Pluripotent Roles for Neuropeptides in Metabolic and Cardiovascular Regulation — Implications for Therapeutic Design

Alastair Ferguson

Department of Biomedical and Molecular Sciences

T

CV Cent

Cardiovascular Control Centers

Met Cent

Metabolic Control Centers

Yeo and Heisler 2012

Met Peptid

CV Peptides

Levay

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

The Paraventricular Nucleus

Org PVN

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

Electrophysiological fingerprinting in the PVN

3V

1. Magnocellular (MNC) 2. Parvocellular Pre-autonomic (LTS) 3. Parvocellular Neuroendocrine (NE)

PVN ATR

Ang a Neurotransmitter in PVN

ANG immunoreactive projections

ANG receptors

Exogenous application

Endogenous release

Ephys MNC

ANG depolarises magnocellular neurons

NE Cells

ANG depolarises NE PVN neurons

NSCC

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

Losartan abolishes body weight gain in DIO rats

Mort

AT1 receptor knockout mice live longer

Benigni et al. JCI 2009

Summ

Messenger VP OT NE PA Channel CV Metabolic

Angiotensin II + + + + NSCC Yes Yes

Orexin

Adiponectin

Nesfatin-1

Summary: ANG – Activation

ORX

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

PVN Orexin Decreases Body Weight

Novak and Levine, Obesity 2009

PVN MNC

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

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

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

-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

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

Messenger VP OT NE PA Channel CV Metabolic

Angiotensin II + + + + NSCC Yes Yes

Orexin + + + + NSCC Yes

Yes

Adiponectin

Nesfatin-1

Summary: ORX - Activation

ADP

Adiponectin

ICV BW

ICV Adiponectin Decreases Body Weight

REC in PVN

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

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

Single Cell rtPCR Magnocellular Neurons

(+) Control 200 bp

400 bp

(-) Control 200 bp

400 bp

suction

PCR sum

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

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

Neuroendocrine CRH neurons

ACTH

-68 mV

10 mV

50 sec

10 mV

100 sec

-73 mV

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

0 60 120 180 240 300

-10

-5

0

Time (sec)

Norm

BP

(m

mH

g)

Adiponectin in PVN Decreases Blood pressure

Summ

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

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

Nesfatin-1 Actions in PVN

Maejima et al. Cell Metab 2009

PVN Neur

Nesfatin-1 Influences PVN Neurons

Ephys cell groups

No Specific Effects on scRTPCR ID’d Cells

T BP PVN

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

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

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

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”

Thanks

Saint Louis University

Rick Samson

Queen’s University

Trainees

Jaideep Bains

Christie DeVille

Matt Follwell

Ted Hoyda

Kevin Latchford

Chris Price

Pauline Smith

"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

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

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

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

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

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

Receptor expression in MNC OT neurons

scRT-PCR

OT 6/7

Parvo OT

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

SFO efferents excite PVN Cells

SFO

PVN

Pituitary Time (ms)

Spik

es/b

in

+

Median

Eminence

IML

Cycling

Mean ORX-A evoked NSCC

-80 -60 -40 -20 0 20

-100

100

-50

50

0

Vm (mV)

Cu

rre

nt

(pA

)

Model

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

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

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

No Specific Effects on Different Cell Groups

PCR

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