 pmole Nesfatin-1 (n=7)
540 pmole Nesfatin-1 (n=6)
0 15 30 45 60 90 120 180 240 300
i.c.v. injections 10 minbefore water bottles replaced
***
****
******
******
*** *** ****
***
*
* versus 180 pmole dose
* versus saline vehicle
*
*
Nesfatin-1 Inhibits Drinking
PVN Neurons
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No Specific Effects on Different Cell Groups
PCR
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PVN
SFO
0 30 60 90 120
Time (sec)
40
60
80
100
120
Blo
od
pre
ssu
re (
mm
Hg
)
Control
Post-losartan
Losartan Blocks Cardiovascular effects of SFO Stim.
20 ng 50ng Dose ANG
0
5
10
15
20
BP
(m
mH
g)
Ch
ang
e
Los PSH