Grossmont Cuyamaca Mediation of the Acute Stress Response Summary Write a 1 to 2 page summary of the article Mediation of the Acute Stress Response by the

Grossmont Cuyamaca Mediation of the Acute Stress Response Summary Write a 1 to 2 page summary of the article Mediation of the Acute Stress Response by the Skeleton by Julian Meyer Berger et al. in Cell Metabolism (November 2019). In addition to your summery, clearly state the author’s question they sought to answer in this paper and comment why the author’s decided to pursue this research. What current information about the mediators of the Acute Stress Response seems inconsistent with the response? Article
Mediation of the Acute Stress Response by the
Skeleton
Graphical Abstract
Authors
Julian Meyer Berger, Parminder Singh,
Lori Khrimian, …, Kamal Rahmouni,
Xiao-Bing Gao, Gerard Karsenty
Correspondence
gk2172@cumc.columbia.edu
In Brief
During the acute stress response in bony
vertebrates, a brain-derived signal
increases glutamate uptake into
osteoblasts, producing a surge in
circulating osteocalcin. Once released,
osteocalcin suppresses the
parasympathetic nervous system,
enabling the stress response.
Highlights
d
The ASR stimulates osteocalcin release from bone within
minutes
d
Glutamate uptake into osteoblasts is required for osteocalcin
release during an ASR
d
Osteocalcin inhibits the parasympathetic tone during an ASR
d
In adrenal insufficiency, increased osteocalcin levels enable
an ASR to occur
Berger et al., 2019, Cell Metabolism 30, 1–13
November 5, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.cmet.2019.08.012
Please cite this article in press as: Berger et al., Mediation of the Acute Stress Response by the Skeleton, Cell Metabolism (2019), https://doi.org/
10.1016/j.cmet.2019.08.012
Cell Metabolism
Article
Mediation of the Acute Stress
Response by the Skeleton
Julian Meyer Berger,1,2 Parminder Singh,3 Lori Khrimian,1 Donald A. Morgan,4 Subrata Chowdhury,1
Emilio Arteaga-Solis,1,5 Tamas L. Horvath,6 Ana I. Domingos,7 Anna L. Marsland,8 Vijay Kumar Yadav,1,3
Kamal Rahmouni,4 Xiao-Bing Gao,6 and Gerard Karsenty1,9,*
1Department
of Genetics and Development, Columbia University Irving Medical Center, New York, NY 10032, USA
in Microbiology, Immunology and Infection, Columbia University Irving Medical Center, New York, NY 10032, USA
3Metabolic Research Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
4Department of Pharmacology, University of Iowa and Veteran Health Care System, Iowa City, IA 52242, USA
5Division of Pediatric Pulmonary, Department of Pediatrics, Columbia University Irving Medical Center, New York, NY 10032, USA
6Program in Integrative Cell Signaling and Neurobiology of Metabolism, Department of Comparative Medicine, Yale University School of
Medicine, New Haven, CT, USA
7Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
8Department of Psychology, University of Pittsburgh, Pittsburgh, PA 15260, USA
9Lead Contact
*Correspondence: gk2172@cumc.columbia.edu
https://doi.org/10.1016/j.cmet.2019.08.012
2Program
SUMMARY
INTRODUCTION
We hypothesized that bone evolved, in part, to
enhance the ability of bony vertebrates to escape
danger in the wild. In support of this notion, we
show here that a bone-derived signal is necessary
to develop an acute stress response (ASR). Indeed,
exposure to various types of stressors in mice, rats
(rodents), and humans leads to a rapid and selective
surge of circulating bioactive osteocalcin because
stressors favor the uptake by osteoblasts of glutamate, which prevents inactivation of osteocalcin
prior to its secretion. Osteocalcin permits manifestations of the ASR to unfold by signaling in post-synaptic parasympathetic neurons to inhibit their activity,
thereby leaving the sympathetic tone unopposed.
Like wild-type animals, adrenalectomized rodents
and adrenal-insufficient patients can develop an
ASR, and genetic studies suggest that this is due to
their high circulating osteocalcin levels. We propose
that osteocalcin defines a bony-vertebrate-specific
endocrine mediation of the ASR.
