The Pennsylvania State University
The Graduate School
Eberly College of Science
REGULATION OF NEUROPEPTIDE Y EXPRESSION AND THE SYMPATHO-
ADRENAL RESPONSE TO STRESS
A Dissertation in
Cell and Developmental Biology
by
Qian Wang
2011 Qian Wang
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
August 2011
The dissertation of Qian Wang was reviewed and approved* by the following:
Douglas Cavener
Professor of Biology
Co-Chair of Committee
Dissertation Advisor
Matthew Whim
Associate Professor of Cell Biology and Anatomy
Louisiana State University Health Science Center
Special member
Co-Chair of Committee
Bernhard Luscher
Professor of Biology, Biochemistry & Molecular Biology, and Psychiatry
Melissa Rolls
Assistant Professor of Biochemistry & Molecular Biology
Byron Jones
Professor of Biobehavioral Health
Zhi-chun Lai
Professor of Biology and Biochemistry & Molecular Biology
Head of Department of Cell and Developmental Biology
*Signatures are on file in the Graduate School
iii
ABSTRACT
Neuropeptide Y (NPY) is a 36 amino acid peptide that is synthesized by many neurons in
the central and peripheral nervous systems. NPY has been implicated in many physiological
functions including the regulation of feeding, anxiety, the stress response and cardiovascular
modulation. A single nucleotide polymorphism (SNP) in the NPY gene has been found to be
associated with elevated serum cholesterol levels, cardiovascular disease and diabetes. This SNP
changes the seventh amino acid in the signal sequence of the NPY preprohormone (preproNPY)
from leucine to proline. It is predicted to alter NPY synthesis, trafficking, or secretion. I tested
this hypothesis by expressing the mutant and wild-type preprohormones tagged with fluorescent
proteins in AtT-20 cells, an endocrine cell line. Prohormone from the mutant preproNPY was
synthesized and processed normally and entered the regulated secretory pathway. When
expressed in endocrine cells, NPY derived from both wild-type and mutant preproNPY were
found in the same large dense core granules. However the SNP altered the degree of co-
packaging and individual granules contained more NPY generated from the mutant preproNPY. I
conclude that the SNP enhances the packaging of NPY. This SNP also led to an increase in the
secretion of NPY, which is likely the consequential effect of the increased biosynthesis of the
prohormone. These findings are consistent with the observation that humans carrying this SNP
have higher levels of plasma NPY after exercise.
Peripheral NPY is derived from the sympathetic nervous system, which includes the
adrenal medulla. This tissue contains modified post-ganglionic neurons and is both part of the
sympathetic system and an endocrine gland. Mammalian adrenal glands consist of two regions,
the cortex and the medulla. Stress activates the HPA axis leading to a secretion of the
glucocorticoids (CORT) from the adrenal cortex. The sympatho-adrenal system is also activated
by stress leading to a release of catecholamines from the adrenal medulla. NPY and tyrosine
iv
hydroxylase (TH, a marker of catecholamine synthesis) are co-expressed in all mouse chromaffin
cells. Adrenal NPY transcription is known to be increased by various stressors and here I
investigate whether NPY has a regulatory role on adrenal activity. To address this question, mice
were exposed to an acute stressor the cold water forced swim test (cold FST) for 5-6 minutes. The
cold FST elevated both the electrical activity of chromaffin cells and the HPA axis. Unexpectedly,
activation of both limbs of the stress response was sustained even 24 hours after the cold FST.
The level of adrenal NPY-ir was significantly increased 24 hours after the cold FST both in vitro
and in situ, and this increase reversed one week later. The stress-induced increase in the levels of
adrenal NPY involved an up-regulation in the activity of the NPY promoter. To determine
whether NPY modulated adrenal activity, both wild type and NPY knockout mice were exposed
to the cold FST. Loss of NPY did not affect the stress-induced increase in plasma Cort, but the
removal of NPY significantly altered the neuronal branch of the stress response. That is, stress
increased the levels of adrenal TH-ir in situ in the wild type mice but the level of adrenal TH-ir
was not altered in the NPY knockout mice. Injection of BIBP 3226, a NPY Y1 antagonist was
associated with an increase in the level of TH-ir in adrenal slices in wild type mice indicating that
NPY negatively regulated adrenal TH via Y1 receptors. I conclude that acute stress has a long-
term effect on the synthetic capacity of the adrenal glands and that NPY plays an important role
in inhibiting the neuronal branch of the stress response.
v
Table of Contents
List of Figures ........................................................................................................................... ix
List of Tables ........................................................................................................................... .xi
Acknowledgements .................................................................................................................. xii
Chapter 1 Introduction ............................................................................................................ 1
1.1 Neurotransmitters in the nervous system ................................................................... 1 1.2 Hypocretin: one example of a prototypical neuropeptide .......................................... 2 1.3 Distribution of neuropeptide Y in the nervous system ............................................... 3 1.4 Processing and biosynthesis of neuropeptide Y ......................................................... 4 1.5 Physiological functions of neuropeptide Y ................................................................ 7
1.5.1 NPY in the cardiovascular system................................................................... 7 1.5.2 NPY in feeding and metabolism ..................................................................... 9 1.5.3 NPY in emotional regulation ........................................................................... 10 1.5.4 NPY in epilepsy .............................................................................................. 11
1.6 My thesis research on neuropeptide Y ....................................................................... 11
Chapter 2 Materials and Methods ........................................................................................... 14
2.1 Specific Methods for Chapter 3 ................................................................................. 14 2.1.1 Site-directed mutagenesis ................................................................................ 14 2.1.2 AtT-20 cells culturing ..................................................................................... 15 2.1.3 Transfection of AtT-20 cells ........................................................................... 15 2.1.4 Immunoprecipiation and Western blot ............................................................ 15 2.1.5 Immunocytochemistry ..................................................................................... 16 2.1.6 Image analysis ................................................................................................. 17
2.2 General Methods for Chapter 4 and Chapter 5 .......................................................... 18 2.2.1 Animals ........................................................................................................... 18 2.2.2 Cold water forced swim test ............................................................................ 18 2.2.3 Chromaffin cell cultures .................................................................................. 19 2.2.4 Immunocytochemistry ..................................................................................... 20 2.2.5 Cryostat frozen sectioning ............................................................................... 20 2.2.6 Immunohistochemistry .................................................................................... 21 2.2.7 Image analysis ................................................................................................. 21 2.2.8 Statistical analysis ........................................................................................... 22 2.2.9 Corticosterone ELISA ..................................................................................... 22
2.3 Specific Methods for chapter 4 .................................................................................. 23 2.3.1 Fox urine exposure .......................................................................................... 23 2.3.2 Semi-quantitative RT-PCR of NPY ................................................................ 23 2.3.3 RT-PCR of PP and PYY ................................................................................. 23
2.4 Specific Methods for chapter 5 .................................................................................. 24 2.4.1 Catecholamine ELISA ..................................................................................... 24 2.4.2 Antagonist injection ........................................................................................ 25 2.4.3 RT-PCR ........................................................................................................... 25
vi
Chapter 3 A Single Nucleotide Polymorphism Alters the Synthesis and Secretion of
Neuropeptide Y ................................................................................................................ 27
3.1 Introduction ................................................................................................................ 27 3.2 Results ........................................................................................................................ 30
3.2.1 Generation of the wild type and mutant NPY fusion protein constructs ......... 30 3.2.2 Expression of NPY prohormones derived from wild type and mutant
preproNPY........................................................................................................ 30 3.2.3 NPY prohormones derived from L7P and wild type preproNPY were
sorted similarly in AtT-20 cells ........................................................................ 31 3.2.4 NPY prohormones derived from L7P and wild type preproNPY entered
the same dense core granules ........................................................................... 32 3.2.5 Prohormones derived from L7P and wild type preproNPY were
differentially co-packaged ................................................................................ 33 3.2.6 NPY prohormones derived from L7P and wild type preproNPY were
sorted similarly in hippocampal neurons .......................................................... 34 3.2.7 Expression of NPY prohormone derived from mutant preproNPY leads to
increased NPY secretion................................................................................... 34 3.3 Discussion .................................................................................................................. 52
3.3.1 The SNP might affect the ER trafficking efficiency of the NPY
preprohormone ................................................................................................. 52 3.3.2 The SNP might cause a more efficient sorting of the NPY prohormone ........ 53 3.3.3 Codon usage does not explain the effect of the SNP ....................................... 54 3.3.4 Comparison of NPY T1128 with SNPs found in other peptide
preprohormones and prohormones ................................................................... 54 3.4 Conclusion ................................................................................................................. 55
Chapter 4 Acute Stress Increases the Levels of Adrenal Neuropeptide Y .............................. 56
4.1 Introduction ................................................................................................................ 56 4.2 Results ........................................................................................................................ 59
4.2.1 The cold FST activated the HPA axis and induced activity in chromaffin
cells ................................................................................................................... 59 4.2.2 GFP is expressed in the hypothalamus, hippocampus and adrenal medulla
in NPY (GFP) transgenic mice ......................................................................... 60 4.2.3 An NPY antibody specifically recognizes NPY in chromaffin cells............... 60 4.2.4 The cold FST led to an up-regulation of NPY-immunoreactivity in
chromaffin cells in vitro ................................................................................... 61 4.2.5 Brief exposure to fox urine also led to an up-regulation of NPY-
immunoreactivity in chromaffin cells in vitro .................................................. 62 4.2.6 The cold FST increased NPY-ir measured in adrenal slices ........................... 62 4.2.7 The cold FST-induced increase of NPY-ir in adrenal slices returned to
baseline one week after stress ........................................................................... 63 4.2.8 The cold FST increased GFP expression in NPY (GFP) transgenic mice ...... 63 4.2.9 Adrenal NPY mRNA expression was increased 3 hours after the cold FST .. 64 4.2.10 Pancreatic polypeptide and peptide YY mRNA expression in the adrenal
medulla after the cold FST ............................................................................... 65 4.3 Discussion .................................................................................................................. 89
4.3.1 Stress increases adrenal NPY transcription and peptide synthesis. ................. 89
vii
4.3.2 The mechanism that is involved in the stress-induced increase of adrenal
NPY. ................................................................................................................. 90 4.3.3 The transcriptional factors that are involved in response of the adrenal
medulla to stress. .............................................................................................. 90 4.3.4 Adrenal epinephrine in the stress response. .................................................... 92 4.3.5 Stress-induced secretion and synthesis coupling ............................................. 93
4.4 Conclusion ................................................................................................................. 94
Chapter 5 NPY Inhibits the Neuronal Branch of the Stress Response.................................... 95
5.1 Introduction ................................................................................................................ 95 5.2 Results ........................................................................................................................ 97
5.2.1 The cold FST led to a sustained activation of the HPA axis ........................... 97 5.2.2 The cold FST led to a sustained activation of the neuronal pathway .............. 97 5.2.3 Co-localization of NPY and TH in the adrenal medulla ................................. 98 5.2.4 The cold FST increased the level of PNMT-immunoreactivity in
chromaffin cells in vitro ................................................................................... 98 5.2.5 The cold FST increased tyrosine hydroxylase-immunoreactivity in adrenal
slices ................................................................................................................. 99 5.2.6 The cold FST-induced increase of TH-ir was reversible in adrenal slices ...... 99 5.2.7 The loss of NPY did not affect the stress-induced increase in plasma
corticosterone ................................................................................................... 100 5.2.8 The loss of NPY altered the neuronal branch of the stress response ............... 100 5.2.9 Expression of NPY Y receptors in the adrenal medulla .................................. 101 5.2.10 Injection of BIBP3226 a NPY Y1 antagonist was associated with an
increase in adrenal TH-ir .................................................................................. 101 5.2.11 Injection of BIIE0246 an NPY Y2 antagonist or L152,804, an NPY Y5
antagonist, did not alter adrenal TH-ir ............................................................. 102 5.2.12 Injection of BIIE0246 was associated with an increase in adrenal NPY-ir... 103 5.2.13 Injection of BIBP3226 or L152,804 did not alter adrenal NPY-ir ................ 103
5.3 Discussion .................................................................................................................. 137 5.3.1 NPY inhibits the expression of adrenal TH and NPY. .................................... 137 5.3.2 NPY does not completely suppress the stress response. ................................. 138 5.3.3 Possible mechanisms underlying the regulatory role of NPY in controlling
the levels of adrenal TH and NPY. ................................................................... 139 5.3.4 Working hypothesis of how adrenal NPY modulates adrenal activity in
the stress response. ........................................................................................... 141 5.4 Conclusion ................................................................................................................. 142
Chapter 6 Discussion .............................................................................................................. 145
6.1 Role of NPY in the cardiovascular system................................................................. 146 6.1.1 Role of peripheral NPY in cardiovascular modulation ................................... 146 6.1.2 Role of the adrenal medulla in cardiac function .............................................. 147 6.1.3 Role of central NPY in the cardiovascular system .......................................... 148
6.2 Role of NPY in metabolic regulation ......................................................................... 148 6.2.1 Role of central NPY in feeding ....................................................................... 148 6.2.2 Role of peripheral NPY in metabolic regulation ............................................. 149
6.3 Role of NPY in the stress response ............................................................................ 150
viii
6.3.1 Role of NPY in controlling the two major stress pathways in the PNS .......... 150 6.3.2 Peripheral NPY mediates stress-induced hypertension ................................... 151 6.3.3 Antagonistic effect of central and peripheral NPY in the cardiac response
may modify the stress response ........................................................................ 152 6.3.4 Interaction between NPY and catecholamines in cardiovascular
modulation may be a survival mechanism in response to stress ...................... 152 6.3.5 Peripheral NPY mediates stress-induced obesity ............................................ 154
6.4 Future directions ........................................................................................................ 155
Appendix A .............................................................................................................................. 156
Appendix B .............................................................................................................................. 157
Appendix C .............................................................................................................................. 158
References ................................................................................................................................ 161
ix
List of Figures
Figure 1-1. Processing pathway of NPY. ................................................................................. 6
Figure 3-1. Schematic of NPY fluorescent fusion protein constructs. ..................................... 36
Figure 3-2. Expression of wild type and mutant prohormones. ............................................... 38
Figure 3-3. Venus fluorescent signals were directly detected on the SDS-PAGE gel using
a Typhoon 8600 gel scanner. ........................................................................................... 40
Figure 3-4. Wild type and L7P NPY prohormones were sorted into the regulated
secretory pathway in AtT-20 cells. .................................................................................. 42
Figure 3-5. L7P and wild type prohormones were packaged into the same dense core
granules in AtT-20 cells. .................................................................................................. 44
Figure 3-6. Mutant and wild type prohormones were differentially co-packaged into
single dense core granules in AtT-20 cells....................................................................... 46
Figure 3-7. Co-expression and co-localization of NPY and FMRFamide in AtT-20 cells. ..... 48
Figure 3-8. NPY T1128 polymorphism leads to an increase in neuropeptide secretion. ......... 50
Figure 4-1. The two major peripheral stress pathways. ........................................................... 57
Figure 4-2. The cold FST activated the HPA axis ................................................................... 67
Figure 4-3. The cold FST activated the neuronal branch of the stress reponse. ...................... 69
Figure 4-4. NPY (GFP)-expressing neurons and neuroendocrine cells in the nervous
system. ............................................................................................................................. 71
Figure 4-5. NPY-ir in chromaffin cells from wild type and NPY knockout mice. .................. 73
Figure 4-6. The cold FST increased the level of NPY-ir in chromaffin cells in vitro. ............ 75
Figure 4-7. Brief exposure to fox urine increased the level of NPY-ir in chromaffin cells
in vitro. ............................................................................................................................. 77
Figure 4-8. The cold FST increased the level of adrenal NPY-ir in situ. ................................ 79
Figure 4-9. The cold FST reversibly increased the level of adrenal NPY in situ. ................... 81
Figure 4-10. The cold FST increase GFP expression in chromaffin cells from NPY (GFP)
mice. ................................................................................................................................. 83
Figure 4-11. The adrenal levels of NPY mRNA increased 3 hours after the cold FST. .......... 85
Figure 4-12. Expression of PP, PYY and NPY mRNA in the adrenal medulla....................... 87
x
Figure 5-1. Catecholamine biosynthetic pathway. ................................................................... 96
Figure 5-2. The cold FST led to a sustained activation of the HPA axis. ................................ 104
Figure 5-3. The cold FST led to a sustained activation of the neuronal axis of the stress
response. ........................................................................................................................... 106
Figure 5-4. Co-localization of NPY and TH in chromaffin cells. ............................................ 108
Figure 5-5. The cold FST increased PNMT-ir in chromaffin cells in vitro. ............................ 110
Figure 5-6. The cold FST increased the level of adrenal TH-ir in situ. ................................... 112
Figure 5-7. The cold FST reversibly increased the level of adrenal TH-ir in situ. .................. 114
Figure 5-8. The cold FST led to a sustained activation of the HPA axis in NPY knockout
mice. ................................................................................................................................. 116
Figure 5-9. The cold FST did not increase the level of adrenal TH-ir in NPY knockout
mice. ................................................................................................................................. 118
Figure 5-10. Expression of NPY Y receptors in the adrenal medulla and whole brain. .......... 120
Figure 5-11. Blocking the NPY Y1 receptor was associated with an increase in the level
of adrenal TH-ir ............................................................................................................... 122
Figure 5-12. Injection per se was sufficiently stressful that it led to an increase in the
level of adrenal TH-ir. ...................................................................................................... 124
Figure 5-13. Blocking the NPY Y1 receptor in NPY knockout mice did not alter the level
of adrenal TH-ir. .............................................................................................................. 126
Figure 5-14. Blocking the NPY Y2 or Y5 receptor did not affect the level of adrenal TH-
ir. ...................................................................................................................................... 128
Figure 5-15. Blocking the NPY Y2 receptor was associated with an increase in the level
of NPY-ir in the adrenal medulla ..................................................................................... 130
Figure 5-16. Injection per se did not alter the level of adrenal NPY-ir ................................... 132
Figure 5-17. Blocking the NPY Y1or Y5 receptor did not affect the level of adrenal NPY-
ir. ...................................................................................................................................... 134
Figure 5-18. Working model. ................................................................................................... 141
xi
List of Tables
Table 1. Primers, annealing temperature and amplifying cycles for NPY Y receptors RT-
PCR.. ..................................................................................................................... 26
xii
Acknowledgements
My Ph.D. thesis cannot be done without the patient guidance, heart-warming
encouragement, inspirational motivation and continuous support from Dr. Matthew Whim, my
supervisor and mentor. I really appreciate Matthew’s careful supervision in my experiments and
writing during my Ph.D. training. I also thank him for his useful advice on my postdoc seeking
and his care in my career plan. I respect his careful attitude and decent accomplishment in science,
which inspires and guides me to continue with my academic career.
I want to show my thanks to Dr. June Liu for her suggestions and support in my Ph.D.
thesis research and her kindly care in my life. My thanks also go to my committee members, Dr.
Douglas Cavener, Dr. Bernhard Luscher, Dr. Melissa Rolls and Dr. Byron Jones for their help
and advice in my research and dissertation writing.
I acknowledge all the previous and current lab members for their suggestions and help in
my research projects.
Finally I would like to thank my Dad Tiejun Wang, Mom Fusheng Bian and Fiancé
Jiehuan huang for their endless love and support to me.
Chapter 1
Introduction
1.1 Neurotransmitters in the nervous system
The nervous system contains two classes of neurotransmitters. The first group contains
classical neurotransmitters and some examples are acetylcholine, the catecholamines, glutamate
and GABA. The other type includes neuropeptide transmitters such as ghrelin, agouti-related
peptide (AgRP) and neuropeptide Y (NPY). Neuropeptides are often located in the same neurons
as classical transmitters (Hokfelt et al., 1980; Hokfelt et al., 1987). Classical neurotransmitters are
usually packaged into small clear-cored vesicles and released from presynaptic varicosities. In
contrast, neuropeptides are peptides consisting of a few up to a hundred amino acids. They are
synthesized in the cell body and stored in dense core secretory granules (DCSGs). DCSGs can
fuse at the soma, presynaptic and postsynaptic sites outside of synaptic active zones (Brigadski et
al., 2005; Hook et al., 2008; Huang and Neher, 1996; Lochner et al., 2006; Sobota et al., 2010;
Whim, 2006).
As neurotransmitters, neuropeptides mediate cell-cell communication in synaptic
transmission and can regulate the excitability of neurons by depolarization or hyperpolarization of
the membrane potential (Hook et al., 2008). As peptide hormones, neuropeptides are required for
endocrine modulation of central and peripheral targets and can alter gene expression, blood flow
and the secretion of other hormones. The same neuropeptides can play important roles both in
neurotransmission and as circulating hormones (Cavadas et al., 2006; Fu et al., 2004; Hook et al.,
2008; Kuo et al., 2007b; Silva et al., 2001; Urban and Randic, 1984). For example as a
neurotransmitter corticotropin-releasing factor (CRF) increases the excitability of hippocampal
2
neurons (Blank et al., 2003). As a hormone CRF triggers ACTH release from the anterior
pituitary leading to the secretion of glucocorticoids into the blood so altering metabolism during
the stress response (Sapolsky et al., 2000).
1.2 Hypocretin: one example of a prototypical neuropeptide
One example of neuropeptide which has been thoroughly studied is hypocretin (Hcrt)
which is also known as orexin. Hcrt 1 and 2 (also called orexin A and B) are two forms of this
neuropeptide which are derived from the same precursor preprohypocretin which is selectively
expressed in the hypothalamus (de Lecea et al., 1998; Gautvik et al., 1996).
Intracerebroventricular (i.c.v) injection of Hcrt during the light cycle when rodents are least
active promoted wakefulness and induced hyperphagia in rats suggesting a role for Hcrt in
controlling sleeping and feeding (Sakurai et al., 1998; Willie et al., 2001). Hcrt knockout mice ate
significantly less compared to wild type controls (Willie et al., 2001). In addition Hcrt-deficient
mice or both Hcrt 1 and Hcrt 2 receptors double knockout mice displayed a narcolepsy phenotype
(Chemelli et al., 1999; Sakurai, 2007). Hcrt evoked a substantial but reversible increase in the
frequency of excitatory postsynaptic currents in rat hypothalamic neurons indicating the
excitatory role of Hcrt in mediating synaptic transmission (de Lecea et al., 1998). These peptides
activated wake-promoting neurons in the hypothalamus and brain stem to maintain a consolidated
awake state (Rolls et al., 2010; Sakurai, 2007).
