Journal of Thermal Biology 74 (2018) 71–76

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Journal of Thermal Biology

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Roles for lysine acetyltransferases during mammalian hibernation T

⁎
Andrew N. Rouble, Liam J. Hawkins, Kenneth B. Storey
Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada

A R T I C LE I N FO A B S T R A C T

Keywords: The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) is a well-known model for studying hibernation.
Hibernation While in a torpid state, these animals globally suppress energy expensive processes, while supporting specialized
Acetyltransferases pathways necessary for survival. Lysine acetyltransferases (KATs) play a crucial role in modulating the ex-
Skeletal muscle pression and activity of a wide-variety of cellular pathways and processes, and therefore, may play a role during
Liver
hibernation when the cell is shifting to an energy conservative, cytoprotective state. Here we measured protein
Adipose tissue
levels of four KATs (CBP, PCAF, GCN5L2, HAT1), total histone acetyltransferase (HAT) activity, and the levels of
acetylation of histone H3 lysine 9 (H3K9ac), in multiple tissues across the torpor-arousal cycle. Our results show
a tissue-specific response of KATs, particularly in the adipose tissues where specific KATs (PCAF and GCN5L2),
HAT activity, and H3K9ac increased in the metabolically active BAT while HAT1, HAT activity and H3K9ac
decreased in WAT. Liver showed significant increases in the KAT PCAF whereas skeletal muscle had decreased
CBP and GCN5L2. Both liver and skeletal muscle showed no change in HAT activity and H3K9me3 increased in
muscle during torpor. Together, these results suggest KATs may play specialized roles in the different tissues of
the ground squirrel to contribute to the hibernator phenotype.

1. Introduction thermogenesis from uncoupled respiration in the specialized mi-

Some mammals have adapted to survive the winter months, when
food resources become limited and temperatures drop below 0 °C, by
entering hibernation. During hibernation, these animals lower their
metabolic rate for weeks to months at a time, with interspersed but
brief periods of arousal where their metabolism temporarily returns to
euthermic levels (French, 1985). While there is considerable variety in
hibernation parameters (e.g. duration, feeding vs. non-feeding, range of
heterothermicity) depending on taxa and climatic region (Giroud et al.,
2008; Nespolo et al., 2010; Weitten et al., 2016), during torpor, animals
such as the thirteen-lined ground squirrel (Ictidomys tridecemlineatus)
display drastic reductions in body temperature (Tb), heart rate,
breathing rate, and organ perfusion rates (Bullard and Funkhouser,
1962; Carey et al., 2003; Frerichs et al., 1994). Congruent with these
physiological changes are alterations to energy metabolism pathways
that result in a shift away from carbohydrate metabolism towards lipid
catabolism, particularly in the liver, and fatty acid oxidation of lipid
reserves in white adipose tissue (WAT) accumulated during the summer
months (Carey et al., 2003; Dark, 2005). These lipid deposits are es-
sential in placental hibernators during periods of arousal, when in-
creases in oxygen consumption and fatty acid oxidation fuel
tochondria of brown adipose tissue (BAT). Remarkably, these animals
endure little to no damage to their tissues, or atrophy of their muscles
(Andres-Mateos et al., 2013), despite prolonged periods of inactivity,
rapid tissue reperfusion, and exposure to the production of high levels
of reactive oxygen species due to heightened oxygen consumption
during arousal (Storey, 2010). Interestingly, it is generally accepted
that these extraordinary metabolic and protective adaptations are the
result of the regulation of gene expression, rather than novel hiberna-
tion-specific genes (Carey et al., 2003). Therefore, the mechanisms of
genetic regulation employed by hibernators have made these animals
particularly interesting subjects in the fields of gerontology, obesity,
diabetes, and neurodegenerative diseases (Carey et al., 2003; Härtig
et al., 2007; Martin, 2008; Storey, 2010; Wu and Storey, 2016).
The molecular regulation of hibernation is in large part dependent
on the reversible post-translational modification of proteins (Bell and
Storey, 2017; Rouble and Storey, 2015; Storey, 2010; Tessier et al.,
2017). We have previously investigated the role of reversible protein
acetylation in this context, with specific focus on the expression and
activity of NAD+-dependent SIRT deacetylases (Rouble and Storey,
2015) and histone deacetylases (HDACs) (Morin and Storey, 2006). The
counter-parts to these deacetylase enzymes are lysine acetyltransferases