The endocrine functions of bone raise the question of why an organ viewed as a scaffold is also an endocrine organ. A possible
answer to this question arises by considering bone physiology in
an evolutionary context. Through its structural properties, bone
protects internal organs in the event of a trauma and allows animals to move and escape danger (Romer, 1933). Bone also mediates hearing, which is a means of detecting danger. A similar
purpose in the context of danger may be ascribed to several
physiological processes enhanced by the bone-derived hormone osteocalcin. Memory is needed in the wild to recall where
food and/or predators are, the ability to increase exercise capacity is an absolute necessity for animals attempting to escape
danger, and circulating testosterone levels rise in the event of
danger (Frankel and Ryan, 1981; Karsenty and Olson, 2016;
Mera et al., 2016; Yuen et al., 2009). From this vantage point,
one could argue that the classical and endocrine functions of
bone concur to equip bony vertebrates with a tool to escape
danger. If this interpretation is, at least in part, correct, bone
should regulate other physiological functions activated in the
presence of danger.
The acute stress response (ASR) is an evolutionarily
conserved physiological process that aims to maintain or restore
Context and Significance
A major question in skeletal biology is to understand why bone, through the hormone osteocalcin, favors energy metabolism, reproduction, memory, and the ability to exercise. Since most of these functions abet survival in unpredictably hostile environments such as the wild, we surmised that bone evolved to enable vertebrates to overcome acute danger. In support of this notion, this study shows that animals need osteocalcin to develop an acute stress response, a function critical to
survival in the wild. When animals encounter an immediate danger, a brain-derived signal stimulates the release of osteocalcin from bone. Once released, osteocalcin turns off the parasympathetic or ‘‘rest-and-digest’’ arm of the autonomic nervous system and thereby allows the acute stress response to proceed.
Cell Metabolism 30, 1–13, November 5, 2019 ª 2019 Elsevier Inc. 1
Please cite this article in press as: Berger et al., Mediation of the Acute Stress Response by the Skeleton, Cell Metabolism (2019), https://doi.org/
10.1016/j.cmet.2019.08.012
homeostasis in animals facing an immediate danger. Such
danger produces an increase in temperature and energy expenditure, higher heart rate, and faster respiration, among other
manifestations (McEwen, 1998; Sapolsky et al., 2000; UlrichLai and Herman, 2009). The sympathetic nervous system that releases catecholamine into peripheral organs is considered to be
the ultimate mediator of the ASR in vertebrates (Axelrod and Reisine, 1984; Ulrich-Lai and Herman, 2009). In addition, circulating
glucocorticoid hormones surge during an ASR, suggesting that
an endocrine mediation of the ASR may exist. As steroids, glucocorticoid hormones act mainly, albeit not only, at the transcriptional level and need hours to regulate physiological processes
(Tsai and O’Malley, 1994), something that seems inconsistent
with the need for an immediate response. Although this certainly
does not rule out that glucocorticoid hormones may be implicated in some capacity in the ASR, it suggests the possibility
that other hormones, possibly peptide ones, could mediate the
ASR. This is why, in considering that an original purpose of
bone was to escape danger, we asked whether bone-derived
hormones contribute to the ASR in bony vertebrates.
Addressing the aforementioned question revealed that osteocalcin is necessary to develop an ASR. Osteocalcin circulating
levels surge within minutes in mice exposed to stressors. This effect of stressors on bone is restricted to osteocalcin, independent
of corticosterone and catecholamine signaling, but requires that
osteoblasts take up glutamate to enhance the release of bioactive
osteocalcin. Osteocalcin is necessary for the development of
several key manifestations of an ASR by signaling through its receptor Gprc6a in post-ganglionic parasympathetic neurons to
inhibit cholinergic activity. Remarkably, adrenalectomized (ADX)
rodents and patients with adrenal insufficiency can still mount a
normal ASR. Genetic experiments indicate that this is due to the
doubling of circulating osteocalcin in these animals. These results
identify bone as a stress organ and osteocalcin as a stress
hormone.