Disruption of sleep is strongly associated with impaired glucose metabolism and obesity
risk (Spiegel et al., 2009). Since hypothalamic Hcrt neurons play significant roles in regulating
both metabolism and wakefulness, it is possible that they centrally coordinate metabolism as well
as arousal to maintain homeostasis (Rolls et al., 2010). Hcrt neurons send projections to many
brain regions including the hippocampus, amygdale and ventral tegmental area (VTA) as well as
3
to the pituitary and sympathetic neurons in the spinal cord suggesting that Hcrt may participate in
autonomic regulation and the stress response (Sakurai, 2007; van den Pol, 1999; Willie et al.,
2001). Both central and peripheral injections of Hcrt increased plasma levels of corticosterone
(Hagan et al., 1999; Malendowicz et al., 1999). Hcrt 1 and 2 receptors are expressed in the
adrenal medulla thus Hcrt may affect epinephrine release and in this way modulate vascular tone
(Lopez et al., 1999). This idea is consistent with the finding that Hcrt injection increased heart
rate and blood pressure (Lopez et al., 1999; Shirasaka et al., 1999) .
Many neuropeptides, like Hcrt, are involved in regulating numerous physiological
processes centrally as well as peripherally. Understanding the function of the nervous system
involves determining the mechanisms for controlling neuropeptide synthesis and secretion.
1.3 Distribution of neuropeptide Y in the nervous system
Neuropeptide Y (NPY) is a 36 amino acid peptide which is widely expressed throughout
the central as well as peripheral nervous systems in mammals (Lundberg et al., 1982b; Tatemoto,
1982b; Tatemoto et al., 1982). It is present in many brain regions including the hippocampus,
hypothalamus, amygdala and cortex (Adrian et al., 1983; Allen et al., 1983; Gray and Morley,
1986), the peripheral nervous system including sympathetic post-ganglionic neurons and the
adrenal medulla (Lundberg et al., 1986; Lundberg et al., 1984; Lundberg et al., 1983; Renshaw
and Hinson, 2001) as well as peripheral organs such as the pancreas and spleen (Ericsson et al.,
1987; Whim, 2011). The primary structure of human NPY differs from porcine NPY in only one
amino acid (Corder et al., 1984; Tatemoto et al., 1982). Studies have revealed that NPY is
strongly conserved throughout evolution in many species from frogs, birds, rodents to humans
(Hoyle, 1999; Larhammar et al., 1993) and this indicates an important physiological function of
NPY.
4
In the central nervous system, substantial amounts of NPY are found in hypothalamic
nuclei including the arcuate nucleus, paraventricular nucleus and dorsomedial nucleus (Allen et
al., 1983; Chronwall, 1985). In 1983 it was first reported that NPY-like immunoreactivity was
present in catecholamine neurons (adrenergic neurons) in the human medulla oblongata (Hokfelt
et al., 1983). Since then other studies have shown that not only catecholamines but also a variety
of other neurotransmitters such as GABA or neuropeptides such as AgRP coexist with NPY
(Aoki and Pickel, 1990; Morton and Schwartz, 2001). Therefore NPY may function in the brain
by interacting with other modulators.
Peripherally it was first demonstrated in 1982 that NPY-like immunoreactivity was found
within neurons that contained tyrosine hydroxylase (TH, the initial and rate-limiting enzyme for
catecholamine biosynthesis) and dopamine-beta-hydroxylase (DβH, an enzyme downstream of
TH which is also required for catecholamine production) (Kvetnansky et al., 2009; Lundberg et
al., 1982b). NPY has been found in peripheral sympathetic neurons and is co-stored and co-
released with the catecholamines (Bischoff and Michel, 1998; Lundberg et al., 1983; Mormede et
al., 1990; Whim, 2006). NPY is also found in peripheral non-neuronal tissues such as the spleen
(Ericsson et al., 1987). This distribution suggested that there are potential interactions between
NPY and adrenergic systems in modulating peripheral physiology.
1.4 Processing and biosynthesis of neuropeptide Y
NPY is derived from a larger protein precursor, the 69 amino-acid peptide
proneuropeptide Y (proNPY). The precursor hormone is first expressed as a 97 amino-acid
peptide preproneuropeptide Y (preproNPY) and the 28 amino-acid signal sequence of preproNPY
is co-translationally removed by cleavage by a membrane-bound signal peptidase when the
preproNPY translocates into the ER lumen. The proNPY is transported to the cis-Golgi network
5
and then from the trans-Golgi network, proNPY is packaged into DCSGs where processing
enzymes including prohormone convertase 2 (PC-2) or prohormone covertase 1/3 (PC 1/3),
peptidylglycine α-amidating monooxygenase (PAM) and carboxypeptidase E (CPE) are co-
packaged (Marx et al., 1999). PC-2 in the superior cervical ganglion neurons or PC 1/3 in the
AtT-20 cell line cleaves the carboxyl-terminal peptide of NPY (CPON)(Paquet et al., 1996). CPE
removes the basic residues and PAM amidates the peptide intermediate to generate the
biologically active 36 amino acid peptide, NPY (Cerda-Reverter and Larhammar, 2000; Dikeakos
and Reudelhuber, 2007; Hegde and Bernstein, 2006; Hook et al., 2008; Marx et al., 1999)(Figure
1-1). Although CPON has been shown to be co-stored and co-released with NPY, no biological
function has been demonstrated for this peptide (Allen et al., 1987; Balbi and Allen, 1994).
NPY secretion from neurons is evoked by action potentials and is released via the
regulated secretory pathway (in contrast to the constitutive secretory pathway)(Dikeakos and
Reudelhuber, 2007; Hexum et al., 1987; Lou et al., 2005; Mitchell et al., 2008; Ramamoorthy and
Whim, 2008; Rudehill et al., 1992; Whim, 2006; Whim and Lloyd, 1989; Zhang et al., 2010).
6
Figure 1-1. Processing pathway of NPY.
NPY is synthesized as a precursor with the signal sequence in the N terminus. The signal
sequence is cleaved by a signal peptidase co-translationally when the preproNPY translocates into
the ER lumen. ProNPY is packaged into DCSGs via the trans-Golgi network. In DSCGs, CPON
is removed by PC 2 (or PC 1/3 in some cells such as AtT-20 cells). The intermediate is amidated
by PAM and two basic residues at the C terminal end of NPY are removed by CPE to generate
the mature NPY.
7
1.5 Physiological functions of neuropeptide Y
The related Y family members include pancreatic polypeptide (PP) which is produced
mainly by the pancreas and peptide YY which is synthesized by cells in the small intestine and
the pancreas. All Y family members share similar peptide sequence and can activate all NPY
receptors (Larhammar, 1996; Larhammar et al., 1993; Lin et al., 2004; Lundberg et al., 1982a;
Tatemoto, 1982a, b; Tatemoto and Mutt, 1980).
The known NPY receptors include Y1, Y2, Y4, Y5 and in the mouse also y6. All Y
receptors are G protein coupled receptors and their activation usually causes inhibitory responses
such as the inhibition of cAMP accumulation. NPY and PYY have similar affinity for all four
functional Y receptors. However PP preferentially activates Y4 receptors (Dumont et al., 1992;
Lin et al., 2004; Michel et al., 1998; Pons et al., 2008).
NPY is implicated in diverse physiological functions including metabolic regulation,
anxiety, depression, seizure, alcoholism and cardiovascular modulation (Abe et al., 2007; Arora
and Anubhuti, 2006; Erickson et al., 1996; Feder et al., 2009; Kuo et al., 2007b; Leggio et al.,
2011; Lin et al., 2004; Nguyen et al., 2011; Palmiter et al., 1998; Rotzinger et al., 2010; Thorsell,
2010).
1.5.1 NPY in the cardiovascular system
In 1982 the group that first isolated NPY from porcine brain found that injection (i.p.) of
this new peptide induced prolonged vasoconstriction. The effect was resistant to α-adrenoceptor
blockade and was not abolished in sympathectomized animals, indicating a direct action of NPY
on vascular smooth muscle. This was the first report of the biological activity of NPY and
suggested the role of peripheral NPY in regulating the cardiovascular system (Lundberg and
8
Tatemoto, 1982). The next year it was shown that central administration of NPY induced
hypotension in the rat so providing evidence of the involvement of central NPY in also regulating
the cardiovascular system (Fuxe et al., 1983). Since then a growing literature has confirmed that
NPY plays an important role in the cardiovascular system.
Conventional NPY transgenic rats that over-express NPY under the control of
endogenous regulatory elements showed decreased blood pressure and reduced catecholamine
release suggesting the role of endogenous NPY in the long-term inhibition of the cardiovascular
system (Michalkiewicz et al., 2003). This is consistent with the effect of central injection of NPY
leading to hypotension (Fuxe et al., 1983). This central NPY-induced hypotension was primarily
mediated by a presynaptic Y2 receptor inhibition (Chen and Westfall, 1993).
Conditional transgenic mice over-expressing NPY in adrenergic and noradrenergic
neurons (i.e. brain noradrenergic neurons and peripheral sympathetic neurons) displayed stress-
induced hypertension indicating a stimulatory role of peripheral NPY in the cardiac response
(Ruohonen et al., 2008). This is also consistent with the effect of injection (i.p) of NPY in
inducing vasoconstriction (Lundberg and Tatemoto, 1982).
Moreover peripheral NPY causes a prolonged vasoconstriction not only by directly
activating NPY Y1 receptors but also potentiates norepinephrine-evoked vasoconstriction via an
increase in intracellular calcium (Abe et al., 2010; Fallgren et al., 1993). The NPY Y1 receptor-
mediated effect was confirmed when it was found that Y1 receptor knockout mice showed an
absence of the NPY-induced increase of blood pressure (Pedrazzini et al., 1998).
In addition peripheral NPY promotes vascular remodeling including angiogenesis. This is
mediated predominantly by Y2/Y5 receptors as well as by vascular smooth muscle proliferation
and hypertrophy via Y1 receptors (Kuo et al., 2007a; Lin et al., 2004; Pedrazzini et al., 1998;
Pons et al., 2004).
9
Since stress triggers the release of NPY together with norepinephrine from sympathetic
nerves, and NPY receptors as well as adrenergic receptors are expressed in heart, NPY and
norepinephrine are believed to be mediators of stress-induced hypertension (Abe et al., 2010;
Chottova Dvorakova et al., 2008).
1.5.2 NPY in feeding and metabolism
In 1984 NPY was found to promote hyperphagia. The first evidence was that i.c.v
administration of NPY significantly induced feeding behavior in rats (Levine and Morley, 1984;
Stanley and Leibowitz, 1984). Since then more evidence has accumulated showing that NPY is
involved in regulating food intake, energy homeostasis as well as body weight.
In the central nervous system, NPY is most highly co-expressed with AgRP in the
hypothalamic arcuate nucleus (ARC), the neural center involved in feeding control and
integration of the signaling of energy balance (Arora and Anubhuti, 2006; Funahashi et al., 2000;
Kotz et al., 1998; Lin et al., 2006; Nguyen et al., 2011; Pedrazzini et al., 1998; Raposinho et al.,
2001; Stanley et al., 1989; Wilding, 2002). These NPY/AgRP neurons in the ARC are major
targets of the food-stimulating hormone ghrelin as well as the food-inhibiting hormones leptin
and insulin via their distinctive receptors (Arora and Anubhuti, 2006; Wilding, 2002). Ghrelin
increases feeding behavior by stimulating the production of NPY and AgRP in the ARC whereas
leptin and insulin inhibit food intake and body weight by decreasing the expression of NPY and
AgRP (Air et al., 2002; Greenman et al., 2004; Kim et al., 2006; Morrison et al., 2005; Nogueiras
et al., 2008; Schwartz et al., 1992; Zigman and Elmquist, 2003).
Using optogenetic manipulation, a recent study has shown that directly stimulating these
NPY/AgRP neurons is sufficient to induce food intake thus providing direct evidence of the
central role of these NPY neurons in regulating feeding behavior (Aponte et al., 2011).
10
In the peripheral system, NPY is thought to directly act on white fat tissue via Y2
receptor. Stress increased NPY/Y2 signaling and thus caused fat cell growth, which resulted in
obesity (Kuo et al., 2008; Kuo et al., 2007b).
1.5.3 NPY in emotional regulation
In 1987 it was found that stress-induced gastric erosion was reduced by approximately 50%
after i.c.v injection of NPY in rats, suggesting an anti-stress role of NPY (Heilig and Murison,
1987). In 1988 Heilig et al. found that anti-depressant drugs increased NPY immunoreactivity in
the brain, and then in 1989 the same group reported that i.c.v administration of NPY showed an
anxiolytic-like effect by interacting with noradrenergic systems in rat anxiety models (Heilig et
al., 1989; Heilig et al., 1988). NPY is considered to be a potent anti-depressant and anxiolytic,
and the role of NPY in regulating emotionality has been studied (Feder et al., 2009; Heilig, 2004;
Morales-Medina et al., 2010; Redrobe et al., 2002; Rotzinger et al., 2010; Sajdyk et al., 2004).
Central administration of NPY inhibits the development of fear conditioning and
promotes extinction whereas NPY Y1 antagonists act to increase the fear response in rats
(Gutman et al., 2008). Furthermore injection of NPY into the amygdala increased resilience to
stress and decreased anxiety-like behaviors in response to acute stress in rats (Sajdyk et al., 2008).
NPY also counteracts the effect of CRH which is expressed in the hippocampus, hypothalamus
and amygdala and promotes anxiety, so maintaining emotional homeostasis during the stress
response (Sajdyk et al., 2004).
11
1.5.4 NPY in epilepsy
The hippocampus is an epileptogenic brain region. NPY was shown to act presynaptically
on hippocampal CA1 neurons to inhibit glutamate-mediated synaptic transmission onto pyramidal
neurons (Colmers et al., 1987). NPY synthesis and release was markedly up-regulated in
hippocampal neurons during epilepsy in rats first suggesting a role of NPY in modulating limbic
excitability and seizure (Marksteiner et al., 1990; Pitkanen et al., 1989).
Mild seizures occurred in 30% of NPY knockout mice but not in littermate wild type
animals. When challenged with the GABA antagonist, pentylenetetrazole (PTZ), NPY-deficient
mice were hyperexcitable and more susceptible to seizures. These results from NPY knockout
mice indicated the essential role of NPY in reducing the levels of excitability in the central
nervous system and in protection from seizures (Erickson et al., 1996).
NPY Y5 receptor knockout mice were found to be more sensitive to kainic acid-induced
seizures and the inhibitory effects of applied NPY in hippocampal slices were absent from these
knockout mice. These data suggested that Y5 receptors were involved in the inhibitory actions of
NPY in the hippocampus (Marsh et al., 1999).
1.6 My thesis research on neuropeptide Y
NPY is abundantly expressed all over the body and is involved in numerous physiological
processes (Abe et al., 2007; Arora and Anubhuti, 2006; Erickson et al., 1996; Feder et al., 2009;
Kuo et al., 2007b; Leggio et al., 2011; Lin et al., 2004; Nguyen et al., 2011; Palmiter et al., 1998;
Rotzinger et al., 2010; Thorsell, 2010). Therefore determining the mechanisms involved in the
regulation of NPY expression and secretion will help us to understand the functioning of the
nervous system. In contrast to central NPY, less attention has been placed on the role of
12
peripheral NPY although NPY is released from sympathetic neurons as well as the adrenal
medulla, and NPY receptors are expressed in peripheral tissues including the heart, liver,
pancreas and adipose tissue (Kuo et al., 2007b; Moltz and McDonald, 1985; Rimland et al., 1991;
Sheriff et al., 1990; Whim, 2006, 2011). My Ph.D. thesis research has been involved in
investigating the regulation and role of peripheral NPY in the sympathetic nervous system.
A single nucleotide polymorphism (SNP) that changes the seventh amino acid in the
signal sequence of the NPY preprohormone (preproNPY) was identified in 1998 (Karvonen et al.,
1998). Humans carrying this T1128 polymorphism had higher levels of plasma NPY after
exercise and this SNP was associated with higher serum cholesterol levels and increased risk for
metabolic and heart diseases (Heilig et al., 2004; Karvonen et al., 1998; Karvonen et al., 2001;
Nordman et al., 2005; Pesonen, 2006, 2008). The data above suggested a role of the peripheral
NPY in modulating metabolism.
Since the location of the identified SNP T1128C is in the preproNPY signal sequence, it
was suggested to lead to a change in the trafficking, synthesis and/or secretion of NPY (Kallio et
al., 2001; Karvonen et al., 1998). I directly tested this hypothesis by comparing the expression of
NPY prohormones derived from mutant and wild type preproNPY. I found that this SNP did not
prevent synthesis and trafficking of the prohormone, but altered the biosynthesis and secretion of
NPY, which suggested a molecular mechanism that may underlie the T1128C phenotype (Heilig
et al., 2004; Karvonen et al., 1998; Karvonen et al., 2001; Nordman et al., 2005; Pesonen, 2006,
2008).
Stress is known to up-regulate sympathetic activity and the stress-induced increase of
peripheral NPY exaggerates high fat diet induced-obesity (Kuo et al., 2007b). This study by Kuo
et al. directly linked sympathetic NPY with the stress response and metabolic changes. As
modified postganglionic neurons and as part of the sympathetic system, the adrenal medulla can
be activated by stress (Hiremagalur et al., 1994; Nankova et al., 1996; Nankova and Sabban, 1999;
13
Sabban et al., 2006). The adrenal glands consist of a cortex and medulla and are part of the
mechanism that allows the organism to cope with stress (Carrasco and Van de Kar, 2003).
The adrenal medulla, where abundant NPY is expressed, is activated during the stress
response (Colomer et al., 2010; Colomer et al., 2008; Ulrich-Lai and Engeland, 2002; Varndell et
al., 1984). Therefore the other aim of my thesis was to investigate how stress modulated adrenal
NPY and the physiological roles of NPY in regulating adrenal functions. My finding suggests that
acute stress increases the levels of adrenal NPY and NPY plays an important role in inhibiting the
neuronal branch of the stress response which may act to prevent a pathological activation of the
fight-or-flight response.
Chapter 2
Materials and Methods
2.1 Specific Methods for Chapter 3
2.1.1 Site-directed mutagenesis
Site-directed mutagenesis was conducted on a pcDNA3.1 vector (Invitrogen, Carlsbad,
CA) which contained preproNPY/fluorescence tag/ FMRFamide using custom-designed
mutagenic primers with the Quick-Change system (Stratagene, La Jolla, CA). The mutant
constructs that used the most frequently employed proline condon CCC were termed “L7P”. This
mutation was generated by using the forward primer:
5’-GCTAGGTAACAAGCGACCCGGGCTGTCCGGACTG-3’
and the reverse primer:
5’-CAGTCCGGACAGCCCGGTCGCTTGTTACCTAGC-3’.
The mutation sites are underlined.
The mutant constructs which contained the actual polymorphism observed by Karvonen et al.
(1998) are referred to “T1128C”. This mutant changed CTG to CCG, a rarely used codon for
proline and it was made with the forward primer:
5’- GCTAGGTAACAAGCGACCGGGGCTGTCCGGACTG-3’
and reverse primer:
5’- CAGTCCGGACAGCCCGGTCGCTTGTTACCTAGC- 3’.
The mutation site is underlined.
15
The constructs were confirmed by sequencing. The primers and sequencing were performed by
the PSU nucleic acid facility (University Park, PA).
2.1.2 AtT-20 cells culturing
AtT-20 cells were grown in 50 ml flasks in 6 ml DMEM containing 10% FCS
(Mediatech, Herndon, VA) and split 1:6 every three days or 1:10 every five to seven days. For
experiments AtT-20 cells were cultured in 35 mm dishes. One hundred microliters of a 1:1 split
cells was plated onto a glass coverslip which was pre-coated with poly-D-lysine (Sigma-Aldrich,
St. Louis, MO) and the dish was flooded with 2 ml DMEM/10%FCS. The cells were maintained
in a humidified incubator with 95% O2/ 5% CO2 at 37OC.
2.1.3 Transfection of AtT-20 cells
Plasmid concentrations were measured using a NanoDrop spectrophotometer. AtT-20
cells were transfected with 100 ng of the plasmids using Lipofectamine 2000 (Invitrogen,
Carlsbad, CA) 24 hours after plating.
2.1.4 Immunoprecipiation and Western blot
AtT-20 cells were transfected with wild type or mutant Venus tagged NPY constructs and
then lysed 48 hours after transfection with 100 μl whole cell extraction buffer (WCEB: 50 mM
Tris-HCl with pH 8.0, 120 mM NaCl, 0.5% v/v NP-40, 10% v/v glycerol, 0.2 mM EDTA, 2 mM
EGTA, 1 mM PMSF and a protease inhibitor mix with 0.1 μg/ml antipain, 1 mU/ml aprotinin ,
0.1 μg/ml chymostatin, 0.1 μg/ml pepstatin A, 0.1 μg/ml leupeptin,). In some of the experiments,
16
the crude cell lysates were subject to Western blot. In other experiments using
immunoprecipitation, lysates were incubated with a mouse monoclonal GFP antibody (1:100;
Abgent, #AM1009a) for 3 hours at 4 OC, followed by incubation for 2 hours with protein G
sepharose beads (Sigma, St. Louis, MO). The beads were then centrifuged at 13,200 rpm for 2
minutes and the pellets were washed twice with WCEB.
The crude lysates or immunoprecipitates were boiled with SDS loading buffer for 5
minutes at 95OC. The boiled samples were separated by electrophoresis on a 15% SDS-PAGE gel,
transferred onto the PVDF membranes, and then immunoblotted with a rabbit polyclonal anti-
GFP antibody (1:1500; Rockland Immunochemicals, #18560). Proteins were detected by a goat
anti-rabbit Cy5 secondary antibody (1:2500, Amersham ECL Plex, #PA45011). Fluorescent
signals were scanned with a Typhoon 8600 scanner and the intensity of the detected bands was
quantified using Image-Pro Plus 5.1 software (Media Cybernetics). Statistical significance was
calculated with a one way ANOVA.
The details of the solutions are listed in Appendix C.
The SDS-PAGE gel with the boiled crude lysates was also directly scanned with a
Typhoon 8600.