Abbreviations: KAT, lysine acetyltransferase; HAT, histone acetyltransferase; H3K9ac, histone H3 lysine 9 acetylation; BAT, brown adipose tissue; WAT, white adipose tissue; Tb, body
temperature; HDAC, histone deacetylase; IA, interbout arousal; EC, euthermic in cold room; EN, entrance into torpor; ET, early torpor; LT, late torpor; EA, early arousal
⁎
Corresponding author.
E-mail address: kenneth_storey@carleton.ca (K.B. Storey).

https://doi.org/10.1016/j.jtherbio.2018.03.013
Received 9 February 2018; Received in revised form 13 March 2018; Accepted 13 March 2018
Available online 15 March 2018
0306-4565/ © 2018 Elsevier Ltd. All rights reserved.
A.N. Rouble et al. Journal of Thermal Biology 74 (2018) 71–76

(KATs), which function to add acetyl-moieties to lysine residues. The

addition of acetyl-groups to target proteins can alter important mole-
cular parameters, such as DNA-binding affinities, enzymatic activities,
protein stability, target specificity, and complex formation (Carrozza
et al., 2003; Chen et al., 2001; Spange et al., 2009). KATs also enable
the histone-acetylation-dependent epigenetic remodeling of chromatin,
thereby controlling the activation of transcription at specific loci
(Carrozza et al., 2003; Lee and Workman, 2007; Verdone et al., 2006).
Four of the best-characterized KATs are CREB Binding Protein
(CBP), P300/CBP-associated factor (PCAF), General Control of Amino
Acid Synthesis Protein 5-Like 2 (GCN5L2), and Histone
Acetyltransferase 1 (HAT1). The functions of these proteins is integral
in processes such as cell death, DNA repair, proliferation, metabolism,
and general transcriptional activation (Chen et al., 2001; Spange et al.,
2009; Xiong and Guan, 2012). Because of these important roles, these
KATs are of great interest to researchers seeking novel approaches to
the treatment of various diseases, and similarly, as potential targets for
the regulation processes that allow hibernating mammals to undergo
metabolic rate depression during torpor. In this study, we explore KATs
in the context of a mammalian hibernator, the thirteen-lined ground
squirrel. Protein levels of CBP, PCAF, GCN5L2, and HAT1 were mea-
Fig. 1. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in liver of I. tride-
sured over the five standardized time points of the torpor-arousal cycle cemlineatus over the torpor-arousal cycle. Representative protein bands are shown for
(Carey et al., 2003; Storey, 2010) with a euthermic control in four selected sampling points (labelled to the left and right of the blots). Histogram shows
tissues: liver, skeletal muscle, BAT, and WAT. These tissues were se- mean standardized band densities ( ± SEM, n = 4). Protein bands were standardized
lected due to their previously mentioned physiology, metabolism, and against the summed intensity of a group of Coomassie-stained protein bands from the
resilience during hibernation. Additionally, total histone acetyl- same sample lane. Data were analyzed using a one-way ANOVA with a post hoc Dunnett's
test. The symbol * indicates significant difference from the respective EC control,
transferase (HAT) activities were measured, comparing euthermic ani-
p < 0.05.
mals to those in deep-torpor. Finally, the levels of acetylated histone H3
lysine 9 (H3K9ac), a KAT-targeted histone residue, were measured
across the torpor-arousal cycle. The results reinforce previous studies
on the role for reversible protein acetylation in the context of mam-
malian hibernation (Rouble and Storey, 2015).