RESULTS
Stressors Trigger a Rapid Surge of Circulating Bioactive
Osteocalcin in Rodents and Humans
Testing whether any protein made in bone is affected during the
ASR revealed that circulating levels of undercarboxylated, i.e.,
bioactive, osteocalcin rose by 50% after a 45-min-long restraint
and by 150% 15 min after electric foot shocks in 2- to 6-monthold mice, in either sex, in two different genetic backgrounds,
and at two different times of day (Figures 1A–1E). Circulating
bioactive osteocalcin levels also rose in rats after restraint and
in humans submitted to a public speaking and cross-examination
stress that increased heart rate and blood pressure (PattersonBuckendahl et al., 1995; Prather et al., 2009) (Figures 1F and
1G). This effect of stressors on bone seen in rodents and humans
is restricted to osteocalcin, as circulating levels of other bonederived hormones and synthesis of type I collagen, the most
abundant protein of the bone matrix, are unaffected in stressorexposed mice (Figures S1A–S1C).
As a third stressor, we also used 2,4,5-trimethyl thiazoline (TMT),
a component of fox urine because it triggers a rapid, innate, and
centrally controlled fear reaction in the mouse (Kim et al., 2016; Kobayakawa et al., 2007; Kondoh et al., 2016; Root et al., 2014). TMT
2 Cell Metabolism 30, 1–13, November 5, 2019
induced a rise in circulating bioactive osteocalcin that began
before that of corticosterone, reached its peak at 2.5 min, and remained steady for at least 3 h. In contrast, urine from a non-predator animal used as a negative control did not affect circulating
bioactive osteocalcin (Figures 1H and 1I). The effect of TMT on
circulating bioactive osteocalcin prompted us to assess through
a chemogenetic designer receptor exclusively activated by
designer drugs (DREADD) approach (Zhu and Roth, 2014) whether
stressors signal in the brain to induce a rise in circulating bioactive
osteocalcin. Adeno-associated virus (AAV) encoding the inhibitory
DREADD hM4Di was injected into the baso-lateral amygdala
(BLA), a brain region proposed to be a fear center in the murine
brain, involved in TMT neuronal relays, and selectively labeled by
viral retrograde tracing from the femur (De?nes et al., 2005; Fadok
€ller and Fendt, 2006; Terburg
et al., 2009; Johansen et al., 2011; Mu
et al., 2018; Tye et al., 2011; Wolff et al., 2014). Subsequent deactivation of the BLA via injection of clozapine-N-oxide (CNO), the
designer drug, prior to exposure to stressors obliterated the surge
of osteocalcin in AAV-injected but not in sham-injected mice (Figures 1J, S1D, and S1E). These data indicate that stressors must
signal in the BLA to trigger the release of bioactive osteocalcin,
though they do not exclude the possibility that other regions of
the brain are also implicated in the development of an ASR.
Bioactive Osteocalcin Is Released from Cells of the
Osteoblast Lineage during an ASR
The stressor-induced surge in circulating bioactive osteocalcin
levels described above occurs independently of sympathetic
signaling through b-adrenergic receptors, a well-known regulator of osteoblast functions (Takeda et al., 2002); circulating adrenal steroid hormones levels; or transcriptional events in bones
or other tissues (Figures 2A–2C and S2A–S2D). This led us to
search for other mechanisms that would account for the release
of bioactive osteocalcin into general circulation during an ASR.
Osteocalcin is activated through decarboxylation, a reaction
that occurs in the bone resorption lacunae (Karsenty and Olson,
2016). However, circulating bioactive osteocalcin (osteocalcin)
levels increased equally well in stressor-exposed oc/oc mice
that lack functional osteoclasts (Scimeca et al., 2000) as in control
mice (Figure 2D). They also increased equally well in stressorexposed wild-type (WT) mice treated with alendronate, an inhibitor of bone resorption (Azuma et al., 1995), or with vehicle (Figures
S2E and S2F). In agreement with these observations, circulating
levels of CTX, a biomarker of bone resorption, or of RankL, an activator of bone resorption, did not increase within minutes in
stressor-exposed WT mice (Figures S2G–S2I). In view of these
observations, we considered the possibility that cells of the osteoblast lineage (osteoblasts and osteocytes) that synthesize osteocalcin are the ones releasing its bioactive form during an
ASR. In support of this notion, we found that exposure to stressors
increased circulating bioactive osteocalcin even further in
Tph1villin / mice, which have twice as many cells of the osteoblast lineage as in control littermates but have normal bone
resorption (Figure 2E) (Yadav et al., 2008). In contrast, exposure
to stressors did not increase circulating bioactive osteocalcin in
a1(I) CollagenDTA mice, which have markedly fewer osteoblasts
than WT mice (Figure 2F) (Yoshikawa et al., 2011).