2.1.5 Immunocytochemistry
AtT-20 cells were transfected with fluorescence- tagged wild type or mutant constructs
for 24 hours and then fixed in 4% paraformaldehyde (in PBS) for 20 min. After 15 min of
permeablization in 0.3% triton-100X (in PBS), cells were incubated with blocking solution (0.25%
BSA and 0.05% Triton-X in PBS) for 30 min and then were incubated with a primary antibody
overnight at 4OC. Primary antibodies included rabbit anti-NPY (1:200; Peninsula Laboratories,
T4070), mouse anti-GM130 (1:1000; BD Transduction Laboratories, #610822), mouse anti-
17
carboxypeptidase E (CPE) (1:600; BD Transduction Laboratories, #610758) and rabbit anti-
FMRFamide (1:200; Peninsula Laboratories, #T4322). Following the incubation with a primary
antibody, cells were washed with PBS for 4 times and then stained with a secondary antibody for
90 minutes, which was followed by washing for 4 times and mounting on a glass slide. Secondary
antibodies were donkey anti-rabbit Alexa 488 (1:200; Invitrogen), goat anti-mouse fluorescein
(FITC), donkey anti-mouse tetramethylrhodamine isothiocyanate (TRITC), donkey anti-rabbit
TRITC (all 1:100; Jackson ImmunoResearch Laboratories).
In the experiments comparing prohormone copackaging, AtT-20 cells were transfected
with the construct plasmids for 48 hours and then incubated with 10 μM cycloheximide for 1 hour
before the fixation to limit ER-derived fluorescence (Sobota et al., 2006).
Cells were fixed for 20 min and then mounted with mounting medium (Vector
Laboratories, # H-1000).
The details of the solutions are listed in Appendix C.
2.1.6 Image analysis
Images were taken using a Nikon TE2000U microscope with a 60 X (1.4 NA) oil-
immersion objective and a Retiga 1300 monochrome camera. Images and analysis were
performed blind to genotype. For the copackaging experiments, puncta were picked in each
fluorescence channel (Venus and RFP) using the same area of interest (AOI, radius of 0.066 μm).
A punctum was first identified in the green channel using an AOI and then the same AOI was
transferred to the red channel. Twenty puncta were selected from each cell along a process and
ten cells on each coverslip from a dish with the transfected cells were analyzed. Two individual
experiments were done using L7P constructs and one individual experiment used the T1128C
construct. Image-Pro Plus 5.1 (Media Cybernetics), Excel 2003, OriginPro7 and ClampFit (Axon)
18
were used for data analysis. Statistical significance was calculated with one way ANOVA and
Kolmogorov–Smirnov tests. Each experiment contained four sister cultures of matched wild type
and T1128C or L7P transfected AtT-20 cells.
2.2 General Methods for Chapter 4 and Chapter 5
2.2.1 Animals
C57BL/6J, C57BL/6J-NPY(GFP) (van den Pol et al., 2009) and 129S-Nptm1Rpa/J NPY
knockout mice (Erickson et al., 1996) were purchased from the Jackson Laboratory. Most of the
animals were male littermates, the only female littermates used were for one group of cell
cultures used for NPY immunostaining in the experiment using the cold forced swim test (cold
FST) (section 2.2.2, 2.2.3 and 2.2.4), one group used for measuring NPY-ir 1 week after the cold
FST (section 2.2.2 and 2.2.5), one group from a BIBP3226 injection experiment (section 2.3.2)
and one group from a L152, 804 injection experiment (section 2.3.2). Animals were housed on a
12:12 hour light-dark cycle (3:00am-3:00 pm) with ad libitum access to water and standard
laboratory rodent chow. All the procedures were approved by the Animal Care Facility and
Committees in the Pennsylvania State University and Louisiana State University Health Sciences
Center.
2.2.2 Cold water forced swim test
Postnatal P21-P23 littermate mice were individually housed for 3 to 5 days to minimize
the stressful effect of weaning. Considering the effects of social isolation, both control and
experimental littermates were housed individually on the same day before exposing an
19
experimental animal with a stressor. After individual housing, experimental mice were placed in
the cold (4-5OC) water for 5 to 6 minutes (cold water forced swim test, cold FST) (Saal et al.,
2003) and then warmed under a heat lamp. Following recovery the animals were returned to their
home cages. Animals used to measure plasma corticosterone levels were decapitated 30 minutes
after the cold FST when the induction of plasma corticosterone is expected to reach the maximal
level (Richard et al., 2010). For c-Fos immunoreactivity mice were decapitated 1 hour after the
cold FST and for the stress reversible effect on NPY- or TH-immunoreactivity animals were
decapitated 24 hours or 1 week after the cold FST. For all the other experiments, animals were
decapitated 24 hours after the cold FST.
2.2.3 Chromaffin cell cultures
Adrenal glands were obtained from mice and the fat tissue was removed. The cortex was
removed from the adrenal glands and the adrenal medulla was placed in dissection medium
(DMEM/8 mM MgCl2). The adrenal medulla was washed twice with HBSS, then digested with
0.1% collagenase (Sigma, # C9891) for 15 minutes in 37OC incubator with O2 and CO2, and then
treated with 0.1% trypsin bovine type XII S (Sigma, # T2271) for 30 minutes at 37OC. Digested
adrenal medulla was transferred into trituration medium to be triturated gently. Dissociated cells
were pelleted by centrifuging at 1,000 g for 4 minutes. Pellets were resuspended with
DMEM/10%FBS and 100 μl of cell suspension was placed onto poly-D-lysine pre-coated
coverslips in culture dishes and incubated for 45 minutes in the 37OC incubator with O2 and CO2
Then the cells were flooded with 2 ml DMEM/10%FBS and maintained in the 37OC incubator
with O2 and CO2.
The details of the solutions are listed in Appendix C.
20
2.2.4 Immunocytochemistry
Two hours after flooding, chromaffin cells were washed with PBS, fixed with 4%
paraformaldehyde for 20 minutes and then permeabilized with 0.3% Triton-X (in PBS) for 15
minutes. Following permeabilization, cells were incubated with blocking solution (0.25% BSA
and 0.05% Triton-X in PBS) for 30 minutes and then were incubated with a primary antibody at
4OC overnight. The following day, cells were washed with PBS for 4 times, stained with a
secondary antibody for 90 minutes, washed with PBS again for 4 times and then mounted on
glass slides. Primary antibodies were rabbit anti-NPY (1:40,000; Peninsula labs, T 4070), guinea
pig anti-PNMT (1: 100; Acris, # EUD 7001), sheep anti-TH (1:200; Millipore, # AB 1542) and
rabbit anti-TH (1:200; Millipore, # AB 152). Secondary antibodies included donkey anti-rabbit
Alexa 488 (1:200; invitrogen), donkey anti-guinea pig DyLight 549, donkey anti-sheep DyLight
549 and donkey anti-rabbit DyLight 549 (all 1:100, Jackson ImmunoResearch Laboratories).
Chromaffin cells from NPY (GFP) animals were fixed for 20 minutes after culturing and
then mounted on glass slides.
The details of the solutions are listed in Appendix C.
2.2.5 Cryostat frozen sectioning
Fat surrounding each adrenal gland was removed and the adrenals were immersed in 4%
paraformaldehyde (in PBS) for overnight fixation at room temperature. The fixed adrenal glands
were washed with PBS and then frozen in dry ice-cold beta-methylbutane for 45 seconds. Frozen
adrenals were then embedded in molds with cryomatrix and kept at -80 OC until use.
21
2.2.6 Immunohistochemistry
Thirty micrometers thick cryo-sections were washed with PBS, fixed with 4%
paraformaldehyde for 20 minutes and then permeabilized with 0.3% Triton-X (in PBS) for 15
minutes. Following permeabilization, sections were quenched with 3% H2O2 for 45-60 minutes
and incubated with blocking solution (blocking reagent in TBS, Perkin Elmer, # FP1020) for 30
minutes. Then sections were stained with a primary antibody at 4 OC overnight. The next day,
sections were washed with PBS-T (0.05% Tween-20 in PBS), incubated with a secondary
antibody peroxidase-conjugated donkey anti-rabbit or anti-sheep IgG (1:500; Jackson
ImmunoResearch Laboratories) for 30 minutes, washed with PBS-T, incubated in TSA-FITC
(Perkin Elmer, # NEL741) or TSA-Cy3 (Perkin Elmer, # NEL 744) for 10 minutes, washed with
PBS-T, rinsed with distilled water and then mounted. The primary antibodies were rabbit anti-
NPY (1:10,000; Peninsula labs, # T4070), sheep anti-TH (1:200; Millipore, # AB 1542), rabbit
anti-TH (1:200; Millipore, # AB 152) and rabbit anti-c-Fos Ab-5 (1:1,000; Calbiochem, # PC38).
2.2.7 Image analysis
All slides were blinded by a non-experimenter to avoid any bias in the analysis. Images
were taken using a TE2000U microscope with a 10X objective for sections and with a 60X oil
immersion objective for cell cultures. ImagePro Plus 5.1 (Media Cybernetics) was used to
analyze the levels of NPY- or TH-immunoreactivity and GFP fluorescence. For cell cultures, a
circular area of interest (AOI) was used to outline each cell. A polygon area of interest was used
to outline the whole adrenal medulla and part of the adrenal cortex (which is negative for NPY-ir
and was used as a background correction). The mean fluorescence intensity (independent of the
AOI size) was used for analysis.
22
For counting the number of c-Fos-ir positive cells in each adrenal section, a circular AOI
with a radius of 66 μm was used to outline the region. The AOI was moved along the adrenal
medulla to cover the whole medulla (without any areas of overlap) and then the average count
from all AOIs in each adrenal section was recorded for group analysis.
2.2.8 Statistical analysis
Statistical significance was calculated using a matched one sample Student’s t test
(normalized data) or paired Student’s t test (raw data) when two groups of values were compared
(the average values from each individual experiment were compared). The Kolmogorov–Smirnov
test was used for calculating P values in the cumulative fraction data sets (the values from all the
cells in all the independent experiments were plotted in one cumulative graph). Three or four
groups of values were compared using a one-way analysis of variance (ANOVA) post-hoc
Tukey’s paired comparison (the average values from each individual experiment were compared).
2.2.9 Corticosterone ELISA
Five hundred microliters of blood was collected from a decapitated animal and
centrifuged at 1,000g at 4 OC for 15 minutes. Plasma was collected in 1mM EDTA containing
tubes and kept at -80 OC until use. Plasma corticosterone levels were measured using a
commercial EIA kit following the manufacturer’s instructions (Assay Designs, Plymouth Meeting,
PA, #900097). Reactions were read using an absorbance microplate reader at 405 nm with a
correction at 570 nm. A standard curve was generated using standard samples provided by the kit
and validated with a linear regression fit in OriginPro7. Duplicates were measured to calculate the
mean of the optical density (OD) values for each blood sample.
23
2.3 Specific Methods for chapter 4
2.3.1 Fox urine exposure
P19-21 mice were kept in new cages for 3 minutes, exposed to fox urine odor for 5
minutes and then were returned to the home cage (Liu et al., 2010). The animals exposed to fox
urine odor were decapitated 16-24 hours later.
2.3.2 Semi-quantitative RT-PCR of NPY
The adrenal cortex was removed from the whole adrenal gland and the adrenal medulla
was collected. Total RNA was extracted from the adrenal medulla using Trizol (Invitrogen, #
15596-026) and purified with an RNeasy mini Kit (Qiagen, # 74104) following the
manufacturer’s instructions. Sequential dilutions of cDNA’s at 1:4, 1:16 and 1:64 were used for
PCR amplification. NPY expression was normalized by actin expression. The forward NPY
primer was 5’-CAC GAT GCT AGG TAA CAA G-3’ and the reverse primer was 5’-CAC ATG
GAA GGG TCT TCA AG-3’. The forward actin primer was 5’-GCC AAC CGT GAA AAG
ATG AC-3’ and the reverse primer was 5’-CAA CGT CAC ACT TCA TGA TG-3’. The PCR
was run for 4 minutes at 94OC, followed by 45 seconds denaturation at 94
OC, 45 seconds
annealing at 55OC and 1 minute of extension at 72
OC for 35 cycles and an additional cycle of
extension at 72OC for 10 minutes.
2.3.3 RT-PCR of PP and PYY
Total RNA was extracted as describe above (section 2.3.2). The cDNA was obtained by
reverse transcription of 3 ng mRNA from pancreatic islets and of 7 ng mRNA from adrenal
24
medulla. The forward insulin primer was 5’-TTT GTC AAG CAG CAC CTT TG-3’ and the
reverse primer was 5’-GCT GGT AGA GGG AGC AGA TG-3’. The forward NPY primer was
5’-CAC GAT GCT AGG TAA CAA G-3’ and the reverse primer was 5’-CAC ATG GAA GGG
TCT TCA AG-3’. The forward PP primer was 5’-CAC GAT GCT AGG TAA CAA G-3’ and the
reverse primer was 5’-CAC ATG GAA GGG TCT TCA AG-3’. The forward PYY primer was
5’-CAC GAT GCT AGG TAA CAA G-3’ and the reverse primer was 5’-CAC ATG GAA GGG
TCT TCA AG-3’. The PCR was run for 4 minutes at 94OC, followed by 45 seconds denaturation
at 94OC, 45 seconds annealing at 55
OC and 1 minute of extension at 72
OC for 35 cycles and an
additional cycle of extension at 72OC for 10 minutes.
2.4 Specific Methods for chapter 5
2.4.1 Catecholamine ELISA
Five hundred microliters of blood was collected from a decapitated animal and
centrifuged at 1,000g at 4 OC for 15 minutes. Plasma was collected in 1mM EDTA-containing
tubes and kept at -80 OC until use. Plasma epinephrine and norepinerphrine from the same blood
samples were measured using commercial ELISA kits following the manufacturer’s instructions
(RM Diagnostics, # BA E5400). Reactions were read by an absorbance microplate reader at 450
nm with a correction at 620 nm. A standard curve was generated using standard samples provided
by the kit and validated with a linear regression fit in OriginPro7. Duplicates were measured to
calculate the mean of the OD values for each blood sample.
25
2.4.2 Antagonist injection
Littermate animals were intraperitoneal (i.p.) injected with 200 μl saline or BIBP 3226
(Tocris Biosciences, 1mg/kg) 15 minutes before the cold FST. A second group of animals were
i.p injected with 200 μl saline or BIIE 0246 (Tocris Biosciences, 1mg/kg) 15 minutes before the
cold FST. The third group were injected with 200 μl saline or L152, 804 (Tocris Biosciences,
10mg/kg) 15 minutes before the cold FST.
2.4.3 RT-PCR
The hippocampus, hypothalamus, cortex and cerebellum were collected from the whole
brain. Collection of mRNA from the adrenal medulla or brain was as described previously
(section 2.3.2). cDNA obtained by reverse transcription of 20 ng mRNA from the adrenal
medulla or brain was used to amplify NPY Y1, Y2, Y5 and Y6 receptors and cDNA from 60 ng
mRNA was used for the NPY Y4 receptor (Ramamoorthy and Whim, 2008). Primers are
presented in Table1. The PCR was run for 4 minutes at 94OC, followed by 45 seconds
denaturation at 94OC, 45 seconds annealing at the temperatures listed in Table1 and 1 minute of
extension at 72OC for the number of cycles as presented in the Table1 and an additional cycle of
extension at 72OC for 10 minutes.
26
Gene Primers Anealing Tm and amplifying cycles
NPY Y1 Forward: 5’ CGG CGT TCA AGG ACA AGT AT 3’ Reverse: 5’ TGA TTC GCT TGG TCT CAC TG 3’
55OC for 35 cycles
NPY Y2 Forward: 5’ TGC CAA TCT GGT TAG GGA AG 3’ Reverse: 5’ GGT GCC AAC TCC TTG TTC TG 3’
55OC for 35 cycles
NPY Y4 Forward: 5’ TTG CAG TTC TCT GGC TAC CCC TG 3’ Reverse: 5’ CTT GCT ACC CAT CCT CAT CGA 3’
56OC for 35 cycles
NPY Y5 Forward: 5’ CAG ATT AAT CCA GCT GTT CTG C 3’ Reverse: 5’ GAA AAC AGC CTT TAT TTG ACA ATG 3’
55OC for 35 cycles
NPY y6 Forward: 5’ TCA CTA AAT AAG ACC ATC GGG TAG 3’ Reverse: 5’ GGG AGG TTT ACC CTA GGA AAT G 3’
55OC for 10 cycles +
53OC for 30 cycles
NPY Forward: 5’ GCT AGG TAA CAA GCG AAT GGG G 3’ Reverse: 5’ CAC ATG GAA GGG TCT TCA AGC 3’
55OC for 35 cycles
Table 1. Primers, annealing temperatures and amplifying cycles for RT-PCR of NPY Y
receptors.
Chapter 3
A Single Nucleotide Polymorphism Alters the Synthesis and Secretion of
Neuropeptide Y
3.1 Introduction
More than one million single nucleotide polymorphisms (SNPs) are reported to be
distributed throughout the human genome and of these it is estimated that 60,000 are found in
exons. Studying these SNPs is thought to be important because genetic differences between
individuals can have a major impact on the response to disease and environmental factors
(Sachidanandam et al., 2001).
Genome-wide mapping analysis is being used to identify SNPs that are associated with
many kinds of complex diseases such as diabetes, cardiovascular disease, breast cancer, prostate
cancer and bladder cancer (Garcia-Closas et al., 2007; Hunter et al., 2007; Larson et al., 2007;
Saxena et al., 2007; Yeager et al., 2007). Establishing an association between SNPs and disease
susceptibility can help to identify biomedically important genes for diagnosis. This is more
advantageous than conventional gene-hunting and by developing gene-targeted medicine,
personalized therapies will ultimately be possible (Sachidanandam et al., 2001).
Following the identification of a functional SNP, it is important to determine the
molecular mechanisms underlying the correlation between the SNP and the disease it causes. In
the nervous system, gene variants encoding transporters, ion channels and neuromodulators have
been associated with clinical deficits or disorders (Egan et al., 2003a; Karaplis et al., 1995;
Mazei-Robison et al., 2005; Vitko et al., 2005).
28
Among these SNPs, one success story is the identification of the molecular mechanisms
that underlie the brain-derived neurotrophic factor (BDNF) Val66Met polymorphism and its
associated phenotypes. In humans this BDNF polymorphism in the 5’ proBDNF domain is
associated with biopolar disorder, depression, memory impairment and schizophrenia (Egan et al.,
2003a; Egan et al., 2003b; Sen et al., 2003). This functional SNP (valine to methionine) has been
reported to lead to abnormal distribution of the prohormone in both the dendrites and cell body of
hippocampal neurons, and to cause decreased activity-dependent secretion (Egan et al., 2003a).
The abnormal trafficking phenotype seems to be due to impaired binding of the mutant BDNF
with a “sorting” protein sortilin, which has been shown to interact with the prodomain of BDNF
(Chen et al., 2005). Chen et al. have generated a transgenic mouse that endogenously expresses
mutant BDNF. The genetic variant of BDNF containing the SNP alters anxiety-related behaviors
in these mice, which correlates well with the phenotype in humans (Chen et al., 2006).
Another good example is the isolation of a SNP in the signal sequence of the human
preproparathyroid hormone (preproPTH) which was obtained from a patient with familial isolated
hypoparathyroidism. A cysteine residue is substituted with arginine in the hydrophobic core of
the signal sequence of preproPTH, which likely impairs the processing of the preprohormone to
prohormone (Arnold et al., 1990). The impaired processing of the mutant preprohormone is likely
due to inefficient membrane targeting and translocation into the ER, since in vitro experiments
show that the mutation disrupts the interaction of the preprohormone with the signal recognition
particle and the translocon. These in vitro results suggest a molecular mechanism that could
explain the pathophysiologic consequences of this SNP in the patient. This disease is
characterized by hypocalcemia which is due to a lack of functional circulating parathyroid
hormone (PTH), the major calcium-regulating hormone (Karaplis et al., 1995).
A functional SNP which changes the seventh amino acid from leucine (Leu) to proline
(Pro) in the signal sequence of the NPY preprohormone was identified by Karvonen et al. in 1998
29
(Karvonen et al., 1998). This SNP is associated with an increased serum cholesterol level, type 2
diabetes as well as heart disease (Heilig et al., 2004; Karvonen et al., 1998; Karvonen et al., 2001;
Nordman et al., 2005; Pesonen, 2006, 2008). An elevated level of plasma NPY was found in
humans carrying this T1128C mutation after exercise and a higher amount of the mature NPY
hormone was present in human endothelial cells that contained the mutant genotype possibly
indicating an increase in the efficiency of cleavage of the mutant preproNPY (Kallio et al., 2001).
In addition rats that received an i.c.v injection of the purified mutant signal peptide and mature
NPY had markedly elevated food intake compared with those animals injected with the wild type
signal peptide and mature NPY suggesting that the signal peptide alone may have biological
activity (Ding et al., 2005). Although these studies provide some evidence showing a differential
effect between the wild type and mutant preproNPY, the underlying molecular and cellular
mechanisms are still not clear.
Since the single nucleotide mutation T1128C is in the signal sequence of the NPY
preprohormone, I hypothesized that this SNP does not change the sequence of the mature NPY,
but may alter the synthesis, sorting and/or secretion of NPY. To test these possibilities,
fluorescent protein-tagged NPY constructs were expressed in AtT-20 cells, a pituitary cell line. I
then directly tested the above hypothesis by comparing the expression of proNPY derived from
the wild type and mutant preproNPY. My results suggest that proNPY from the wild type and
mutant preproNPY entered the regulated secretory pathway in a similar manner. However
individual dense core granules contained significantly more prohormone derived from mutant
than wild type preproNPY. This polymorphism also increased the secretion of NPY which is
likely due to the increased synthesis. My findings suggest a molecular mechanism that may
underlie the phenotypes associated with the T1128 polymorphism.
3.2 Results
3.2.1 Generation of the wild type and mutant NPY fusion protein constructs
To express and monitor the distribution of NPY prohormone derived from the wild type
and mutant preproNPY, I first generated wild type and mutant fluorescent protein-tagged NPY
constructs (Figure 3-1). The identified T1128C polymorphism changes the DNA sequence from
CTG to CCG (Karvonen et al., 1998). I termed this mutant “T1128C”. The wild type NPY
preprohormone has CTG which is a frequently used leucine codon whereas the T1128C mutant
contains CCG which is the least frequently used proline codon. To minimize any potential effects
caused by the difference in codon usage and examine the solely effect of the amino acid
substitution, I also made additional mutant constructs using the most frequently proline codon
(CCC) and this mutant was termed “L7P”. I used these constructs to examine their expression and
monitor their intracellular trafficking pathways in the following experiments.