2. Results

2.1. Analysis of KAT protein levels over the torpor-arousal cycle

Relative protein levels of select KATs (CBP, PCAF, GCN5L2, and

HAT1) were measured in the liver (Fig. 1), skeletal muscle (Fig. 2), BAT
(Fig. 3), and WAT (Fig. 4) of the ground squirrel over the six time-points
of the torpor arousal cycle. In liver, protein levels of CBP were sig-
nificantly reduced to 0.4 ± 0.08 during interbout arousal (IA) as
compared to values for animals that were euthermic in the cold room
(EC), while levels of PCAF were significantly elevated over controls
during four stages: entrance into torpor (EN), early torpor (ET), late
torpor (LT) and early arousal (EA) (by 4.7 ± 0.2, 2.2 ± 0.09,
3.0 ± 0.2, and 4.6 ± 0.3-fold, respectively). Protein levels of GCN5L2
were also significantly decreased to 0.6 ± 0.03 during LT when com-
pared to EC, and subsequently increased during EA by 1.4 ± 0.2-fold
over controls. No significant fluctuations were observed for protein Fig. 2. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in skeletal muscle of
I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1.
levels of HAT1 in liver over the torpor-arousal cycle.
In skeletal muscle, CBP protein levels were significantly reduced
during LT to 0.3 ± 0.06 of the corresponding EC values, while levels of did not change significantly at any of the sampled time points.
GCN5L2 were also significantly lowered during EN, LT, EA and IA (to
0.7 ± 0.03, 0.6 ± 0.03, 0.7 ± 0.1, and 0.7 ± 0.06 of EC, respec- 2.2. Analysis of total HAT activity
tively), but not during ET. Protein levels of PCAF and HAT1 did not
change significantly over torpor-arousal. Total relative HAT activity was measured in the total soluble protein
In BAT, protein levels of PCAF were significantly elevated during EN fraction of liver, skeletal muscle, BAT, and WAT, comparing EC and LT
by 1.9 ± 0.1-fold over EC, while levels of GCN5L2 were also elevated stages (Fig. 5). In liver and skeletal muscle, relative HAT activity did
during LT by 1.4 ± 0.04-fold before significantly decreasing during IA not change significantly during LT as compared to EC control (Liver EC
to 0.7 ± 0.01 of EC. Protein levels of HAT1 were also significantly – 2869 ± 743 ng/h/mg, LT – 2377 ± 297 ng/h/mg; Muscle EC –
reduced during ET and IA to 0.6 ± 0.02 and 0.4 ± 0.05 of EC values, 749 ± 39 ng/h/mg, LT – 808 ± 105 ng/h/mg). In contrast, relative
respectively. In contrast, levels of CBP did not fluctuate significantly HAT activity in BAT was significantly elevated during LT by 2.0 ± 0.2-
over the course of the torpor-arousal cycle. fold as compared to EC (EC – 2407 ± 581 ng/h/mg, LT –
In WAT, protein levels of HAT1 decreased significantly during LT to 4754 ± 497 ng/h/mg), whereas total KAT activity in WAT was sig-
0.5 ± 0.04 of the EC value, whereas levels of CBP, PCAF and GCN5L2 nificantly reduced during LT to 0.5 ± 0.07 of the EC value (EC –

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A.N. Rouble et al. Journal of Thermal Biology 74 (2018) 71–76

Fig. 5. Total relative HAT activity in liver, skeletal muscle, brown adipose tissue and
white adipose tissue of I. tridecemlineatus comparing euthermic control (EC) and late
torpor (LT) points of the torpor-arousal cycle. Histograms show means ± SEM, n = 4.
Data were analyzed using the Student's t-test. The symbol * indicates significant differ-
ence from the respective EC control, p < 0.05.

Fig. 3. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in brown adipose
tissue of I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1.

Fig. 6. Relative protein expression of histone H3 acetylated at lysine 9 (H3K9ac) in liver,

skeletal muscle, brown adipose tissue and white adipose tissue of I. tridecemlineatus over
the torpor-arousal cycle. All other information as in Fig. 1.