To determine if the release of bioactive osteocalcin is triggered
by molecule(s) present in the general circulation and that signal in
Please cite this article in press as: Berger et al., Mediation of the Acute Stress Response by the Skeleton, Cell Metabolism (2019), https://doi.org/
10.1016/j.cmet.2019.08.012
Ocn (% baseline)
**
Bioactive Ocn (ng/mL)
**
Bioactive Ocn (ng/mL)
50
100
0
0
Stressor
Stressor
J
*
150
*
50
*
*
150
*
*
*
*
*
*
0
50
0
0
5
10 15
60 100 140 180
AAV8.2-hEF1?hM4Di-mCherry
100
75
50
ns
250
*
Bioactive Ocn (ng/mL)
TMT
250
25
3
2
1
0
-1
0
Stressor
Time post-TMT (minutes)
Rate of Ocn release
(ng/mL/min)
Vehicle (n=6)
CNO (n=6)
Ocn (n=6)
Corticosterone (n=6)
*
hi
cl
C e
N
O
Odorant
100
Stressor
I
Bioactive Ocn (ng/mL)
ns
0
0
Stressor
*
Ve
0
10
35
Time post-stress
(min)
Bioactive Ocn (ng/mL)
0
50
50
Rat (n=6)
200
**
100
100
**
Ocn (% baseline)
*
TMT (n=6)
Rabbit urine (n=6)
100
Corticosterone (ng/mL)
*
0
F
C57Bl/6 (n=10)
129SV (n=10)
**
H
Mouse (n=10)
Human (n=20)
200
0
50
Stressor
Stressor
E
12 pm (n=8)
8 pm (n=8)
**
0
50
100
**
**
50
100
**
**
Bioactive Ocn (ng/mL)
100
G
D
Female (n=10)
Male (n=6)
**
Bioactive Ocn (ng/mL)
(n=10)
(n=10)
C
2-month-old (n=10)
6-month-old (n=10)
**
B
Restraint Foot shock
Bioactive Ocn (ng/mL)
A
Figure 1. Stressors Trigger a Rapid Surge of Circulating Bioactive Osteocalcin (Ocn) in Rodents and Humans
(A) Serum Ocn levels in 3-month-old WT mice after restraint or foot shocks.
(B) Serum Ocn levels in 2- and 6-month-old WT mice before and after foot shocks.
(C) Serum Ocn levels in male and female WT mice before and after foot shocks.
(D) Serum Ocn levels at 12 p.m. and 8 p.m. before and after foot shocks.
(E) Serum Ocn levels in C57BL/6 or 129SV WT mice before and after foot shocks.
(F) Serum Ocn levels in WT female rats after restraint.
(G) Serum Ocn levels in WT mice after foot shock and humans after public speaking stress.
(H) Serum Ocn levels in WT mice after TMT or rabbit urine exposure.
(I) Serum Ocn and corticosterone levels in TMT-exposed WT mice (gray).
(J) Serum Ocn levels and the rate of Ocn release in TMT-exposed WT mice expressing hM4Di in the BLA after injection of CNO or vehicle.