AtT-20 cells are an endocrine cell line derived from a mouse pituitary tumor. They do not
endogenously express NPY but are capable of synthesizing NPY (Dickerson et al., 1987). The
advantage of using AtT-20 cells is that they contain dense core granules that can be identified
clearly (Quinn et al., 1991; Sobota et al., 2006).
3.2.2 Expression of NPY prohormones derived from wild type and mutant preproNPY
To determine whether the mutation affected the processing or expression levels of the
NPY prohormone, I used Western blotting to test the expression of proNPY derived from wild
type and mutant preproNPY in AtT-20 cells. The cells were transfected with the wild type NPY-
31
Venus, L7P NPY-Venus or T1128C NPY-Venus. The cell lysates were immunoblotted with a
GFP antibody and the two bands shown correspond to proNPY (37kD) and mature NPY (31kD)
in both wild type and mutant NPY-Venus transfected cells (Figure 3-2 A). The presence of Venus
in each cleavage product was also confirmed by directly scanning a gel for the presence of a
fluorescent protein (Figure 3-3). Therefore the polymorphism did not prevent the synthesis of the
NPY prohormone and mature peptide.
The ratio of NPY vs. proNPY was quantified and no detectable difference was found
between hormones from wild type and mutant preproNPY (Figure 3-2 B). When the tagged
peptides were purified by immunoprecipition and then detected by immunoblotting with a GFP
antibody, only one band was found which corresponded to proNPY (Figure 3-2 C upper band).
The intensity of the bands corresponding to proNPY was normalized by an unspecific band
(Figure 3-2 C lower band). Quantitative analysis showed no detectable difference among all the
groups (Figure 3-2 D). Therefore the polymorphism did not change the expression levels of
proNPY and the relative ratios between mature NPY and the prohormone.
3.2.3 NPY prohormones derived from L7P and wild type preproNPY were sorted similarly
in AtT-20 cells
AtT-20 cells were transfected with the wild type or L7P IRES-GFP constructs and the
transfected cells were identified by cytoplasmic GFP expression. The cells expressing
prohormones derived from wild type or mutant preproNPY showed punctuate NPY-
immunoreactivity (NPY-ir) when stained with an NPY antibody (Figure 3-4 A). When the cells
were transfected with wild type NPY-RFP or L7P NPY-RFP, they showed punctuate distribution
of RFP that co-localized with the NPY-ir (Figure 3-4 B) indicating that these fluorescent fusion
proteins were reliable markers for tracking the distribution of the prohormones.
32
Neuropeptides like NPY usually enter the regulated secretory pathway via the
endoplasmic reticulum (ER), trans-Golgi network and dense core granules (Dikeakos and
Reudelhuber, 2007; Hexum et al., 1987; Rudehill et al., 1992). To test whether the SNP affects
the trafficking pathway of the NPY prohormone, AtT-20 cells were transfected with the wild type
NPY-Venus or L7P NPY-Venus and immunostained with a GM130 (cis-Golgi marker) antibody.
Both wild type NPY-Venus and L7P NPY-Venus co-localized with the GM130-ir (Figure 3-4 C).
Furthermore when the cells were transfected with the wild type NPY-RFP or L7P NPY-RFP,
their punctuate distribution overlapped precisely with carboxypeptidase E-ir (CPE-ir; Figure 3-4
D). CPE is a dense core granule marker and is required for the cleavage of many peptide
prohormones (Fricker, 1988; Hook et al., 2008). Thus prohormones derived from L7P and wild
type preproNPY were targeted into the regulatory secretory pathway.
3.2.4 NPY prohormones derived from L7P and wild type preproNPY entered the same
dense core granules
Most of the NPY SNP carriers are heterozygous thus NPY neurons in vivo are likely to
contain both wild type and mutant preprohormones (Karvonen et al., 1998). To mimic this
situation AtT-20 cells were co-transfected with wild type NPY-Venus and L7P NPY-RFP. Co-
transfection of the cells with wild type NPY-Venus and wild type NPY-RFP was used as a
control. A complete overlap of the Venus and RFP puncta was found in both cases indicating that
prohormones derived from wild type and L7P preproNPY are co-packaged into the same dense
core granules. A two channel intensity line scan confirmed the overlap (Figure 3-5).
From these results, the processing of prohormones from wild type and L7P preproNPY
were indistinguishable. This is in contrast to the effects of SNPs in some other peptides such as
BDNF (Egan et al., 2003a; Karaplis et al., 1995). However sometimes differences in peptide
33
packaging may be quantitative. This possibility was examined in the following series of
experiments.
3.2.5 Prohormones derived from L7P and wild type preproNPY were differentially co-
packaged
AtT-20 cells were co-transfected with wild type and mutant preprohormones tagged with
either Venus or RFP (four possible combinations). Because Venus and RFP might be synthesized
at different intrinsic rates, the control cells were co-transfected with wild type NPY-Venus and
wild type NPY-RFP or with L7P NPY-Venus and L7P NPY-RFP. I measured the intensity of
single puncta in both the green and red channels, and then plotted the data as a cumulative
distribution of the green vs. red ratio (Figure 3-6 A). As shown in the cumulative fraction figure,
the two controls (i.e. wild type NPY-Venus + wild type NPY-RFP vs. L7P NPY-Venus + L7P
NPY-RFP) overlapped, which validated the approach and confirmed that swapping the
prohormones did not alter the synthesis rate of Venus and RFP. The most right-shifted curve with
the “greenest” puncta was from the cells co-transfected with L7P NPY-Venus and wild type
NPY-RFP. Therefore the NPY prohormone from L7P preproNPY accumulated to a higher level
than the NPY prohormone from wild type preproNPY in single granules. On the other hand, the
most left-shifted curve with the “reddest” puncta was from the cells expressing L7P NPY-RFP
and wild type NPY-Venus so these cells contained relatively more L7P-RFP. Thus, I can
conclude that the dense core granules contain higher levels of NPY prohormone derived from
L7P preproNPY than that from wild type preprohormone.
As mentioned in section 3.2.1 and Figure 3-1, the actual observed polymorphism in NPY
is “T1128C” (i.e. CTG mutated to CCG) rather than “L7P” ( i.e. CTG mutated to CCC). I used
the L7P constructs in most of my studies to minimize any potential confounding effects due to
34
codon usage. However in order to test whether the low frequency proline codon (CCG) actually
found in the SNP (i.e. T1128C) had a similar phenotype to L7P, I repeated the experiment with
T1128C-Venus and T1128C-RFP. A similar type of prohormone differential co-packaging was
found as seen in the cumulative distribution (Figure 3-6 B). Therefore the dense core granules
contained higher levels of NPY prohormone derived from mutant preproNPY and this is not due
to codon bias but to the change in the amino acid sequence.
3.2.6 NPY prohormones derived from L7P and wild type preproNPY were sorted similarly
in hippocampal neurons
To examine prohormone trafficking in an acutely isolated cell type, polarized
hippocampal neurons that endogenously synthesize NPY were transfected with both wild type
and mutant preprohormones (Higuchi et al., 1988a; Higuchi et al., 1988b). Similar to the situation
in AtT-20 cells, the SNP did not alter the distribution of the NPY-containing granules but
increased the packaging of NPY prohormone derived from mutant preproNPY in the dense core
granules in hippocampal neurons. The work in this section was mainly contributed by Dr. Prabhu
Ramamoorthy and Mr. Gregory Mitchell. I made the wild type and mutant constructs used for the
neuronal transfections.
3.2.7 Expression of NPY prohormone derived from mutant preproNPY leads to increased
NPY secretion
In order to investigate whether the polymorphism can affect the secretion of NPY,
peptide release was measured from chromaffin cells using the FMRFamide tagging technique.
Chromaffin cells are good model for measuring NPY secretion since these cells endogenously
express and secrete NPY. Chromaffin cells fire action potentials and secretion can be triggered by
35
depolarizing the cells (Whim, 2006; Whim and Moss, 2001). Two copies of FMRFamide were
added to the NPY IRES constructs (Figure 3-1). The release of FMRFamide will activate the
ionotropic FMRFamide receptors that are expressed on the surface of the same cell causing an
inward sodium current. Because the FMRFamide peptide and NPY are co-packaged into the same
dense core granules, the release of FMRFamide is a surrogate for the release of NPY (Whim and
Moss, 2001).
Punctate wild type NPY-Venus or T1128C-Venus was co-localized with FMRFamide-ir
in transfected AtT-20 cells and the overlap was confirmed with an intensity line scan (Figure 3-7).
Thus the FMRFamide peptide and proNPY derived from wild type or mutant preproNPY are
packaged in the same dense core granule and the secretion of FMRFamide can be used to monitor
the release of NPY derived from both wild type and mutant preprohormones.
Chromaffin cells transfected with wild type and mutant NPY-Venus showed punctate
fluorescence confirming the synthesis of proNPY from both wild type and mutant
preprohormones. Chromaffin cells were then transfected with either the wild type or T1128C
NPY preprohormone (IRES constructs, Figure 3-1 and Figure 3-8 A). Since NPY is secreted
through the regulated pathway, stimulation with a train of depolarizations was used to evoke
secretion. The average amplitude of the peptidergic secretory events was larger from the cells
expressing the T1128C NPY preprohormone compared to the wild type NPY preprohormone (72
± 32 pA vs. 29 ± 9 pA, mean ± SD; p < 0.05; Figure 3-8 A and B). The cumulative amplitude
distribution of the release events from the cells expressing T1128C was significantly right-shifted
compared to the NPY peptide derived from wild type preproNPY (Fig 3-8 C and D). These
experiments indicated that the SNP led to an increase in peptide secretion. Transfection of the
chromaffin cells with Venus tagged NPY was done by Mr. Gregory Mitchell; the
electrophysiology was performed by Dr. Matthew Whim; I made the plasmid constructs and
examined the expression of the FMRFamide peptide in NPY-Venus transfected AtT-20 cells.
36
Figure 3-1. Schematic of NPY fluorescent fusion protein constructs.
The SNP is located in the signal sequence (arrows indicate the position of the mutation.)
“T1128C” constructs are the observed polymorphism that changes the 7th amino acid from
leucine to proline (Karvonen et al., 1998). “L7P” constructs contain the same amino acid
substitution but use the most frequently used proline codon. Venus is a yellow fluorescent protein
and RFP is a red fluorescent protein. IRES-GFP expresses cytoplasmic GFP. CPON stands for
carboxyl peptide of NPY. Two copies of the FMRFamide peptide are attached to the end of
CPON. Thanks to the initial L7P constructs generated by Mr. Gregory Mitchell.
37
38
Figure 3-2. Expression of wild type and mutant prohormones.
(A). AtT-20 cells were transfected with NPY preprohormone constructs tagged with Venus. The
bands corresponding to proNPY-Venus (37 kDa) and the cleavage product, NPY-Venus (31 kDa)
are indicated. (B). Processing of NPY prohormone was quantified as the ratio of the cleavage
product to the prohormone. The group data indicates that there is no detectable difference in the
processing ratios (mean ± SD, n = 6, P = 0.19, one way ANOVA). (C). AtT-20 cells were
transfected with wild type NPY-Venus, L7P-Venus, T1128C NPY-Venus or were non-transfected.
The cell lysates were subjected to immunoprecipitation with a GFP antibody. Only the
prohormone (37 kDa) could be detected. (D). NPY synthesis was quantified as the ratio of the
NPY prohormone to the lower non-specific band which was used as a loading control. The group
data indicates that there was no detectable difference in the synthesis of proNPY among the
different constructs (mean ± SD, P = 0.97, one-way ANOVA).
39
40
Figure 3-3. Venus fluorescent signals were directly detected on the SDS-PAGE gel using a
Typhoon 8600 gel scanner.
At the emission wavelength of 520 nm, three fluorescent bands corresponding to preproNPY-
Venus, proNPY-Venus and NPY-Venus can be detected on the gel from the wild type and mutant
NPY-Venus transfected cells but not from the non-transfected cells.
41
42
Figure 3-4. Wild type and L7P NPY prohormones were sorted into the regulated secretory
pathway in AtT-20 cells.
(A). AtT-20 cells were transfected with wild type or L7P-IRES-GFP. Transfected cells identified
by cytoplasmic GFP showed punctuate NPY-ir indicating the synthesis of NPY prohormones
derived from wild type or L7P preproNPY. The lower images showed GFP negative controls.
Scale bar 10 µm. (B). The cells expressing wild type NPY-RFP or L7P NPY-RFP proteins
showed punctuate fluorescence that co-localized with NPY-ir. (C). Wild type NPY-Venus and
L7P NPY-Venus fluorescent fusion proteins partially overlapped with the GM130-ir. (D). Wild
type NPY-RFP and L7P NPY-RFP fluorescence co-localized with CPE-ir. Scale bars 20 µm.
43
44
Figure 3-5. L7P and wild type prohormones were packaged into the same dense core
granules in AtT-20 cells.
Cells were co-transfected with wild type NPY-Venus and wild type NPY-RFP (upper panel) or
with wild type NPY-Venus and L7P NPY-RFP (lower panel). The line scan (right panel) was
through the center of the white boxed region. Scale bar 15 µm.
45
46
Figure 3-6. Mutant and wild type prohormones were differentially co-packaged into single
dense core granules in AtT-20 cells.
(A). The cells were co-transfected with wild type and L7P NPY preprohormones tagged with all
four combinations of Venus and RFP fluorescent proteins. The green vs. red intensity ratio in the
single granules from the co-transfected cells was measured and presented as a cumulative graph.
Twenty puncta were selected from each cell along one process and ten cells were analyzed from
each transfected combination. The graph shown was from a representative experiment (n=2). (B).
Cells were co-transfected with wild type and T1128C NPY preprohormones tagged with all four
combinations of Venus and RFP fluorescent proteins. NS, not significant, **p < 0.01, *p < 0.05,
Kolmogorov-Smirnov test.
47
48
Figure 3-7. Co-expression and co-localization of NPY and FMRFamide in AtT-20 cells.
AtT-20 cells were transfected with wild type NPY-Venus or T1128C NPY-Venus preprohormone.
Both wild type NPY-Venus and T1128C NPY-Venus co-localized with FMRFamide
immunoreactivity (Fa-ir). The line scans (right panels) were taken through the center of the boxed
regions. Scale bar 20µm.
49
50
Figure 3-8. NPY T1128C polymorphism leads to an increase in neuropeptide secretion.
(A). Peptide release was detected using FMRFamide tagging. The transfected chromaffin cells
were identified by cytoplasmic GFP expression. The middle and right panels are examples of the
peptidergic secretory events. Secretion was evoked with a brief depolarization from 80 to 0mV
for 20 ms repeated 200 X at 5 Hz. (B). The average secretory current from the cells expressing
wild type or T1128C-NPY preprohormone (mean ± SD, *p < 0.05, single factor ANOVA). (C).
Cumulative distribution of the peptidergic secretory events from the cells expressing wild type
and T1128C-NPY preprohormones ( p < 0.01, Kolmogorov-Smirnov test). (D). Amplitude
frequency histogram of the secretory events shown in (D).
51
52
3.3 Discussion
In transfected AtT-20 endocrine cells, NPY prohormones derived from wild type and
mutant preproNPY entered the regulated secretory pathway in a similar manner and were co-
packaged in the same dense core granules. However when the levels of proNPY were quantified,
the individual granules were found to contain more NPY prohormone derived from mutant
preproNPY. This is consistent with the observation that the cells expressing the mutant
preprohormone had an increased peptide secretion. TheT1128C polymorphism is therefore a
gain-of-function mutation, which contrasts with many other prohormones such as BDNF which
generally leads to misprocessing and a reduction in secretion (Egan et al., 2003a; Karaplis et al.,
1995).
3.3.1 The SNP might affect the ER trafficking efficiency of the NPY preprohormone
How can the altered biosynthesis and secretion of the hormone by a single amino acid
substitution be explained? The T1128 SNP changes the seventh amino acid of the preproNPY
signal sequence from leucine to proline. However the signal sequence is removed co-
translationally when preproNPY translocates into the endoplasmic reticulum (ER) lumen. In
silico analysis using SignalP suggests that the T1128 polymorphism would not prevent signal
sequence recognition and would not change the cleavage site (Bendtsen et al., 2004). This is
consistent with our finding of the regulated secretion of NPY from the mutant-transfected cells.
Eukaryotic signal sequences typically contain a basic N domain, a hydrophobic core, and
a slightly polar C terminus (Hegde and Bernstein, 2006). Leucine is a hydrophobic amino acid,
however proline falls on the hydrophobic-polar border and the distinctive cyclic structure of the
proline side chain gives proline an exceptional conformational rigidity. A SNP in the signal
53
sequence of preproPTH alters the interaction of the nascent protein with the signal recognition
particle (SRP, ensuring the delivery of the peptide to the ER) and the signal peptidase, which
removes the signal sequence, allowing the preprohormone to translocate into the ER lumen. Like
the mutation in preproPTH, the difference between the two amino acids in the signal sequence of
preproNPY might affect its interaction with the SRP and the signal peptidase (Hegde and
Bernstein, 2006; Karaplis et al., 1995).
The differential packaging of NPY prohormones derived from the wild type and mutant
preproNPY into dense core granules was only detected when the two preprohormones were co-
expressed. This observation may indicate that competition between wild type and mutant
preprohormones during the translocation process might occur. A differential preference for
interaction with the SRP or signal peptidase between the wild type and mutant preproNPY might
account for the different degrees of co-packaging. Signal sequences are multifunctional and can
regulate not only the location of the mature protein but also the efficiency of ER translocation
(Hegde and Bernstein, 2006).
3.3.2 The SNP might cause a more efficient sorting of the NPY prohormone
The postulated bias in the efficiency of ER translocation may lead to a situation in which
the transport of proNPY from mutant preproNPY is faster than wild type within the trans-Golgi
network. This may allow the proNPY from the mutant preprohormone to be sorted into DCSGs
earlier and more efficiently than the wild type.
In addition the signal peptide might have an independent function after cleavage (Hegde
and Bernstein, 2006). It is possible that the mutated signal peptide could facilitate the trafficking
of proNPY.
54
3.3.3 Codon usage does not explain the effect of the SNP
The leucine codon in the wild type preproNPY is CTG which is the most commonly used
version, whereas the T1128C polymorphism, which encodes proline (CCG) is the least frequently
used proline codon. Non-optimal codons in some signal peptides can lead to translational pausing
which helps to correct protein folding and thus increases peptide synthesis (Zalucki et al., 2008). I
found a similar phenotype when I expressed either the optimal codon CCC (L7P) or the non-
optimal proline codon CCG (T1128C) in preproNPY. This suggests that codon usage does not
account for the differential packaging and secretion observed in the NPY prohormone derived
from mutant preproNPY.
3.3.4 Comparison of NPY T1128 with SNPs found in other peptide preprohormones and
prohormones
SNPs have been identified in a variety of peptide prohormones and these are generally
associated with a loss-of-function phenotype. The Val66Met in proBDNF leads to impaired
trafficking and reduced secretion (Egan et al., 2003). The Cys18Arg mutation in the signal
sequence of preproPTH isolated from a patient with hypocalcemia causes inefficient prohormone
processing (Karaplis et al., 1995). The Leu16Arg substitution in the signal sequence of hypocretin
was identified in a human case of early onset narcolepsy and this SNP causes incomplete
prohormone cleavage and retention in the soma (Peyron et al., 2000).
In contrast the T1128C polymorphism led to an increase in the biosynthesis of proNPY
and up-regulation of NPY regulated secretion. These results parallel the finding that humans
carrying this SNP have higher levels of plasma NPY during exercise (Kallio et al., 2001). NPY is
involved in regulating lipid metabolism; for example it can inhibit lipolysis (Turtzo et al., 2001).
Elevation in NPY signaling positively regulates the stress-induced adipocyte growth that
55
develops during obesity (Kuo et al., 2007b). This may contribute to the phenotype of an elevated
serum cholesterol level, increased plasma triglycerides and a higher risk of developing obesity as
well as type 2 diabetes which are found in T1128C carriers (Karvonen et al., 1998; Pesonen,
2008).
3.4 Conclusion
In conclusion my experiments demonstrate that the L7P SNP does not alter the
trafficking pattern of proNPY but leads to an increase in prohormone packaging into dense core
granules and an elevation in NPY peptide secretion. The results also emphasize that functional
SNPs can have readily detectable effects at the level of single granules. Most of this work has
been published in the Journal of Neuroscience (Mitchell *, Wang *, Ramamoorthy and Whim
(2008) 28: 14428-34; * co-first author).
Chapter 4
Acute Stress Increases the Levels of Adrenal Neuropeptide Y
4.1 Introduction
Every individual experiences stressful life events and the ability to cope with stress is
extremely important to prevent harmful outcomes. The beneficial aspect of the acute stress
response is that it triggers an adaptive response which allows the organism to cope with the
stressor. The harmful aspect can be evident during exposure to a chronic stress which can elicit a
pathological response (McEwen, 1998; Sabban et al., 2006).
People with poor stress coping ability are highly susceptible to numerous diseases such
as depression and other psychiatric disorders (Feder et al., 2009). Chronic stress also leads to the
development of cardiovascular disease (Kuo et al., 2007a) and increases the risks of immune
dysfunction and cancer (Johnstone and Baylin, 2010; McEwen, 1998; Wong, 2006). In addition
chronic stress is associated with an increased susceptibility to obesity and drug addiction (Kuo et
al., 2007b; Ungless et al., 2010).
Exposure to either an acute or chronic stressor results in a series of coordinated responses
consisting of changes in behavior, neuronal plasticity, neuroendocrine functions and secretion of
hormones (Lupien et al., 2009; Roozendaal et al., 2009; Van de Kar and Blair, 1999). In the CNS
the stress response takes place in many brain regions including the hippocampus (Pittenger and
Duman, 2008), hypothalamus (Hewitt et al., 2009), amygdala (Knoll et al., 2011), prefrontal
cortex (Yuen et al., 2011), ventral tegmental area (Krishnan et al., 2007) and cerebellum (Liu et
al., 2010). In the PNS the stress response involves two major pathways, both of which are
initiated by hypothalamic activity (Axelrod and Reisine, 1984; Sapolsky et al., 2000; Wong,
57
2006). One is the “hormonal pathway” in which stress activates the hypothalamus-pituitary
gland-adrenal cortex axis (known as the HPA axis) and leads to a release of corticotropin-
releasing hormone (CRH) from the hypothalamus. CRH induces secretion of adrenocorticotropin
hormone (ACTH) from the pituitary gland. The release of ACTH stimulates the adrenal cortex to
secrete glucocorticoids and then glucocorticoids participate in a negative feedback loop, which
inhibits the activity of the hypothalamus and pituitary to prevent the over-activation of the HPA
axis (Figure 4-1 A) (Axelrod and Reisine, 1984; Sapolsky et al., 2000). The other peripheral
stress pathway is the “neuronal pathway” characterized by the activation of the sympatho-adrenal
medullary system. Descending input from the hypothalamus activates spinal preganglionic
neurons. Some of these neurons project to their targets via splanchnic nerves. Acetylcholine,
which is the primary neurotransmitter in the splanchnic nerves, is released, depolarizing the
chromaffin cells in the adrenal medulla leading to the release of the catecholamines (mainly
epinephrine), which alters heart rate and blood pressure (Figure 4-1 B) (Axelrod and Reisine,
1984; Wong, 2006).
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Figure 4-1.The two major peripheral stress pathways.
(A). The hormonal pathway; also known as the HPA axis. (B). The neuronal pathway; the
sympatho-adenal medullary system.
The stress-induced release of hormones including ACTH from the pituitary gland,
glucocorticoids from the adrenal cortex, epinephrine from the adrenal medulla and
norepinephrine from the sympathetic nerves (with a minor contribution from the adrenal medulla)
helps to achieve stability which prevents the development of a pathological response to stress
(Axelrod and Reisine, 1984).
Each adrenal gland consists of a cortex and a medulla, both of which are involved in the
stress response. Therefore the adrenal glands are part of the mechanisms that allow the organism
to cope with stress (Carrasco and Van de Kar, 2003). The functional unit of the adrenal medulla is
the chromaffin cell, a modified postganglionic neuron which synthesizes and secretes hormones,
not only the catecholamines, but also NPY (Varndell et al., 1984).
Expression of adrenal NPY mRNA is elevated by various stressors including cold and
immobilization (Hiremagalur et al., 1994; Hiremagalur and Sabban, 1995; Raghuraman et al.,
2011). The levels of adrenal NPY mRNA are increased by a relatively short period of
immobilization and the elevation is abolished by the transcriptional inhibitor actinomycin D.
Furthermore this stress-induced rise of NPY mRNA expression requires splanchnic innervation
suggesting that the neuronal activation of chromaffin cells is essential for the immobilization-
induced increase of NPY transcription (Hiremagalur et al., 1994).
Although the adrenal mRNA levels of NPY are increased by several stressors, it is still
not clear whether acute stress increases the levels of the NPY peptide in chromaffin cells and if
the response is seen in all chromaffin cells.
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Here I investigate these questions by exposing mice to an acute stressor, the cold water
forced swim test (cold FST) or fox urine odor. I find that the cold FST and exposure to the odor
of fox urine leads to an increase in NPY immunoreactivity (NPY-ir) in chromaffin cells. Stress
increases the number of chromaffin cells that express relatively high levels of NPY peptide and
decreases the number of cells that contain relatively low levels of NPY-ir. The increase in NPY
synthesis in chromaffin cells also involves an increase in NPY transcription. In addition the cold
FST increases the levels of NPY in adrenal slices and the increase has reversed one week later.
4.2 Results
4.2.1 The cold FST activated the HPA axis and induced activity in chromaffin cells
The cold FST is a common stressor used in investigating the effect of acute stress and
was shown to enhance synaptic transmission in dopamine neurons in the CNS (Huber et al., 2001;
Saal et al., 2003). However it has not been used widely in the context of the peripheral stress
response. To examine its validity as a peripheral stressor, mice were exposed to the cold FST for
5-6 minutes and decapitated thirty minutes later (Figure 4-2 A). Blood was collected for
measurement of the levels of plasma corticosterone, the adrenal component of the HPA axis.
Plasma corticosterone levels were significantly higher in the stressed animals compared to the
matched controls (Figure 4-2 B) indicating that the cold FST led to an activation of the HPA axis.
I then asked if the cold FST activated the neuronal branch of the stress response in
addition to the hormonal axis. c-Fos is an immediate early gene whose expression is an indirect
marker of neuronal activity (Jhou et al., 2009; Lobo et al., 2010). C-Fos-ir in adrenal slices was
examined 60 minutes after the cold FST and compared to the matched controls (Figure 4-3 A).
The number of c-Fos-ir cells was significantly increased in slices of the adrenal medulla after
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stress (Figure 4-3 B and C) indicating that the cold FST elevated the electrical activity of
chromaffin cells.
Thus the cold FST activated both the HPA axis and the neuronal branches of the stress
response.
4.2.2 GFP is expressed in the hypothalamus, hippocampus and adrenal medulla in NPY
(GFP) transgenic mice
I obtained a NPY-hrGFP transgenic mouse line that was generated using a large bacterial
artificial chromosome (BAC) containing the NPY promoter and upstream regulatory elements
that drives the expression of Renilla GFP. These mice were shown to express GFP in most of the
known NPY neurons in the brain (van den Pol et al., 2009). In these mice I confirmed that GFP
was richly distributed in the hypothalamus and the hippocampus (Figure 4-4 A and B). I found
that GFP was also expressed in the adrenal medulla but not in the adrenal cortex suggesting that
NPY in the adrenal glands was only made by chromaffin cells (Figure 4-4 C). Moreover GFP was
found in all chromaffin cells.
4.2.3 An NPY antibody specifically recognizes NPY in chromaffin cells
To test the specificity of an NPY antibody that would be used in later experiments, I
stained chromaffin cell cultures from wild type and NPY knockout mice with the NPY antibody.
Chromaffin cells from the wild type animal were stained with the NPY antibody (Figure 4-5 A)
In contrast chromaffin cells from the knockout animal were not stained with the NPY antibody
(Figure 4-5 B). The lack of NPY-ir in chromaffin cells from the NPY knockout mouse indicates
that the NPY antibody I used specifically recognizes NPY in the adrenal medulla.
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4.2.4 The cold FST led to an up-regulation of NPY-immunoreactivity in chromaffin cells in
vitro
To determine whether the cold FST regulated the levels of adrenal NPY in chromaffin
cells, animals were exposed to the cold FST for 5 to 6 minutes. After 24 hours the chromaffin
cells were dissociated from stressed and matched control animals (Figure 4-6 A). Quantification
of the levels of NPY-ir in single chromaffin cells from stressed and littermate control mice
indicated that stress increased NPY-ir (Figure 4-6 B and C). The frequency histograms of the
levels of NPY-ir in chromaffin cells from both matched control and stressed mice was better
fitted with two Gaussian distributions compared to one (Figure 4-6 D). This indicates that there
are two sub-populations of chromaffin cells, one with relatively high levels of NPY-ir and the
other with lower levels of NPY-ir. The distributions of NPY-ir in chromaffin cells showed that
the cold FST increased the number of cells expressing high levels of NPY but decreased the
proportion of cells with low NPY-ir compared with the matched controls (Figure 4-6 D). The
rightward shift in the cumulative distribution of intensities from the cold FST-exposed animals
suggested that chromaffin cells from stressed mice contained higher levels of NPY (Figure 4-6 E).
My findings suggest that the cold FST-induced stress increases the levels of NPY-ir due to a shift
between two sub-populations of chromaffin cells.
The levels of NPY-ir in chromaffin cells dissociated from a mouse which was sacrificed
30 minutes after the cold FST was not different from the matched control (Appendix A). This
result indicated that the stress-induced increase in the levels of NPY-ir in chromaffin cells
required an extended period of time to become evident.
Previous studies have shown that the mRNA levels of NPY in whole adrenal glands were
altered by immobilization or cold (Hiremagalur et al., 1994; Hiremagalur and Sabban, 1995;
Raghuraman et al., 2011). The present work shows that the levels of NPY rise in single
chromaffin cells.
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4.2.5 Brief exposure to fox urine also led to an up-regulation of NPY-immunoreactivity in
chromaffin cells in vitro
To investigate whether the effect of acute stress on the levels of adrenal NPY was
universal or restricted to one specific stressor, I exposed mice to a different stressor, fox urine
odor for 5 minutes after which the animals were returned to their home cage (Figure 4-7 A). Fox
urine exposure induces an innate fear in mice as indicated by a freezing behavior (Hu et al., 2007;
Liu et al., 2010). Sixteen to twenty four hours after the fox urine exposure, I measured the levels
of NPY-ir in single chromaffin cells dissociated from stressed and matched control animals.
Similar to the cold FST-induced stress, the fox urine exposure also increased NPY-ir in
chromaffin cells (Figure 4-7 B, C and E). In a similar fashion to the cold FST, the fox urine
exposure led to a sub-population shift of NPY-ir in chromaffin cells (Figure 4-7 D).
Thus two different types of acute stressor both increased NPY-ir in chromaffin cells in a
similar manner. This suggests that acute stress leads to a coordinated up-regulation of the levels
of adrenal NPY.
4.2.6 The cold FST increased NPY-ir measured in adrenal slices
Having found that acute stress increased NPY-ir in chromaffin cell cultures in vitro, I
next examined the levels of NPY-ir in adrenal slices from cold FST- exposed mice and matched
controls. The advantage of this approach is that the structure of the gland is preserved. Frozen
sections were made from control and stressed animals 24 hours after the cold FST, and stained
with the NPY antibody. The cold FST significantly increased the levels of NPY-ir in the adrenal
medulla (Figure 4-8 A and B). Therefore acute stress increases the levels of adrenal NPY in situ
which is consistent with the results from chromaffin cells in vitro.
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The levels of NPY-ir in adrenal slices from a mouse which was sacrificed 30 minutes
after the cold FST was not different from the matched control (Appendix A). This is consistent
with the result that NPY-ir in chromaffin cells in vitro was not changed 30 minutes after the cold
FST (section 4.2.4).
4.2.7 The cold FST-induced increase of NPY-ir in adrenal slices returned to baseline one
week after stress
Acute stress is believed to be part of an adaptive mechanism that enables an organism to
cope with stress, in contrast to chronic stress which may cause depression. I next asked whether
the stress-induced increase of NPY was reversible. This is important because over-activation of
the adrenal system in the stress response can be pathological. Animals were exposed to the cold
FST then sacrificed either 24 hours or one week later. Matched control animals were treated
identically except they were not exposed to stress (Figure 4-9 A). I measured and compared NPY-
ir in the adrenal medulla from these three groups of animals. NPY-ir in the adrenal medulla was
significantly higher at 24 hours after the cold FST but returned to control values by one week
after acute stress (Figure 4-9 B and C). The return of adrenal NPY-ir back to baseline after stress
may imply that a negative feedback regulatory pathway is involved in the adrenal response to
stress.
4.2.8 The cold FST increased GFP expression in NPY (GFP) transgenic mice
Since stress increased the adrenal levels of the NPY peptide, I then investigated whether
stress increased the levels of adrenal NPY via a process that involved an increase of NPY gene
transcription or a reduction of NPY secretion (which would lead to increased levels of
intracellular NPY). To test these possibilities I used an NPY (GFP) transgenic mouse, in which
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GFP is linked to the NPY promoter and the NPY signal sequence is interrupted. These BAC NPY
(GFP) transgenic mice still make endogenous NPY (van den Pol et al., 2009). The NPY (GFP)
mice were exposed to the cold FST and 24 hours later chromaffin cells were dissociated from
stressed mice and matched controls. I measured the expression of the reporter gene by the
fluorescence levels of GFP. GFP fluorescence was significantly higher in chromaffin cells from
stressed animals compared to matched controls (Figure 4-10 A, B and D). The frequency
histograms of GFP fluorescence in chromaffin cells from both matched control and stressed mice
was fitted with two Gaussian distributions better than one (Figure 4-10 C). Comparison of the
frequency histograms of GFP fluorescence from control and stressed animals showed a sub-
population shift in the GFP fluorescence after stress in a similar manner to that seen with NPY-ir
(Figure 4-10 C).
The increased GFP expression after stress suggested that the cold FST elevated NPY
promoter activity in chromaffin cells since the GFP expression was driven by the NPY promoter.
Unlike NPY, GFP is a non-secreted protein so the increase of GFP expression after stress was due
to an increased synthesis rather than reduced secretion (although I still cannot rule out the
possibility that a reduced NPY secretion contributes to the increase of the adrenal peptide). The
data from the NPY (GFP) animals suggested that stress increased the levels of adrenal NPY by a
process that involved an increase of gene transcription and peptide production.
4.2.9 Adrenal NPY mRNA expression was increased 3 hours after the cold FST
Since adrenal NPY promoter activity was shown to increase in the NPY (GFP) animals, I
then measured the levels of NPY mRNA in the adrenal medulla from the wild type animals by
semi-quantitative RT-PCR. The results indicated an increase in NPY mRNA expression 3 hours
after the cold FST (Figure 4-11 A and B). This supports the data from the NPY (GFP) transgenic
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mice that the NPY promoter activity was increased after stress. Therefore the cold FST-induced
increase of adrenal NPY is likely to involve transcriptional regulation.
4.2.10 Pancreatic polypeptide and peptide YY mRNA expression in the adrenal medulla
after the cold FST
The NPY family of peptides includes two other members, pancreatic polypeptide (PP)
and peptide YY (PYY), which are primarily found in the pancreas and intestine, respectively
(Ekblad and Sundler, 2002). No PYY-ir or mRNA has been reported in the rat adrenal medulla.
However PYY-ir was shown to be present in the cat adrenal medulla (Ekblad and Sundler, 2002;
Pieribone et al., 1992). PP-like immunoreactivity in the rat adrenal medulla was reported but the
antibody used was not well characterized (Pieribone et al., 1992; Vaillant and Taylor, 1981).
Later studies using a well-defined antibody and a radioimmunoassay failed to detect the
expression of PP in the rat adrenal medulla (Miyazaki and Funakoshi, 1988; Pieribone et al.,
1992).
To test whether PP and PYY are expressed in the mouse adrenal medulla and whether
stress can affect their expression, I utilized RT-PCR to examine the mRNA levels of PP and PYY.
mRNA encoding PP and PYY was present in mouse pancreatic islets validating the primers that I
used. Insulin was used as a positive control for islet tissue (Figure 4-12 A). Amplification of NPY
in the adrenal medulla indicated the successful extraction of mRNA and generation of cDNA.
Neither PP nor PYY expression was detected in the adrenal medulla of control mice in three
independent experiments (Figure 4-12 B and C). No PP expression was present in the animals 24
hours after the cold FST (Figure 4-12 B and C). However 24 hours after stress, in one experiment
PYY mRNA was detected in the adrenal medulla whereas the other two experiments did not
reveal the amplification of PYY (Figure 4-12 B and C). The apparent variability in adrenal PYY
66
expression in the stressed mice may depend on the different individuals or the intensity of the
stress. The result implies that PYY might be expressed in the mouse adrenal medulla, perhaps
under extremely stressful circumstances.
In conclusion, among the NPY family of peptides, only NPY was consistently expressed
in the mouse adrenal medulla and only this member of the Y peptide family was increased by
exposure to different stressors.
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Figure 4-2. The cold FST activated the HPA axis.
(A). Stress paradigm. P21-P23 male littermates were used in each individual experiment. (B).
Plasma corticosterone levels increased 30 minutes after the cold FST (mean ± SEM, n = 5). **p <
0.01, Student’s paired t test. The open black circles indicate the value from each individual
animal. The black lines link the matched control and stressed animals from each individual
experiment.
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Figure 4-3. The cold FST activated the neuronal branch of the stress response.
(A). Stress paradigm. P21-P23 male littermates were used in each individual experiment. (B).
Adrenal glands stained for c-Fos from control and stressed animals. The number of c-Fos-ir cells
in the medulla (within the white dashed lines) were quantified using multiple areas of interest
(white circles). The enlarged inset represented the region that was outlined by the white square.
Scale bars 100 μm. (C). Group data showing that stress increased the number of c-Fos-ir cells in
the medulla (mean ± SEM, n = 3). *p < 0.05; Student’s paired t test. Seven to ten sections were
used to measure c-Fos-ir from each animal. The open black circles indicate the value from each
individual animal. The black lines link the matched control and stressed animals from each
individual experiment.
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Figure 4-4. NPY (GFP)-expressing neurons and neuroendocrine cells in the nervous system.
(A). NPY (GFP) neurons in the mouse hippocampus. (B). NPY (GFP) neurons in the mouse
hypothalamus. (C). NPY (GFP) is expressed in the mouse adrenal medulla.
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Figure 4-5. NPY-ir in chromaffin cells from wild type and NPY knockout mice.
(A). Dissociated chromaffin cells from a wild type animal were stained with an NPY antibody.
(B). Chromaffin cells from an NPY knockout animal did not exhibit any NPY-ir when stained
with the NPY antibody. The enlarged inset is a bright field image of chromaffin cells. The white
dashed circles outline chromaffin cells in the NPY knockout animal. Scale bar 5 μm.
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Figure 4-6. The cold FST increased the level of NPY-ir in chromaffin cells in vitro.
(A). Stress paradigm. P21-P23 male littermates were used in six of the seven independent
experiments, and one paired group were P21 female littermates. (B). Examples of chromaffin
cells from control and stressed animals. Scale bar 10 μm. (C). Group data indicated that NPY-ir is
higher in chromaffin cells from stressed animals compared to matched controls (mean±SEM, n =
7). **p < 0.01; Student’s paired t test. Seventy cells were used from each animal to measure the
levels of NPY-ir. The open black circles indicate the value from each individual animal. The
black lines link the matched control and stressed animals from each individual experiment. (D).
Representative frequency histogram from either matched control or stressed mice fitted with two
Gaussian distributions (red dashed curve vs. navy blue dashed curve). The representative
frequency histogram showed that stress resulted in a decrease in the proportion of low NPY-ir
cells and an increase in the proportion of high NPY-ir cells. (E). The cumulative distributions
indicated chromaffin cells from stressed animals contained higher levels of NPY. ***p < 0.001;
Kolmogorov-Smirnov test.
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Figure 4-7. Brief exposure to fox urine increased the level of NPY-ir in chromaffin cells in
vitro.
(A). Stress paradigm. P19-P21 male or female littermates were used. (B). Chromaffin cells from
control and stressed animals. Scale bar 10 μm. (C). Group data indicated that NPY-ir is higher in
chromaffin cells from stressed animals compared to matched controls (mean ± SEM, n = 6). *p <
0.05; Student’s paired t test. Sixty to seventy cells were used from each animal to measure NPY-
ir. The open black circles indicate the value from each individual animal. The black lines link the
matched control and stressed animals from each individual experiment. (D). A representative
frequency histogram from either matched control or stressed mice fitted with two Gaussian
distributions (red dashed curve vs. navy blue dashed curve). The representative frequency
histogram showed that stress led to a decrease in the proportion of low NPY-ir cells and an
increase in the proportion of high NPY-ir cells. (E). The cumulative distributions indicated
chromaffin cells from stressed animals contained higher levels of NPY. ***p < 0.001;
Kolmogorov-Smirnov test.
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Figure 4-8. The cold FST increased the level of adrenal NPY-ir in situ.
(A). Adrenal sections from a control animal and 24 hours after the cold FST. Scale bars 100 μm.
(B). Group data showing that NPY-ir was significantly higher in the adrenal medulla from
stressed animals compared to the matched controls. NPY-ir was normalized to the values of
controls (mean ± SEM, n = 3; raw data: 94.15 ± 23.42 vs. 173.75 ± 34.01 arbitrary units). *p <
0.05; Student’s matched one sample t test. P21-P23 male littermates were used in each individual
experiment. Seven to thirteen sections were used to measure NPY-ir from each animal. The open
black circles indicate the value from each individual animal. The black lines link the matched
control and stressed animals from each individual experiment.
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Figure 4-9. The cold FST reversibly increased the level of adrenal NPY-ir in situ.
(A). Stress paradigm. P21-P23 male littermates were used in two of the three independent
experiments, and one paired group were P21 female littermates. (B). Adrenal sections from a
control animal, 24 hrs and 1 week after the FST. Scale bars 100 μm. (C). Group data showed that
NPY-ir was significantly higher in the adrenal medulla at 24 hrs after the cold FST but declined
to control values by 1 week after the FST (mean ± SEM, n = 3). *p < 0.05; **p < 0.01; one-way
ANOVA (post-hoc Tukey’s paired comparison; F (2, 6) = 16.31). Eight to ten sections were used
to measure the levels of NPY-ir from each animal. The open black circles indicate the value from
each individual animal. The black lines link the matched control and stressed animals from each
individual experiment.
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Figure 4-10. The cold FST increased GFP expression in chromaffin cells from NPY (GFP)
mice.
(A). GFP expression in chromaffin cells from a control and stressed animal. Scale bar 10 μm. (B).
Group data indicated that GFP expression was significantly higher in chromaffin cells from the
stressed animals compared to the matched controls (mean±SEM, n = 3). **p < 0.01; Student’s
paired t test. P21-P22 male littermates were used in each individual experiment. Ninety cells were
used from each animal to measure GFP fluorescence. The open black circles indicate the value
from each individual animal. The black lines link the matched control and stressed animals from
each individual experiment. (C). A representative frequency histogram from either matched
control or stressed mice fitted with two Gaussian distributions (red dashed curve vs. navy blue
dashed curve). The representative frequency histogram showed that stress led to a decrease in the
proportion of low GFP-expressing cells and an increase in the proportion of high GFP-expressing
cells. (D). Cumulative distributions indicated that chromaffin cells from the stressed animals
contained higher levels of GFP. ***p < 0.001; Kolmogorov-Smirnov test.
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Figure 4-11. The adrenal levels of NPY mRNA increased 3 hours after the cold FST.
(A). An example of a RT-PCR experiment for NPY and actin from a control animal and 3 hours
after the cold FST. The negative controls were RT reactions without reverse transcriptase. (B).
Group data indicated that the level of NPY mRNA was significantly higher in the stressed
animals. Actin expression was used as an internal control. Results from the stressed animals were
normalized to matched controls (n = 3). *p < 0.05; Student’s matched one sample t test. P21-P23
male mice were used. The open black circles indicate the value from each individual animal. The
black lines link the matched control and stressed animals from each individual experiment.
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Figure 4-12. Expression of PP, PYY and NPY mRNA in the adrenal medulla.
(A). RT-PCR analysis showed the expression of PP (196 bp), PYY (260 bp) and insulin (241 bp)
in pancreatic islets. (B). An example of RT-PCR for PP, PYY and NPY (288 bp) from the adrenal
medulla in one group of control and stressed animals. (C). An example of RT-PCR of PP, PYY
and NPY from the adrenal medulla in a second group of control and stressed animals. The
negative controls were RT reactions without reverse transcriptase. P21 male littermates were used.