Mice are 3-month-old females unless otherwise specified. Values are mean ± SEM. ns, not significant; *p < 0.05; **p < 0.01; by Student’s t test or one-way ANOVA with Bonferroni post hoc test. cells of the osteoblast lineage, we cultured osteoblasts obtained from WT mice in the presence of sera obtained from either unstressed or stressed Osteocalcin / (Ocn / ) mice. In this experimental setting, the osteocalcin molecules detected in the supernatant can only originate from the cultured osteoblasts. We found that WT osteoblasts produced similar amounts of bioactive osteocalcin whether they were cultured in the presence of sera from unstressed or stressed Ocn / mice, and none of the specific hormones tested in serum-free conditions increased the release of bioactive osteocalcin by osteoblasts (Figures S2J and S2K). In contrast, blocking neuronal activity with the peripheral ganglionic blocker chlorisondamine prevented the increase of circulating bioactive osteocalcin in stressor-exposed WT mice, thus suggesting a neuronal mediation of the effect of stressors on circulating bioactive osteocalcin levels (Figure 2G). Glutamate Mediates the Stressor-Induced Release of Bioactive Osteocalcin from Osteoblasts Among all the neurotransmitters tested, only glutamate (Talman et al., 1980; Toyota et al., 2018) significantly increased the amount of bioactive osteocalcin found in the supernatant of mouse osteoblasts (Figure 3A). In agreement with the data presented in Figure S2J, circulating glutamate does not change in WT mice exposed to stressors (Figure 3B). On the other hand, ablating peripheral glutamatergic neurites using a pegylated diphtheria toxin (PEG-DT) that does not cross the blood brain barrier (Pereira et al., 2017) injected into mice with a conditional diphtheria toxin receptor driven by Vglut2-Cre (Vong et al., 2011) (Vglut2iDTR mice) prevented the stressor-induced increase in circulating bioactive osteocalcin (Figures 3C and S3A–S3C). In addition, we observed that glutamatergic neurites are present in bone and abut osteoblasts (Serre et al., 1999) (Figure 3D). These results suggest that glutamate action on cells of the osteoblast lineage can originate from neurites within bone. These VGLUT2-positive neurites present in bones are tyrosine hydroxylase (TH) negative and do not express any of the markers of sensory neurons tested (Figures 3D and 3E). We note that the existence of a subpopulation of VGLUT2-positive, TH-negative neurons has been previously reported (Brumovsky et al., 2011a, 2011b; Furlan et al., 2016). A single glutamate transporter, Eaat1 or Glast (Mason et al., 1997; Rothstein et al., 1996), is expressed three orders of Cell Metabolism 30, 1–13, November 5, 2019 3 Please cite this article in press as: Berger et al., Mediation of the Acute Stress Response by the Skeleton, Cell Metabolism (2019), https://doi.org/ 10.1016/j.cmet.2019.08.012 A B C D E F G Figure 2. Bioactive Osteocalcin Is Released from Cells of the Osteoblast Lineage during an Acute Stress Response (A) Serum Ocn levels and the rate of Ocn release in nadolol- and vehicle-treated WT mice exposed to TMT. (B) Serum Ocn levels and the rate of Ocn release in adrenalectomized (ADX) and sham-operated TMT-exposed WT mice. (C) Northern blot analysis of Ocn (top) and L19 (bottom) expression after foot shock. (D) Serum Ocn levels and the rate of Ocn release in oc/oc and WT mice before and after TMT exposure. (E) Serum Ocn levels and the rate of Ocn release in Tphvil / and Tphf/f mice before and after TMT exposure. (F) Serum Ocn levels and rate of Ocn release in a1(I) CollagenDTA/+ and a1(I) Collagen+/+ mice before and after TMT exposure. (G) Serum Ocn levels and the rate of Ocn release in chlorisondamine- and vehicle-treated WT mice before and after TMT exposure. Mice are 3-month-old females. Rats are 4-month-old females. Values are mean ± SEM. ns, not significant; *p < 0.05; **p < 0.01; by Student’s t test or one-way ANOVA with Bonferroni post hoc test. magnitude higher in osteoblasts than any other glutamate transporter and any other cell type tested, including osteoclasts (Figures 3E and S3F). The role of glutamate transport in osteoblasts through Glast in the release of osteocalcin was delineated in several ways. First, incubation with radiolabeled glutamate significantly increased the intracellular concentration of glutamate in WT but not Glast / osteoblasts; second, UCPH102, a specific inhibitor of Glast function (Haym et al., 2016), prevented the uptake of radiolabeled glutamate by WT mouse osteoblasts; third and unlike the case in WT osteoblasts, glutamate could not stimulate the release of osteocalcin from UCPH102treated WT or Glast / osteoblasts; fourth and in vivo, circulating bioactive osteocal... Purchase answer to see full attachment

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