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4.3 Discussion
4.3.1 Stress increases adrenal NPY transcription and peptide synthesis.
Various stressors such as cold and immobilization are known to elevate NPY gene
expression in the adrenal medulla (Hiremagalur et al., 1994; Hiremagalur and Sabban, 1995;
Raghuraman et al., 2011). The stress-induced increase of adrenal NPY is transcription-dependent
since the transcriptional inhibitor actinomycin D abolished the effect of stress on adrenal NPY
gene expression (Hiremagalur et al., 1994). In my study I also found that the cold FST-induced
stress increased NPY promoter activity in chromaffin cells.
Although the levels of adrenal NPY mRNA were up-regulated by stress, the effect of
stress on NPY peptide synthesis and how it is mediated at the single cell level is not clear. My
study demonstrates a stress-induced increase of NPY peptide synthesis in chromaffin cells. Since
I am not able to directly measure the secretion of endogenous NPY from single chromaffin cells,
I compared GFP expression in control and stressed NPY (GFP) transgenic mice as a way to
determine whether stress increased NPY synthesis. The stress-induced increase of GFP
expression in chromaffin cells from the NPY (GFP) mice confirmed that the increased levels of
adrenal NPY 24 hours after stress involved an up-regulation of NPY production rather than a
reduction in secretion. In the previous chapter (section 3.2.7), I showed that an increased level of
NPY biosynthesis likely leads to an elevation of evoked NPY peptide release. Thus the stress-
induced long-term up-regulation of adrenal NPY synthesis may lead to an increase of NPY
secretion and have profound effects on metabolic and cardiovascular regulation.
I also investigated the cellular actions involved in the response of the adrenal medulla to
stress and found a shift in the expression of NPY between two sub-populations of chromaffin
cells after stress. Chromaffin cells from control animals express both low and high levels of NPY.
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Stress increases the number of cells that express high levels of NPY but does not up-regulate the
NPY levels in all of the chromaffin cells. I speculate a possible mechanism for this stress-induced
sub-population shift may involve the following: (1) some cells that contain a high level of NPY
have reached a threshold and NPY cannot be further increased; (2) a portion of NPY-containing
cells that express low levels of NPY lack the components that are needed to sense the signaling
pathways triggered by stress; (3) other cells with low levels of NPY can be activated by stress and
these cells switch between expressing low and high levels of NPY during the stress response. It is
known that there are sub-populations in chromaffin cells: for example the epinephrine-containing
vs. norepinephrine-containing cells (Cahill et al., 1996). Since all mouse chromaffin cells express
NPY, the different sub-populations in the levels of NPY may function in regulating the
expression of other hormones in chromaffin cells.
4.3.2 The mechanism that is involved in the stress-induced increase of adrenal NPY.
The immobilization-induced increase of adrenal NPY requires splanchnic innervation
which is the neuronal input into the adrenal medulla (Hiremagalur et al., 1994). The evidence for
the necessary involvement of the sympathetic innervation in the stress-induced increase of NPY
mRNA is that either denervation by splanchnicectomy or injection of the nicotinic receptor
antagonist chlorisondamine blocks the immobilization-induced elevation of NPY mRNA level
(Hiremagalur et al., 1994; Hiremagalur et al., 1995).
4.3.3 The transcriptional factors that are involved in response of the adrenal medulla to
stress.
Acute stress elicits a global change of gene expression and the greatest number of
changes in the adrenal medulla are observed within transcription factors (Liu et al., 2008). c-Fos
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is not only an indirect marker of neuronal activation, but also a transcriptional factor which is
activated by stress (Figure 4-2 A and B)(Kovacs, 1998). The NPY promoter region contains an
AP1 binding site motif which is potentially induced by c-Fos activation (Titolo et al., 2008).
However direct binding of c-Fos with the NPY promoter still lacks experimental evidence.
Delta FosB is a truncated splice isoform of FosB, another Fos family member which
participates in forming the transcriptional factor complex AP1 (Nestler, 2008). Delta FosB in the
nucleus accumbens, an important brain rewarding circuit was shown to mediate resilience in the
chronic social defeat stress response. A genome-wide analysis of gene expression in the nucleus
accumbens indicated an increase of NPY gene expression in the mice with an overexpression of
delta FosB compared to the controls (Vialou et al., 2010). Delta FosB expression in the adrenal
medulla was significantly increased by repeated immobilization stress (Nankova et al., 2000).
Overexpression of delta FosB in PC12 cells (a cell line derived from a tumor of the rat adrenal
medulla) resulted in an increase of tyrosine hydroxylase promoter (containing an AP1- like
binding motif) activity (Nankova et al., 2000). Therefore delta FosB may also potentiate adrenal
NPY transcription.
Besides the Fos family of proteins, the adrenal cAMP response element-binding protein
(CREB) is significantly activated by stress and MAPK-ERK1/2 signaling, an upstream regulator
of CREB is also induced (Sabban et al., 2006). There is a half-CRE site in the NPY promoter
region and CREB directly binds to the -118 to +22 bp region of the NPY promoter in the N-38
neuronal cell line as shown by a chromatin immunoprecipitation assay (Titolo et al., 2008).
Since the immobilization stress-induced increase in adrenal NPY transcription requires
neuronal innervation of chromaffin cells (Hiremagalur et al., 1994; Hiremagalur et al., 1995), c-
Fos which is activated during neuronal firing is a good candidate to mediate the effect of stress on
an increase of adrenal NPY transcription (Labiner et al., 1993). In addition the activation of
chromaffin cells during the stress response causes an elevation of Ca2+
influx. Therefore the
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pathways activated by Ca2+
influx could account for the stress-induced increase of NPY and this
may involve CREB activation (Labiner et al., 1993).
Although the transcriptional factors c-Fos (section 4.2.1), delta Fos B (Nankova et al.,
2000) and CREB (Sabban et al., 2006) in the adrenal medulla are activated by stress and
potentially induce NPY gene expression, there is no direct evidence to link them with the stress-
induced increase of adrenal NPY gene expression. Therefore the molecular mechanisms and
signaling molecules involved in the stress-induced elevation of adrenal NPY transcription need to
be determined in future experiments.
4.3.4 Adrenal epinephrine in the stress response.
The activation of chromaffin cells after stress leads to an increase of catecholamine
release within seconds (Axelrod and Reisine, 1984). The elevation of plasma catecholamines
might be part of the pathway that leads to the increase of adrenal NPY transcription. Splanchnic
denervation which blocks catecholamine release abolishes the stress-induced adrenal NPY up-
regulation (Hiremagalur et al., 1994; Hiremagalur et al., 1995).
In preliminary experiments I found that injection of epinephrine increased adrenal NPY-
ir (Appendix B) and direct incubation of adrenal slices with epinephrine also elevated NPY-ir
(Appendix B). This data may indicate that the stress-induced increase in plasma catecholamines is
an upstream signaling event which activates adrenal NPY gene expression. Both alpha and beta
adrenergic receptors are expressed in the adrenal medulla and they are all G protein coupled
receptors (Cesetti et al., 2003; Kleppisch et al., 1992). A regulatory mechanism involving adrenal
catecholamine secretion has been described that acts via autocrine feedback inhibition of α2
adrenergic receptors in chromaffin cells (Lymperopoulos et al., 2007). Since the activation of beta
adrenergic receptor leads to an increase of cAMP (Cesetti et al., 2003), adrenal catecholamine
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release may exert autocrine activation through beta adrenergic receptors to induce the activation
of CREB thereby regulating adrenal NPY gene expression.
4.3.5 Stress-induced secretion and synthesis coupling
The metabolic stress that is triggered by insulin-induced hypoglycemia stimulates the
splanchnic nerve and catecholamine release from the adrenal medulla leading to a depletion of
catecholamines in chromaffin cells (Laslop et al., 1989). The increased plasma catecholamines
ensure that a compensatory glycogenolysis and gluconeogenesis takes place to maintain glucose
homeostasis. The induction of adrenal tyrosine hydroxylase during an insulin challenge enables a
refilling of the depleted pool of catecholamines in chromaffin cells, which is important for
maintaining a continual secretion of catecholamines and thus regulating the levels of plasma
glucose (Hamelink et al., 2002). In pituitary adenylate cyclase-activating polypeptide (PACAP)
knockout mice, there is a failure to increase adrenal catecholamine synthesis during an insulin
challenge. These animals showed a more long-lasting insulin-induced hypoglycemia and an
insulin dose-related lethality compared to wild type animals (Hamelink et al., 2002). These results
suggest an important role for the coupling of secretion and biosynthesis of catecholamine
hormones in response to stress.
Stress stimulates the secretion of NPY from sympathetic neurons and chromaffin cells
(Kuo et al., 2007a). In my experiments I showed that NPY synthesis in chromaffin cells was up-
regulated by stress. The coupling of the stress-induced increase of NPY release and synthesis in
chromaffin cells may be a survival mechanism that allows the organism to maintain physiological
homeostasis.
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4.4 Conclusion
In conclusion my experiments showed that acute stress increased the levels of the NPY
peptide in the adrenal medulla and that this is due to an increased number of cells expressing high
levels of NPY and a reduced number of cells containing low levels of NPY. The increase of the
NPY peptide involved an up-regulation of NPY gene transcription and the increase reversed one
week after the stress.
Chapter 5
NPY Inhibits the Neuronal Branch of the Stress Response
5.1 Introduction
In the previous chapter I described how the stress response is regulated by two major
pathways which are the hormonal pathway (the “HPA axis”) and the neuronal pathway (the
“sympathetic-adrenal medullary system”). Activation of these two pathways leads to the release
of the hormones cortisol (corticosterone in rodents) from the adrenal cortex and the
catecholamines from the adrenal medulla (Axelrod and Reisine, 1984; Sapolsky et al., 2000;
Wong, 2006).
Adrenal cortisol has many important metabolic actions such as promoting hepatic
gluconeogenesis and lipogenesis, and inhibiting both acute and chronic inflammation (Heitzer et
al., 2007; Landys et al., 2006). Adrenal catecholamines are essential during the fight-or-flight
response altering blood pressure, increasing hepatic gluconeogenesis, elevating thermogenesis
and enhancing lipolysis in fat cells (Bachman et al., 2002; Keys and Koch, 2004; Song et al.,
2010; Zhang et al., 2010a).
The catecholamines are synthesized from the precursor amino acid tyrosine. The enzyme
tyrosine hydroxylase (TH) converts tyrosine to dihydroxyphenylalanine (DOPA) and then DOPA
is converted to dopamine (a catecholamine) by aromatic amino acid decarboxylase (DDC).
Dopamine-β-hydroxylase (DβH) catalyzes the generation of norepinephrine (NE, another
catecholamine), which can be converted to epinephrine (Epi, a third type of catecholamine) by
phenylethanolamine N-methyltransferase (PNMT, Figure 5-1). NE and Epi are the
catecholamines found in chromaffin cells. Since TH is the first and rate-limiting enzyme for
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making the catecholamines, it is often used as a marker of catecholamine biosynthesis
(Kvetnansky et al., 2009). A typical response of the adrenal medulla to stress involves not only an
increase in the secretion of catecholamines within seconds but also an induction of TH expression
(Sabban et al., 1995).
Figure 5-1. Catecholamine biosynthetic pathway.
Tyrosine is the precursor of the catecholamines. TH is the rate-limiting enzyme. Dopamine, NE
and Epi are different types of catecholamines but only NE and Epi are found in chromaffin cells.
NPY another important hormone produced and secreted by the adrenal medulla, is up-
regulated by stress (Hiremagalur et al., 1994). However the physiological functions of NPY in
modulating adrenal activity during the stress response are still unknown. Here I ask whether NPY
regulates the adrenal components in either branch of the stress response.
Previous in vitro studies showed that the catecholamines and NPY were co-released
(Whim, 2006) and most studies reported that NPY stimulated catecholamine secretion (Cavadas
et al., 2002; Cavadas et al., 2001). It was also reported that NPY inhibited TH promoter activity
in the SK-N-MC cell line through NPY Y1 receptors (Cavadas et al., 2006). These in vitro
experiments indicate a strong association between NPY and catecholamines, both of which are
components of the neuronal branch of the stress response.
In this chapter I test the hypothesis that NPY inhibits the expression of adrenal TH thus
repressing the neuronal branch of the stress response. In the following experiments I employed
NPY knockout mice and also pharmacologically blocked the actions of NPY by injecting
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antagonists of NPY receptors into mice. I found that (1). NPY selectively inhibited the neuronal
branch of the stress response by down-regulating the levels of adrenal TH via the Y1 receptor; (2).
NPY also reduced adrenal NPY expression via Y2 receptors.
5.2 Results
5.2.1 The cold FST led to a sustained activation of the HPA axis
In chapter 4.2.1, I showed that the cold FST activated the HPA axis 30 minutes after
stress. Here I measured mouse plasma corticosterone 24 hours after the cold FST. Unexpectedly,
plasma corticosterone was significantly higher in animals 24 hours after the cold FST compared
to control animals suggesting a sustained activation of the HPA axis (Figure 5-2).
The mice I used to exposure to the cold FST were between P24 and P31, which is an
early adolescent period in rodents (Lupien et al., 2009). In rodents brain development occurs
during this time and the HPA axis shows a prolonged activation in response to stress due to the
incomplete maturation of negative-feedback systems (Goldman et al., 1973; Vazquez and Akil,
1993). This may be the reason for the long-lasting effect of the cold FST on plasma
corticosterone levels in these animals.
5.2.2 The cold FST led to a sustained activation of the neuronal pathway
In chapter 4.2.2 the increase of c-Fos expression 1 hour after the cold FST indicated an
activation of the neuronal pathway. In the following experiment I measured the plasma
catecholamine levels 24 hours after the cold FST. The majority of plasma epinephrine comes
from the adrenal medulla whereas most norepinephrine is from sympathetic neurons (Kvetnansky
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et al., 2009). The fold change in plasma levels of the catecholamines showed that plasma
epinephrine was consistently elevated even 24 hours after the cold FST (Figure 5-3) suggesting
that the cold FST induced a sustained activation of chromaffin cells.
Therefore the cold FST led to a maintained activity in both the HPA and the neuronal
axis of the stress response.
5.2.3 Co-localization of NPY and TH in the adrenal medulla
NPY-ir and TH-ir are co-localized in the chromaffin cells (Figure 5-4 A). I then used an
NPY (GFP) transgenic mouse to examine more precisely the expression of NPY and TH in
chromaffin cells. GFP was expressed in all chromaffin cells and was a reliable reporter of NPY
expression since it colocalized with NPY-ir both in vitro and in situ (Figure 5-4 B). TH-ir co-
localized with GFP in all chromaffin cells and adrenal slices indicating that TH and NPY are co-
expressed in all chromaffin cells (Figure 5-4 C).
5.2.4 The cold FST increased the level of PNMT-immunoreactivity in chromaffin cells in
vitro
Chromaffin cells contain two types of catecholamines, either epinephrine or
norepinephrine. PNMT is the enzyme that converts norepinephrine to epinephrine so it is used as
a marker of epinephrine-containing chromaffin cells. Since NPY was found in all mouse
chromaffin cells, NPY is co-expressed with both types of catecholamines in chromaffin cells.
To further investigate the expression of NPY in the two populations of chromaffin cells
(i.e. epinephrine-containing and norepinephrine-containing cells), I measured the levels of both
NPY-ir and PNMT-ir in chromaffin cells from stressed and matched control mice. The levels of
PNMT-ir in chromaffin cells had a continuous distribution (Figure 5-5 A and C). I observed
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chromaffin cells with low and high PNMT-ir (fitting in two Gaussian distributions) in both
control and stressed mice (Figure 5-5 C and D). No correlation was found between the two NPY-
ir sub-populations in chromaffin cells (i.e. low NPY-ir vs. high NPY-ir) and the two PNMT-ir
sub-populations (i.e. low PNMT-ir vs. high PNMT-ir) (Figure 5-5 C).
Stress increased PNMT-ir in single chromaffin cells (Figure 5-5 A, B and E) and more
cells expressed high levels of PNMT-ir and fewer cells contained lower levels of PNMT-irafter
stress (Figure 5-5 D). However it is not clear whether stress increased the number of cells
expressing epinephrine and decrease the number of cells containing norepinephrine, or if stress
only elevated the expression of PNMT in the cells that contained epinephrine.
5.2.5 The cold FST increased tyrosine hydroxylase-immunoreactivity in adrenal slices
To determine the effect of stress on the biosynthesis of the catecholamines in the adrenal
medulla, TH-immunoreactivity (TH-ir) in adrenal slices from stressed mice and matched controls
was examined 24 hours after the cold FST. As mentioned previously TH is the rate-limiting
enzyme for producing the catecholamines, so TH is used as a marker of catecholamine
biosynthesis. I found that the cold FST significantly increased TH-ir in the adrenal medulla
indicating an elevation of catecholamine synthetic capacity in the adrenal medulla in stressed
mice (Figure 5-6 A and B).
5.2.6 The cold FST-induced increase of TH-ir was reversible in adrenal slices
To test whether the increase of TH expression was reversible, I measured TH-ir in mouse
adrenal slices one week after stress compared to matched controls and animals 24 hours after
stress. I found that TH-ir was significantly higher in the adrenal medulla at 24 hours after the cold
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FST but returned towards control values by one week after acute stress (Figure 5-7 A and B). The
increase of adrenal TH-ir reversed 1 week after stress and this is similar to the result that the
stress-induced increased levels of adrenal NPY returned to control values 1 week after stress
(section 4.2.7, Figure 4-9).
5.2.7 The loss of NPY did not affect the stress-induced increase in plasma corticosterone
To investigate the role of NPY in regulating the stress response, I exposed NPY knockout
mice (Erickson et al., 1996) to the cold FST. Twenty-four hours later, plasma corticosterone
levels from the cold FST-exposed mice were significantly higher compared to the control
knockout mice (Figure 5-8), which was a similar response to that seen in wild type animals
(Figure 5-2). These results indicate that the loss of NPY does not obviously affect the
adrenocortical response to stress.
5.2.8 The loss of NPY altered the neuronal branch of the stress response
Although NPY did not affect the stress-induced increase in plasma glucocorticoid, it may
change the neuronal branch of the stress response. Since the catecholamines are one of the key
components in the neuronal pathway, I measured TH-ir in adrenal slices from control and cold
FST-exposed NPY knockout mice. In contrast to the wild type animals, in which the cold FST
increased TH-ir in the adrenal medulla (Figure 5-6 A and B; Figure 5-9 A and B), stress did not
increase TH-ir in the NPY knockout mice. This lack of effect may be due to a basal increase of
TH-ir in the NPY knockout control animals (Figure 5-9 A). Unfortunately the wild type animals
were not littermates of the knockout mice since the NPY knockout mice I obtained are
homozygous knockout with a different genetic background to the C57/BL6 wild type mice. I
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therefore next tested my hypothesis using an alternative strategy as described in the following
experiments.
5.2.9 Expression of NPY Y receptors in the adrenal medulla
To determine which Y receptors are expressed in the adrenal medulla, I used reverse
transcription PCR (RT-PCR) to detect the mRNA expression of the Y receptors. The primers for
Y1, Y2, Y4, Y5 and Y6 were validated using mRNA from whole brain (Figure 5-10 A). RT-PCR
results (using the same amounts of adrenal medulla mRNA and whole brain mRNA) indicated
that Y1, Y2, Y5 and some Y4 were expressed in the adrenal medulla but no y6 (Figure 5-10 B).
Y4 is activated by pancreatic peptide rather than NPY (Michel et al., 1998; Lin et al., 2006), so I
next tested the effects of blocking Y1, Y2 and Y5 by injecting antagonists to these receptors in
the following experiments.
5.2.10 Injection of BIBP3226 a NPY Y1 antagonist was associated with an increase in
adrenal TH-ir
Mice were injected (i.p.) with either saline or BIBP3226 (1mg/kg), a selective NPY Y1
antagonist (Fu et al., 2004) and 15 minutes after the injection, one animal from each injected
group was exposed to the cold FST (Figure 5-11 A). Twenty-four hours later, animals were
decapitated and the level of adrenal TH-ir was examined. I found that TH-ir was significantly
higher in the adrenal medulla from the BIBP3226-injected animals compared to saline-injected
animals (Figure 5-11 B and C).
However the cold FST no longer increased adrenal TH-ir in the saline or BIBP3226-
injected animals. Thus injection per se was sufficiently stressful that it led to an increase in the
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levels of adrenal TH-ir (Figure 5-12). My data suggested that NPY inhibited TH expression via
Y1 receptors, which supports the idea of a basal activation of TH-ir in the NPY knockout mice.
To test whether the effect of blocking Y1 on the increase in adrenal TH expression was
mediated via the actions of NPY, I injected the NPY knockout mice (i.p.) with either saline or
BIBP3226. No difference in the levels of adrenal TH-ir between the saline-injected and
BIBP3226- injected NPY knockout animals was found (Figure 5-13). This result confirmed that
there was a basal activation of TH in the NPY-deficient animals and the inhibitory effect of Y1
on adrenal TH expression was mediated via the actions of NPY. Therefore NPY plays an
important role in down-regulating the levels of adrenal TH via a Y1 receptor.
5.2.11 Injection of BIIE0246 an NPY Y2 antagonist or L152,804, an NPY Y5 antagonist, did
not alter adrenal TH-ir
To examine whether other Y receptors were also involved in regulating adrenal TH, I
blocked the other two Y receptors Y2 and Y5, respectively. I injected animals (i.p.) with either
saline or BIIE0246 (1mg/kg) a selective NPY Y2 antagonist (Dumont et al., 2000) and 15
minutes after the injection, one animal from each injected group was exposed to the cold FST. I
found no difference in the levels of TH-ir in the adrenal medulla between the BIIE0246-injected
animals and saline-injected animals (Figure 5-14 A and B).
I then injected animals (i.p.) with either saline or L152, 804 (10mg/kg) a selective NPY
Y5 antagonist (Kanatani et al., 2000). No difference in the levels of adrenal TH-ir was found in
the L152, 804-injected animals and saline-injected animals (Figure 5-14 C).
Therefore NPY inhibited adrenal TH via NPY Y1 receptors selectively rather than via Y2
or Y5.
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5.2.12 Injection of BIIE0246 was associated with an increase in adrenal NPY-ir
Since NPY is a negative regulator of adrenal TH, a component of the neuronal branch of
the stress response, I next asked whether NPY was also involved in regulating its own expression
since this is another important component of the neuronal pathway. I found that the level of
adrenal NPY-ir from the BIIE0246-injected animals was significantly higher compared to the
saline-injected controls but was not different from the saline-injected cold FST-exposed animals
(Figure 5-15 A and B) indicating that NPY repressed the expression of NPY in the adrenal
medulla via aY2 receptor.
However unlike adrenal TH, the cold FST increased the levels of adrenal NPY-ir in the
saline-injected animals. Thus the injection per se did not lead to an increase of adrenal NPY-ir
levels (Figure 5-16). The different effects of injection on the levels of adrenal TH-ir and NPY-ir
suggest that different stressors regulate NPY and TH expression differently perhaps depending on
the types and/or the intensity of the stress.
5.2.13 Injection of BIBP3226 or L152,804 did not alter adrenal NPY-ir
I then compared the levels of adrenal NPY-ir from the saline-injected and BIBP3226-
injected animals. No difference in the levels of adrenal NPY-ir between the saline-injected and
BIBP0246-injected animals was found (Figure 5-17 A and B).
Then I measured adrenal NPY-ir from the saline-injected and L152, 804-injected animals
and found no difference in the levels of adrenal NPY-ir between the saline-injected and L152,
804-injected animals (Figure 5-17 C). Therefore NPY inhibited the expression of NPY in the
adrenal medulla via Y2 receptors selectively rather than via aY1 or Y5 receptor.
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In conclusion blocking Y1 and Y2 receptors is associated with an increase in the levels of
adrenal TH-ir and NPY-ir respectively, indicating that NPY is a negative regulator of adrenal
functioning by suppressing the synthesis of the catecholamines and NPY. It suggests that the
functional role of NPY may be to prevent a pathological activation of the fight-or-flight response.
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Figure 5-2. The cold FST led to a sustained activation of the HPA axis.
Plasma corticosterone levels were significantly increased 24 hours after the cold FST (mean ±
SEM, n = 8). *p < 0.05; Student’s paired t test. P21-P23 male littermates were used in each
individual experiment. The open black circles indicate the value from each individual animal. The
black lines link the matched control and stressed animals from each individual experiment.
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Figure 5-3. The cold FST led to a sustained activation of the neuronal axis of the stress
response.
(A). Fold change of plasma levels of epinephrine 24 hrs after the FST. *p < 0.05; Student’s
matched one sample t test. P21-P23 male littermates were used in each individual experiment.
Data normalized to control (mean ± SEM, n = 9; raw data: 10.05 ± 2.11 ng/ml vs.13.05 ± 1.00
ng/ml). (B). Fold change of plasma levels of norepinephrine 24 hours after the cold FST. Data
normalized to control (mean ± SEM, n = 9; raw data: 21.38 ± 2.89 ng/ml vs.26.48 ± 4.09 ng/ml).
The open black circles indicate the value from each individual animal. The black lines link the
matched control and stressed animals from each individual experiment.
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Figure 5-4. Co-localization of NPY and TH in chromaffin cells.
(A). Co-staining of NPY-ir and TH-ir in single chromaffin cells. Scale bars 10μm. (B). Co-
localization of GFP and NPY-ir in chromaffin cells from an NPY (GFP) mouse. Upper panel
scale bars 100μm. Lower panel scale bars 10μm. (C). Co-localization of GFP and NPY-ir in
adrenal slices in the NPY (GFP) mouse. Scale bars 100 μm. (D). Co-localization of GFP and TH-
ir in chromaffin cells of the NPY (GFP) mouse. Upper panel scale bars 100μm. Lower panel scale
bars 10μm. (E). Co-localization of GFP and TH-ir in adrenal slices in the NPY (GFP) mouse.
Scale bars 100 μm.
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Figure 5-5. The cold FST increased PNMT-ir in chromaffin cells in vitro.
(A). Examples of chromaffin cells from a control and stressed animal. Scale bar 10 μm. (B). The
levels of PNMT-ir were measured in seventy cells from each animal. The levels of PNMT-ir in
single chromaffin cells were ranked from the lowest value to the highest value and the cells were
numbered from 1 to 70 according to the ranking. NPY-ir (gray dots) and PNMT-ir (blue in
control and red in stress) from the same chromaffin cell were plotted against the number of each
cell. (B). The results of a typical experiment. (C). Group normalized data showed that the level of
PNMT-ir was higher in chromaffin cells from stressed animals compared to matched controls
(mean±SEM, n = 3). *p < 0.05; Student’s matched one sample t test. P21 male littermates were
used in each individual experiment. (D). The representative frequency histogram from either
matched control or stressed mouse fitted in two Gaussian distributions (red dashed curve vs. navy
blue dashed curve). The frequency histogram showed that stress resulted in a decrease in the
proportion of low PNMT-ir cells and an increase in the proportion of intensely PNMT-ir cells. (E).
A plot of the cumulative distributions indicated that chromaffin cells from stressed animals
contained higher levels of PNMT-ir. ***p < 0.001; Kolmogorov-Smirnov test.
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*
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Figure 5-6. The cold FST increased the level of adrenal TH-ir in situ.
(A). Adrenal sections from a control animal and an animal 24 hours after the cold FST. Scale bars
100 μm. (B). Group data showed that the level of TH-ir was significantly higher in the adrenal
medulla from stressed animals compared to matched controls. Data was normalized to control
(mean ± SEM, n = 3; raw data: 105.10 ± 21.49 vs. 230.88 ± 11.48). *p < 0.05; Student’s matched
one sample t test. P21-P23 male littermates were used in each individual experiment. The levels
of TH-ir were measured in eight to ten sections from each animal. The open black circles indicate
the value from each individual animal. The black lines link the matched control and stressed
animals from each individual experiment.
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Figure 5-7. The cold FST reversibly increased the level of adrenal TH-ir in situ.
(A). Adrenal sections from a control animal, 24 hrs and 1 week after the FST. Scale bars 100 μm.
(B). Group data showed that the level of TH-ir was significantly higher in the adrenal medulla at
24 hrs after the cold FST but declined towards control values by 1 week after the FST (mean ±
SEM, n=3). *p < 0.05; **p < 0.01; one-way ANOVA (post-hoc Tukey’s paired comparison;
F (2, 6) = 41.64). P21-P23 male littermates were used in each individual experiment. The levels
of TH-ir were measure in eight to ten sections from each animal. The open black circles indicate
the value from each individual animal. The black lines link the matched control and stressed
animals from each individual experiment.
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Figure 5-8. The cold FST led to a sustained activation of the HPA axis in NPY knockout
mice.
Plasma corticosterone levels were significantly increased 24 hours after the cold FST in the NPY
knockout mice (mean ± SEM, n = 5). *p < 0.05; Student’s paired t test. P21 male littermates were
used in each individual experiment. The open black circles indicate the value from each
individual animal. The black lines link the matched control and stressed animals from each
individual experiment.
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Figure 5-9. The cold FST did not increase the level of adrenal TH-ir in NPY knockout mice.
(A). Adrenal sections from a control animal and 24 hrs after the FST; wild type and NPY
knockout animals are shown. Scale bars 100 μm. (B). TH-ir was significantly higher in the
adrenal medulla from stressed wild type animals compared to matched controls. Data was
normalized to control values (mean ± SEM, n = 3; raw data: 180.00 ± 47.17 vs. 336.85 ± 73.77
arbitrary units). *p < 0.05; Student’s matched one sample t test. However stress did not elevate
TH-ir in the NPY KO animals. Data was normalized to control values (mean ± SEM, n = 3; raw
data: 329.94 ± 73.80 vs. 374.52 ± 65.21 arbitrary units). P21 male wild type littermates or
knockout littermates were used in each individual experiment. The level of TH-ir was measure in
eight to ten sections from each animal. The open black circles indicate the value from each
individual animal. The black lines link the matched control and stressed animals from each
individual experiment.
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Figure 5-10. Expression of NPY Y receptors in the adrenal medulla and whole brain.
(A). RT-PCR experiment examining the expression of Y1, Y2, Y4, Y5, Y6 and NPY in whole
brain. The negative control in the RT-PCR is derived from a reaction using NPY primers but
without reverse transcriptase. (B). RT-PCR experiment examining the expression of Y1, Y2, Y4,
Y5 and NPY mRNA in the mouse adrenal medulla. The same amount of starting mRNA was
used as in (A). The negative control in the RT-PCR is derived from a reaction using NPY primers
but without reverse transcriptase.
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Figure 5-11. Blocking the NPY Y1 receptor was associated with an increase in the level of
adrenal TH-ir.
(A).Stress paradigm. P21 male littermates were used in two of the three independent experiments,
and one paired group were P21 female littermates. (B). Adrenal sections from a control animal
and 24 hours after the cold FST; Mice were injected (i.p.) either with 200 μl saline or BIBP3226
(a Y1 receptor antagonist). Scale bars 100 μm. (C). The level of TH-ir was significantly higher in
the adrenal medulla from the BIBP 3226-injected animals compared to controls. Note that the
cold FST no longer increased TH-ir in the saline or BIBP3226-injected animals. Data are mean ±
SEM, n=3; *p < 0.05, **p < 0.01; one-way ANOVA (post-hoc Tukey’s paired comparison;
F (3, 8) = 6.05). The levels of TH-ir were measured in eight to ten sections from each animal. The
open black circles indicate the value from each individual animal. The black lines link the
matched control and stressed animals from each individual experiment.
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Figure 5-12. Injection per se was sufficiently stressful that it led to an increase in the level of
adrenal TH-ir.
The level of TH-ir was higher in the adrenal medulla from the saline-injected animals
compared to uninjected controls or even to the cold FST-exposed animals (n=2). P21 male
littermates were used in each individual experiment. The levels of TH-ir were measured from
eight to ten sections from each animal. The open black circles indicate the value from each
individual animal. The black lines link the matched control and stressed animals from each
individual experiment.
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Figure 5-13. Blocking the NPY Y1 receptor in NPY knockout mice did not alter the level of
adrenal TH-ir.
The level of TH-ir in the adrenal medulla was not changed when NPY knockout mice were
injected with BIBP3226 compared to saline injection (right).TH-ir was elevated in the cold FST-
exposed wild type animals compared to control (n=1). P21 male littermates were used. The levels
of TH-ir were measured in eight sections from each animal.
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Figure 5-14. Blocking the NPY Y2 or Y5 receptor did not affect the level of adrenal TH-ir.
(A). Adrenal sections from a control animal and 24 hrs after the cold FST; mice were
injected (i.p.) either with 200 μl saline or BIIE0246 (a Y2 receptor antagonist). Scale bars 100 μm.
(B). No difference in the level of TH-ir was detected in the adrenal medulla from the BIIE0246-
injected animals compared to controls. P21 male littermates were used in each individual
experiment. (C). No difference in the level of TH-ir was detected in the adrenal medulla from the
L125,804 (a Y5 receptor antagonist)-injected animals compared to controls. P21 male littermates
were used in two of the three independent experiments, and one paired group were P21 female
littermates. Data are mean ± SEM, n = 3; one-way ANOVA (post-hoc Tukey’s paired comparison;
for (B), F (3, 8) = 0.029; for (C), F (3, 8) = 0.283). The levels of TH-ir were measured from eight
to ten sections from each animal. The open black circles indicate the value from each individual
animal. The black lines link the matched control and stressed animals from each individual
experiment.
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Figure 5-15. Blocking the NPY Y2 receptor was associated with an increase in the level of
NPY-ir in the adrenal medulla.
(A). Adrenal sections from a control animal and 24 hrs after the cold FST; mice were
injected (i.p.) either with saline or BIIE0246. Scale bars 100 μm. (B). BIIE0246-injected animals
had significantly higher level of NPY-ir in the adrenal medulla compared to saline-injected
controls but the same levels as saline-injected cold FST-exposed animals. Note that unlike TH-ir,
the cold FST increased the level of adrenal NPY-ir in the saline or BIIE0246-injected animals.
Data are mean ± SEM, n = 3; *p < 0.05, **p < 0.01; one-way ANOVA (post-hoc Tukey’s paired
comparison; F (3, 8) = 3.46). P21 male littermates were used in each individual experiment. The
levels of NPY-ir were measured in eight to ten sections from each animal. The open black circles
indicate the value from each individual animal. The black lines link the matched control and
stressed animals from each individual experiment.
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Figure 5-16. Injection per se did not alter the level of adrenal NPY-ir.
The level of NPY-ir was not changed in the adrenal medulla from the saline-injected
animals compared to uninjected controls (n = 2). P21 male littermates were used in each
individual experiment. The levels of NPY-ir were measured from eight to ten sections from each
animal. The open black circles indicate the value from each individual animal. The black lines
link the matched control and stressed animals from each individual experiment.
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Figure 5-17. Blocking the NPY Y1 or Y5 receptor did not affect the level of adrenal NPY-ir.
(A). Adrenal sections from a control animal and 24 hrs after the cold FST; Mice were
injected (i.p.) either with saline or BIBP3226. Scale bars 100 μm. (B). No difference in the level
of NPY-ir was detected in the adrenal medulla from the BIIE-injected animals compared to
saline-injected animals. P21 male littermates were used in two of the three independent
experiments, and one paired group were P21 female littermates. (C). No difference in the level of
NPY-ir was detected in the adrenal medulla from L125,804 (Y5 receptor antagonist)-injected
animals compared to saline-injected animals. P21 male littermates were used in two of the three
independent experiments, and one paired group were P21 female littermates. Data are mean ±
SEM, n = 3; *p < 0.05; one-way ANOVA (post-hoc Tukey’s paired comparison; for (B), F (3, 8)
= 1.95; for (C), F (3, 8) = 0.823). The levels of NPY-ir were measured in eight to ten sections
from each animal. The open black circles indicate the value from each individual animal. The
black lines link the matched control and stressed animals from each individual experiment.
136
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5.3 Discussion
5.3.1 NPY inhibits the expression of adrenal TH and NPY.
NPY and the catecholamines are the most abundant hormones expressed in the adrenal
medulla and they coexist in all mouse chromaffin cells. Although it is known that stress increases
both the levels of adrenal NPY (Hiremagalur and Sabban, 1995) and TH, a marker of
catecholamine biosynthesis (Sabban et al., 1995), the role of the stress-induced increase in
adrenal NPY was not clear.
NPY was reported to inhibit TH promoter activity in the SK-N-MC cell line through a Y1
receptor and to decrease TH synthesis in PC12 cells (Cavadas et al., 2006; McCullough and
Westfall, 1996). Using an in vivo approach I have shown that NPY inhibits the expression of
adrenal TH via a Y1 receptor.
Intraperitoneal injection of BIBP 3226 (a Y1 antagonist) and BIIE0246 (a Y2 antagonist)
were associated with increased levels of adrenal TH and NPY-ir respectively. Both BIBP 3226
and BIIE 0246 cannot cross the brain barrier (Doods et al., 1999; Doods et al., 1996; Shoblock et
al., 2010) so the intraperitoneal injection of these two antagonists underlies the effect of
peripheral NPY on inhibiting adrenal activity.
Adrenal catecholamine secretion is partially regulated through an inhibitory autocrine
feedback effect that is mediated by α2 adrenergic receptors expressed in chromaffin cells
(Lymperopoulos et al., 2007). In my experiments I found a complete overlap of NPY and TH in
all chromaffin cells and an expression of Y1, Y2 and Y5 receptors in the adrenal medulla.
Although I cannot exclude the involvement of NPY arising from peripheral sources other than the
adrenal medulla , it is likely that it is adrenal NPY that is responsible for regulating the expression
of adrenal TH and neuropeptide Y in an autocrine manner.
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5.3.2 NPY does not completely suppress the stress response.
The level of adrenal NPY was increased 24 hours after stress (chapter 4) and the level of
adrenal TH was also up-regulated at the same time point after stress (this chapter). Although NPY
was shown to suppress the expression of adrenal TH, the stress-induced increase in the level of
adrenal NPY did not block the elevation in the level of adrenal TH twenty four hours after stress.
This suggests that the effect of NPY in repressing the level of adrenal TH is a chronic effect.
Besides the inhibitory role of NPY on adrenal catecholamine synthesis, most studies have
reported that NPY can stimulate the secretion of catecholamines from chromaffin cells (Cavadas
et al., 2006; Cavadas et al., 2002; Cavadas et al., 2001). A coupled increase of secretion and
biosynthesis of the catecholamines in response to stress is required for the fight-or-flight response
and to compensate for the changes in physiology (Hamelink et al., 2002; Discussion 4.3.5). This
first step of the stress response may involve the stimulatory action of NPY on adrenal
catecholamine release. Then in the second step of the stress response, NPY appears to participate
in a negative feedback loop suppressing the synthesis of adrenal catecholamines and expression
of itself. This may be in order to prevent an overactivation of the adrenal medulla.
In contrast to its role in regulating the neuronal branch of the stress response, NPY is not
essential for adrenocortical function during the stress response. The effects of NPY on
glucocorticoid secretion in other studies are controversial. Several in vivo studies suggested that
NPY had no acute effect on the level of plasma glucocorticoids (Mazzocchi et al., 1996;
Mazzocchi and Nussdorfer, 1987). An in vitro study indicated an inhibitory role of NPY on
corticosterone secretion from adrenocortical cells (Malendowicz et al., 1990) whereas another in
vitro experiment revealed an stimulatory role of NPY on corticosterone release in adrenocortical
cells (Neri et al., 1990). My results indicate that NPY may be not involved in regulating
corticosterone secretion which fits with other in vivo studies (Mazzocchi et al., 1996; Mazzocchi
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and Nussdorfer, 1987) and is also consistent with the result that loss of NPY did not affect the
adrenocortical response during metabolic stress fasting (Erickson et al., 1997; Erickson et al.,
1996).
However I cannot exclude the possibility that stress increases plasma glucocorticoids in
NPY knockout mice to a different extent compared to that in wild type animals and it is not clear
whether loss of NPY affects the other two components of the HPA axis, the hypothalamus and
pituitary gland.
In addition mice were not handled before an exposure to the cold FST and control
animals were not handled at all since they were individually housed until sacrificed. Therefore the
memory of a stressful experience might contribute to the sustained increase in circulating
corticosterone that was observed in the stressed animals.
5.3.3 Possible mechanisms underlying the regulatory role of NPY in controlling the levels of
adrenal TH and NPY.
An in vitro SK-N-MC cell line study indicated that NPY downregulated TH promoter
activity via a Y1 receptor (Cavadas et al., 2006). All Y receptors are G-protein coupled receptors
and their activation usually causes inhibitory responses including the inhibition of cAMP
accumulation (Lin et al., 2004). Therefore blocking Y1 receptors is predicted to lead to an
increase in cAMP accumulation. The TH promoter region contains a perfect motif for a cAMP
response element (CRE) at -45 to -38 bp, so the transcription of the TH gene is potentially
stimulated by pathways activated by cAMP (Cambi et al., 1989; Kilbourne et al., 1992; Kim et al.,
1994; Sabban et al., 2006).
NPY also inhibits the expression of itself in the adrenal medulla via a different receptor,
the Y2 receptor. The NPY promoter binds CREB and the Y2 receptor is also a G-protein coupled
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receptor so the activation of a Y2 receptor is expected to inhibit cAMP accumulation (Titolo et al.,
2008). Therefore the mechanism underlying the NPY gene regulation may also involve a cAMP-
potentiated pathway.
However it is difficult to explain how distinct Y receptors on chromaffin cells regulate
NPY and TH by the same signaling pathway. For example why does the Y1 receptor not regulate
NPY and why does the Y2 receptor not modulate TH?
NPY regulated the secretion of catecholamines via a Y1 receptor-coupled mitogen-
activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathway (Rosmaninho-
Salgado et al., 2007a; Rosmaninho-Salgado et al., 2007b). Therefore it is possible that a different
signaling pathway rather than the classical cAMP-CREB pathway might be involved in regulating
the synthesis of TH via the Y1 receptor.
In the CNS it was reported that NPY mediated a presynaptic inhibition through the Y2
receptor and a postsynaptic inhibition through the Y1 receptor in the hypothalamus (Fu et al.,
2004). In the PNS the splanchnic nerve innervating the adrenal medulla is the presynaptic input. I
speculate that NPY may mediate presynaptic inhibition via the Y2 receptor by suppressing
acetylcholine release from the splanchnic nerve and thus inhibiting the expression of adrenal
NPY. This speculation is supported by evidence showing that NPY reduced the calcium current
and inhibited acetylcholine release from rat vagal afferent neurons (Wiley et al., 1990). NPY may
suppress the activity of chromaffin cells by postsynaptic inhibition through the adrenal Y1
receptor and thus inhibit TH expression in chromaffin cells. These suggestions are consistent with
the finding that the splanchnic nerve innervation is required for the stress-induced increase of
adrenal NPY transcription but not for the induction of adrenal TH during s restraint-induced
stress response (Hiremagalur et al., 1994; Hiremagalur et al., 1995).
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5.3.4 Working hypothesis of how adrenal NPY modulates adrenal activity in the stress
response.
Although I have shown that loss of NPY and blocking NPY receptors was associated
with an increase in the level of adrenal TH, the functional consequence of the stress-induced
increase in the level of adrenal NPY is still not clear. My proposed working model is (1). stress
increases both adrenal NPY and TH expression one day after stress as part of an acute stress
response; (2). an increased release of NPY due to increased adrenal NPY then represses the
expression of both adrenal TH and NPY as part of a chronic stress response (Figure 5-18 A). In
chapter 3.2.7, it was shown that an increased synthesis of NPY prohormone could lead to a
consequential increase in the secretion of NPY. Moreover recent data in our lab suggests that the
stress-induced upregulation of adrenal catecholamine synthesis leads to a consequential elevation
in catecholamine release. These results indicate that an increased synthesis of hormones could
result in an elevated level of secretion and thus support the second point in my working
hypothesis.
Adrenal NPY may participate in a negative feedback loop to maintain adrenal
homeostasis and to prevent a pathological activation of the fight-or-flight response during stress.
The physiological function of NPY in modulating the neuronal branch of the stress response may
parallel the role of glucocorticoids in negatively regulating the HPA axis (Figure 5-18 B).
To investigate the functional consequences of the stress-induced increase in the level of
adrenal NPY, two methods including denervation of the adrenal gland by splanchnicectomy and
injection of a nicotinic receptor antagonist chlorisondamine could be used. These would
potentially block the stress-induced increase in the level of adrenal NPY (Hiremagalur et al., 1994;
Hiremagalur et al., 1995). Possible future research would be to compare the level of adrenal TH
and other sympathetic parameters such as heart rate, blood pressure and thermogenesis in stressed
intact and stressed denervated animals.
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5.4 Conclusion
I conclude that NPY negatively regulates the expression of adrenal TH and NPY. NPY
plays an important role in inhibiting the neuronal branch of the stress response but does not affect
the adrenocortical response during stress.
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Figure 5-18. Working model.
(A). A working model depicting the role of adrenal NPY in modulating adrenal
functioning during the stress response. (B). NPY may function in a negative feedback loop
regulating the activation of the sympatho-adrenal system during the stress response.
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Chapter 6
Discussion
In this thesis I first described experiments that determined how a functional single
nucleotide polymorphism (T1128) in the NPY signal sequence altered NPY prohormone
biosynthesis and secretion (chapter 3). This SNP is associated with metabolic and cardiovascular
disease and humans containing this SNP have higher levels of plasma NPY after exercise
(Pesonen, 2006, 2008).
Plasma NPY is thought to originate from post-ganglionic sympathetic neurons, since
simulation of these neurons will lead to a secretion of NPY into the blood (Kuo et al., 2007a;
Morris et al., 1986). In the remainder of my thesis research I have studied the regulation of NPY
synthesis in the sympathetic nervous system (SNS) using chromaffin cells (modified post-
ganglionic neurons) and the adrenal medulla as a model system. I have shown that stress
increases the activity of chromaffin cells and that the expression of adrenal NPY is up-regulated
by two different stressors (chapter 4). NPY receptors are found in the heart, smooth muscle cells,
fat tissue, pancreas and the adrenal gland (Dumont et al., 1992), so peripheral NPY is thought to
regulate both the cardiovascular system and metabolism (Kuo et al., 2007a; Kuo et al., 2008).
In the peripheral nervous system NPY post-synaptically stimulates, and presynaptically
inhibits, norepinephrine release from sympathetic nerves (Kuo et al., 2007a) , and my data
(chapter 5) as well as the results from other studies suggests that NPY regulates both the secretion
and synthesis of the catecholamines in chromaffin cells (Cavadas et al., 2006; Cavadas et al.,
2002; Cavadas et al., 2001) . The catecholamines are importantly involved in modulating the
cardiovascular system (Lymperopoulos et al., 2007) and metabolism (Bachman et al., 2002;
Bowers et al., 2004).
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Therefore in this chapter I will discuss the role of NPY in both the cardiovascular system
and in metabolic regulation. Besides the function of peripheral NPY in these two systems, I will
outline how the role of NPY in the central nervous system is coordinated with the SNS to regulate
the cardiovascular system and metabolism. I will also discuss the role of NPY in the stress
response. In particular I will discuss the function of NPY in controlling the cardiovascular system
during stress and the role of peripheral NPY in stress-induced metabolic syndrome.
6.1 Role of NPY in the cardiovascular system
6.1.1 Role of peripheral NPY in cardiovascular modulation
The first identified biological activity of NPY was to induce a prolonged vasoconstriction
and this effect was resistant to α-adrenergic receptor blockage (Lundberg and Tatemoto, 1982).
Later it was determined that theY1 receptor mediated this process since in Y1 knockout mice, the
NPY-induced increase in blood pressure was abolished (Pedrazzini et al., 1998).
Indirectly NPY enhanced norepinephrine-evoked vasoconstriction in an intracellular
calcium-dependent manner, and the effect of NPY application was through a post-synaptic Y1
receptor (Abe et al., 2010; Fallgren et al., 1993; Kuo et al., 2007a; Pedrazzini et al., 1998). NPY
also suppressed norepinephrine release by a presynaptic Y2 receptor via a negative feedback
mechanism (Donoso et al., 1988; Kuo et al., 2007a; Maynard and Burnstock, 1994). An
interaction between the NPY system and catecholamine signaling is thus important in controlling
the cardiovascular system.
The role of peripheral NPY in elevating blood pressure is consistent with the finding that
the T1128C SNP in the NPY preprohormone is associated with a higher risk of heart disease
(Pesonen, 2006; 2008).
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6.1.2 Role of the adrenal medulla in cardiac function
Chromaffin cells in the adrenal medulla are modified post-ganglionic sympathetic
neurons and are part of the sympathetic nervous system. Chromaffin cells can fire action
potentials and are innervated by preganglionic sympathetic fibers. Nicotinic acetylcholine
receptors expressed by chromaffin cells are activated by acetylcholine released from
preganglionic nerves leading to depolarization of the chromaffin cells. Chromaffin cells
synthesize both NPY and the catecholamines (Whim, 2006). They are the major source of plasma
epinephrine. Upon depolarization the catecholamines and NPY are released into the blood
(Axelrod and Reisine, 1984; Whim, 2006).
As a source of plasma NPY and the catecholamines, the adrenal medulla is expected to
have a role in cardiac function. A transcriptome study in a genetic hypertension and hypotension
mouse model showed that mRNA levels of adrenal NPY and TH (a marker of catecholamine
synthesis) were significantly higher in the hypertensive mouse strain compared with the
hypotensive strain (Fries et al., 2004).
Direct evidence that the adrenal medulla modulates cardiac functions comes from a study
of adrenal G protein-coupled receptor kinase 2 (GRK2) upregulation in mediating a sympathetic
overdrive that promotes heart failure (Lymperopoulos et al., 2007). Hyperactivity of the
sympathetic nervous system and increased catecholamine release was found in two heart failure
models. These overactive responses were caused by an upregulation of adrenal GRK2. The
increase of adrenal GRK2 disrupted α2-adrenergic receptor (α2AR) signaling in the adrenal
medulla and this disruption impaired the autocrine feedback inhibition of catecholamine secretion
by α2AR’s in chromaffin cells (Lymperopoulos et al., 2007).
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6.1.3 Role of central NPY in the cardiovascular system
Central injection of NPY induced hypotension in rat (Fuxe et al., 1983). This is the
opposite effect to the vasoconstriction that is produced by peripheral NPY injection in rats
(Lundberg and Tatemoto, 1982). A transgenic rat line in which NPY is overexpressed under the
control of its natural regulatory elements has been developed. These animals showed decreased
blood pressure and reduced catecholamine release suggesting that the role of endogenous NPY is
a long-term inhibition of the sympathetic nervous system (Michalkiewicz et al., 2003). The
central NPY vasodepressor effect was primarily mediated by a presynaptic Y2 receptor inhibition
and thus a decreased sympathetic outflow to the heart (Chen and Westfall, 1993). This conclusion
is further supported by evidence that the heart rate was increased in NPY Y2-deficient mice
(Naveilhan et al., 1999).
6.2 Role of NPY in metabolic regulation
6.2.1 Role of central NPY in feeding
NPY has been noted for its stimulatory effect on feeding (Lin et al., 2004). Central
administration of NPY significantly increases food intake and chronic infusion of NPY can
induce obesity from overeating (Flier and Maratos-Flier, 1998).
The first conventional NPY knockout mouse on a 129/SvCp-J background did not show
a phenotype of reduced feeding, body weight or adiposity under normal conditions (Erickson et
al., 1996). This report was surprising since for decades neuroscientists believed the central role of
NPY was in promoting hyperphagia but this NPY knockout mouse line had no phenotype with
respect to body weight regulation (Dube et al., 1994; Levine and Morley, 1984; Stanley et al.,
1986). However when the NPY knockout mice were backcrossed onto a C57BL/6 background,
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they showed decreased re-feeding after fasting. This result suggested an altered behavior of food
intake in the mice lacking NPY (Bannon et al., 2000). The lack of a clear phenotype related to
energy balance in NPY germline knockout mice might be explained by compensatory
mechanisms in development (Trivedi et al., 2001).
Y1 knockout mice display slightly diminished feeding and strongly reduced fasting-
induced re-feeding, however body fat and serum leptin was increased in these mice (Pedrazzini et
al., 1998). Y5-deficent mice display normal food intake and body weight but developed obesity
when they were older than 30 weeks (Marsh et al., 1998). The data generated from these
knockout mice suggested that the Y1 and Y5 receptors were important for maintaining energy
homeostasis.
One Y2 knockout mouse model displayed decreased bodyweight gain (Sainsbury et al.,
2002b) whereas another Y2 knockout mouse line showed increased body weight as well as food
intake (Naveilhan et al., 1999). The deletion of Y2 receptors in the adult mouse hypothalamus led
to transiently decreased body weight and increased food intake indicating the functional role of
the hypothalamic Y2 receptors in controlling feeding. This also illustrates the importance of site-
specific knockout models compared to a conventional knockout, as developmental and
compensation mechanisms may mask such effects in germline knockout mice (Sainsbury et al.,
2002a).
6.2.2 Role of peripheral NPY in metabolic regulation
Compared to the studies of hypothalamic NPY, less attention has been given to the role
of extrahypothalamic NPY in metabolic regulation. Peripheral NPY is suggested to be a marker
of sympathetic nervous system activity (Dumont et al., 1992; Heilig, 2004). NPY and the
catecholamines are co-released from sympathetic neurons and both transmitters regulate the
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activity of peripheral tissues including the heart, liver, pancreas, adrenal glands and adipose tissue
since these are innervated by the sympathetic nervous system (Heilig, 2004; Kuo et al., 2007a).
Transgenic mice overexpressing NPY in adrenergic and noradrenergic neurons (i.e. brain
noradrenergic neurons and peripheral sympathetic neurons) showed an increase of adiposity,
impaired glucose tolerance and liver triglyceride accumulation (Ruohonen et al., 2008).
Humans carrying the T1128C polymorphism in the NPY preprohormone have higher
levels of plasma NPY after exercise (Kallio et al., 2001). Our studies suggest that this SNP
augments the packaging of the NPY prohormone into DCSGs and thus led to an up-regulation of
NPY secretion (Mitchell et al., 2008). This data may suggest a molecular mechanism underlying
the observation that a higher level of exercise-induced plasma NPY was found in the T1128
carriers (Kallio et al., 2001) and that the SNP was associated with higher levels of serum
cholesterol and increased risk for metabolic disease (Karvonen et al., 1998; Karvonen et al., 2001;
Pesonen, 2006, 2008). These phenotypes observed in humans with this SNP are also consistent
with the role of NPY in mediating stress-induced obesity (Kuo et al., 2007b). However the role
of NPY in regulating lipid metabolism is complex since it also inhibits lipolysis (Heilig et al.,
2004; Nordman et al., 2005).
6.3 Role of NPY in the stress response
6.3.1 Role of NPY in controlling the two major stress pathways in the PNS
One characteristic of the stress response is the activation of the HPA axis leading to a
marked secretion of the glucocorticoids into the blood (Axelrod and Reisine, 1984; Sapolsky et
al., 2000). In my experiments the cold FST induced-stress led to an increased level of plasma
corticosterone (CORT) even 24 hours after the stress and this effect was maintained in the NPY
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knockout mice (chapter 5). An increase in plasma CORT was also observed in both the NPY
deficient mice and wild type animals during the response to acute fasting (Erickson et al., 1997;
Erickson et al., 1996). Furthermore plasma CORT was elevated during restraint or cold stress in
the conditional NPY transgenic animals (Ruohonen et al., 2009).These findings suggest that NPY
is not essential for an appropriate functioning of the adrenal cortex during stress.
Besides the HPA axis, sympathetic activity is enhanced in the stress response and NPY is
released from the sympathetic nerves (Kuo et al., 2007a). In my experiments, I found that NPY
expression in the adrenal medulla was elevated by diverse stressors including exposure to fox
urine, cold FST and a metabolic stress, food restriction (data not shown). In addition peripheral
NPY is a negative regulator of adrenal NPY expression and catecholamine synthesis indicating
that NPY is involved in inhibiting neuronal activation during the stress response.
6.3.2 Peripheral NPY mediates stress-induced hypertension
Stress activates the sympathetic nervous system leading to a secretion of NPY from
sympathetic nerves, and produces hypertension which can be blocked by a Y1 antagonist (Han et
al., 1998). This study indicated that NPY is a mediator of stress-induced hypertension.
This hypothesis was further supported by a study using conditional NPY transgenic mice
(Ruohonen et al., 2009). Transgenic mice with an overexpression of NPY in catecholaminergic
neurons showed an increase in arterial pressure during the night after surgery stress but no
difference during the recovery phase (Ruohonen et al., 2009). This result suggests that
overexpression of NPY in catecholaminergic neurons increases stress-induced sympathetic
activity and hypertension.
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6.3.3 Antagonistic effect of central and peripheral NPY in the cardiac response may modify
the stress response
The data from two NPY transgenic models, both of which over-express NPY was found
to be contradictory (Michalkiewicz et al., 2003; Ruohonen et al., 2009). The main difference
between these two transgenic models was (1). excess NPY was expressed in numerous brain
regions including the hypothalamus which regulates the sympathetic output to the peripheral
system in the conventional transgenic rats. (2). however NPY was only over-expressed in brain
noradrenergic neurons and in the sympathetic nervous system in the conditional transgenic mice.
It is possible that the overexpression of NPY in the transgenic rats inhibited the sympathetic drive
by a CNS-mediated mechanism whereas the increased sympathetic activity in the NPY transgenic
mice was mainly due to the actions of peripheral NPY.
The results in the two different NPY transgenic models are reminiscent of the opposite
effects from the peripheral and central injections of NPY which increased and decreased blood
pressure, respectively. The data suggests a central inhibitory, but peripheral stimulatory, action of
NPY in regulating the sympathetic outflow (Kuo et al., 2007a).
The antagonistic effects of central and peripheral NPY in controlling sympathetic outflow
to the heart might be a survival mechanism in response to stress such that the increased
vasoconstriction by peripheral NPY during stress is an adaptive response, whereas repression of
the sympathetic drive by central NPY would prevent a pathological activation.
6.3.4 Interaction between NPY and catecholamines in cardiovascular modulation may be a
survival mechanism in response to stress
Many in vitro studies have investigated the effects of NPY on catecholamine release and
synthesis in chromaffin cells. The catecholamines and NPY are co-released (Whim, 2006). Most
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studies reported that NPY stimulated catecholamine secretion from chromaffin cell cultures
(Cavadas et al., 2002; Cavadas et al., 2001). This effect involved the Y1 receptor since the NPY-
induced release of the catecholamines was abolished in chromaffin cells from Y1-deficient mice
(Cavadas et al., 2006).
The basal level of catecholamine synthesis in the adrenal medulla was up-regulated in Y1
knockout mice (Cavadas et al., 2006). The inhibitory role of NPY in adrenal catecholamine
synthesis is further supported by my results showing that the expression of TH in the adrenal
medulla was also basally activated in the NPY knockout mice (chapter 5). Moreover I found that
i.p. injection of BIBP3226 (a Y1 antagonist) and BIIE0246 (a Y2 antagonist) led to an increase in
the levels of adrenal TH and NPY respectively (chapter 5). Since neither of the antagonists cross
the brain barrier (Doods et al., 1999; Doods et al., 1996; Shoblock et al., 2010), my data suggests
that the role of NPY in the periphery is to directly down-regulate the levels of adrenal
catecholamines and NPY.
My speculation for explaining the stimulatory action of NPY on catecholamine release
and the inhibitory effect of NPY on the synthesis of adrenal catecholamines is as following: (1)
an initial up-regulation of catecholamine signaling by NPY and the cooperative effects of these
transmitters in enhancing the cardiac system are adaptive processes triggered by the fight-or-
flight response during stress; (2). as the stress-induced activation of the sympathetic and
cardiovascular systems proceeds, NPY participates in a negative feedback loop that represses the
synthesis of the catecholamines and also of NPY. The function of this loop may be to prevent the
system from becoming maladaptive due to an over-activation of the stress response.
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6.3.5 Peripheral NPY mediates stress-induced obesity
Stress exaggerated the high fat diet-induced obesity which was mediated by peripheral
NPY signaling (Kuo et al., 2007b). Stress increased the release of NPY and glucocorticoids from
sympathetic nerves, and the adrenal cortex, respectively. In a glucocorticoid-dependent manner,
the expression of sympathetic NPY and Y2 receptors in the white adipose tissue (WAT) were up-
regulated by stress. The activation of the Y2 receptor stimulated the growth of abdominal fat
leading to metabolic syndrome (Kuo et al., 2007b). The data above not only suggest a direct
effect of the peripheral NPY in modulating metabolism but also link stress with obesity in
peripheral NPY/ Y2 signaling.
Stress activates the sympatho-adrenomedullary system stimulating the secretion of
epinephrine and to a lesser extent, norepinephrine, into the blood. This process is fundamental for
the fight-or-flight response. By activating β adrenergic receptors, the catecholamines enhance
lipolysis as well as inhibiting adipocyte proliferation in the WAT, and stimulate thermogenesis in
the brown fat tissue (Bachman et al., 2002; Bowers et al., 2004). In my experiments injection (i.p.)
of BIBP3226 (a Y1 antagonist) was associated with an increase in the levels of adrenal TH (a
marker of catecholamine synthesis). BIBP3226 does not cross the brain-barrier (Doods et al.,
1996; Shoblock et al., 2010), so the inhibitory effect of NPY on catecholamine synthesis in the
adrenal medulla was mediated by a peripheral mechanism. Since catecholamine signaling
enhances energy expenditure, peripheral NPY may do the opposite by slowing down metabolism
not only via Y2 signaling directly but also by negatively regulating catecholamine synthesis.
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6.4 Future directions
In my current study I have demonstrated that the cold FST leads to a long-term increase
of adrenal NPY synthesis. A metabolic stress, food restriction, also elevates the levels of adrenal
NPY (data not shown). In future work I hope to directly investigate the role of adrenal NPY in
altering metabolism and the cardiovascular response to stress
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Appendix A
Appendix A. The cold FST did not alter the level of adrenal NPY-ir in vitro or in situ 30 min
after stress.
(A). The level of NPY-ir was not altered in chromaffin cells 30 min after stress compared with
the control (n = 1). The levels of NPY-ir were measured in seventy cells from each animal (N.S.,
not significant). (B). The level of NPY-ir was not changed in adrenal sections 30 min after stress
compared with the control (n = 1). The levels of NPY-ir were measured in eight sections from
each animal (N.S., not significant).
157
Appendix B
Appendix B. The level of adrenal NPY-ir was increased in epinephrine-injected animals and
epinephrine-treated acute slices.
(A). The level of NPY-ir in adrenal frozen sections was increased 24 hours after epinephrine
injection compared with saline injection (n=2). The levels of NPY-ir were measured in eight
sections from each animal (p < 0.05 in each individual experiment). (B). NPY-ir was increased 24
hours after treating 400 μm thick acute adrenal slices with 100 mM epinephrine (n=1). Methods:
20 μm frozen adrenal sections were prepared by re-sectioning the treated acute slices. The levels
of NPY-ir were measured in eight sections from each animal (p < 0.05).
158
Appendix C
(A). Phosphate buffered salt solution (PBS), pH 7.4
NaCl: 137 mM
KCl: 2.7 mM
Na2HPO4.7H2O: 10 mM
KH2PO4: 1.8 mM
(B). Blocking solution (0.25%)
PBS: 20 mL
Bovine serum albumin (BSA): 0.05 g
Triton X-100: 10 μl
(C). HBSS+20mM HEPES (20H HBSS), pH 7.25
KCl: 5.3 mM
KH2PO4: 0.44 mM
NaHCO3: 4 mM
NaCl: 138 mM
Na2HPO4.7H2O: 0.3 mM
Glucose: 5.5 mM
HEPES: 20 mM
(D). Trituration medium
HBSS: 10 mL
159
BSA: 1 mg/ml BSA
MgCl2: 8 mM
(E). Collagenase solution
Collagenase: 1 mg
BSA: 6 mg
20H HBSS: 1 mL
(F). Trypsin solution
Trypsin: 1 mg
BSA: 6 mg
20H HBSS: 1 mL
(G). 10 X Tris-Glycine (1 X Tris-Glycine; SDS-PAGE running buffer)
Tris base: 15.14 g
Glycine: 72.07 g
SDS: 5 g
To final volume of 500 mL
(H). 5 X Transfer buffer
Tris base: 7.5 g
Glysine: 35.625 g
To final volume of 500 mL
(I) 1 X transfer buffer
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5 X transfer buffer: 100 mL
ddH2O: 300 mL
Methanol: 100 mL
(J). 10 X PBS
NaCl: 40 g
KCl: 1 g
KH2PO4: 1 g
Na2HPO4.7H2O: 10.8 g
To a final volume of 500 mL
(K) 1 X PBS-T (Western blot washing buffer)
10 X PBS: 50 mL
ddH2O: 450 mL
Tween-20: 0.3%
161
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Vita
Qian Wang
Education Ph.D. Candidate in Cell and Developmental Biology Sep 2006-Present
The Pennsylvania State University, USA
Dissertation Regulation of neuropeptide Y expression and the sympathoadrenal response to
stress
Bachelor of Science, major in Biotechnology Sep 2002- Jul 2006
Shanghai Jiao Tong University, P.R.China
Publications, Abstracts and Manuscripts
Wang Q and Whim MD. Acute stress leads to a long-term change of adrenal neuropeptide
Y. Manuscript in preparation, 2011.
Wang Q and Whim MD. Neuropeptide Y inhibits the neuronal branch of the stress
response. Manuscript in preparation, 2011.
Ramamoorthy P, Wang Q, and Whim MD. Cell type-dependent trafficking of neuropeptide
Y- containing dense core granules in CNS neurons. Under revision, 2011.
Wang Q and Whim MD. Acute stress leads to a long-term change in the adrenal levels of
neuropeptide Y (2010). Annual meeting of Society for Neuroscience.
Wang Q and Whim MD. Acute stress alters the levels of neuropeptide Y in a
sub-population of adrenal chromaffin cells (2009). Annual meeting of Society for
Neuroscience.
Wang Q, Mitchell GC, Ramamoorthy P, Whim MD (2008). A single nucleotide
polymorphism associated with a change in serum cholesterol levels increases synthesis and
secretion of neuropeptide Y. Annual meeting of Society for Neuroscience.
Mitchell GC*, Wang Q*, Ramamoorthy P, Whim MD (2008). A common single
nucleotide polymorphism alters the synthesis and secretion of neuropeptide Y. J Neurosci.
28:14428-14434. * Co-first author.
Honors and Awards
2009 J Ben and Helen D. Hill Memorial Fund Award
2006 Huck Institutes of Life Sciences Fellowship, PSU