Fig. 4. Relative protein expression of CBP, PCAF, GCN5L2 and HAT1 in white adipose
tissue of I. tridecemlineatus over the torpor-arousal cycle. All other information as in Fig. 1. action in transcriptional complexes (Chan and La Thangue, 2001; Nagy
and Tora, 2007; Roth et al., 2001). Since transcription is tightly con-
96 ± 14 ng/h/mg, LT – 46 ± 7 ng/h/mg). trolled in hibernating mammals, it may be the case that KATs such as
CBP, PCAF, GNC5L2, and HAT1 are also regulated or involved in the
regulation of this energy intensive process. Our previous studies have
2.3. Analysis of acetylation status of histone H3 lysine 9 during hibernation
identified possible roles for HDACs and SIRT-deacetylases in metabolic
rate depression and cellular protective pathways during hibernation
Relative protein levels of histone H3 acetylated at lysine 9 (H3K9ac)
(Morin and Storey, 2006; Rouble and Storey, 2015). However, given
(Fig. 6) were measured in ground squirrel liver, muscle, BAT and WAT,
that KATs have never previously been characterized in hibernators,
over the six sampling-points of the torpor-arousal cycle. Relative
their regulation in the context of this form of metabolic suppression has
amounts of H3K9ac protein increased significantly in BAT during ET
been unknown, representing a major gap in knowledge within the field
and LT (by 2.4 ± 0.2 and 2.2 ± 0.2-fold, respectively) and in muscle
of hibernation research. With the goal of filling this gap, this study has
during ET (by 1.8 ± 0.1-fold) as compared to the respective EC con-
attempted to provide an initial characterization of KAT involvement in
trols. In WAT, H3K9 levels decreased significantly during ET to
hibernation, and succeeds in providing evidence to suggest a role for
0.4 ± 0.02 before increasing by 2.2 ± 0.2-fold during IA, as com-
these enzymes within this context.
pared to EC. In liver, no significant fluctuations in H3K9 levels were
observed.
3.1. Hibernation and KAT protein levels
3. Discussion
Our results show that, of the four KATs considered, these enzymes
KATs have been implicated in the regulation of a wide variety of are differentially expressed in a tissue-specific manner at various points
cellular processes through their modification of histones and direct during hibernation, but do not exhibit an overall pattern of universal

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A.N. Rouble et al.
protein suppression or activation (Figs. 1–4). This observation would
suggest that, in these animals, each of the studied factors likely serves
some distinct role(s) at different times, and in different tissues,
throughout hibernation. For example, PCAF protein expression was
strongly enhanced in liver from EN through to EA (returning to eu-
thermic levels during the interbout period), while this same pattern was
not observed in any other tissue. This suggests that the increased ex-
pression of PCAF serves a specific role in liver during torpor that does
not appear to be required in other tissues. Interestingly, under condi-
tions that inhibit glycolysis, PCAF is known to acetylate and reduce the
activity of pyruvate kinase (Lv et al., 2011), a major glycolytic enzyme
that is also suppressed by RPP during hibernation (Storey and Storey,
2010). The increased expression of PCAF in liver throughout hiberna-
tion may therefore contribute to the inhibition of certain metabolic
processes such as glycolysis, in a manner analogous to the increased
activities of specific kinases/phosphatases that also occur during this
time in the liver. Protein levels of CBP also demonstrated a tissue-spe-
cific response, most notably being suppressed during late torpor only in
muscle tissue. Given CBP's role as a diverse transcriptional co-activator
(Chan and La Thangue, 2001; Kalkhoven, 2004), this fluctuation may
serve an important purpose in the regulation of the metabolic sup-
pression in hibernator muscle. This is because its presence and acetyl-
transferase activity in certain transcriptional complexes is responsible
and necessary for the regulation of many different genes; therefore, a
decrease in CBP expression could contribute to the widespread tran-
scriptional suppression that is characteristic of the torpid state. In fact,
this proposed function would also complement current evidence that
suggests an epigenetic contribution to global transcriptional arrest. In
hibernator muscle, the acetylation of histone H3 at lysine residue 23
(H3K23) is known to be significantly reduced during torpor, which is
consistent with the notion of suppressed transcriptional activity at loci
associated with this histone modification (Morin and Storey, 2006).
This change is also correlated with an increase in the expression levels
of several HDACs which target this residue, in addition to levels of total
HDAC activity which was demonstrated as a proof of concept in
Hawkins and Storey (2017) and Morin and Storey (2006). CBP also
targets this residue as a substrate for its acetyltransferase activity
(Henry et al., 2013). Thus, the observed decrease in CBP expression
during late torpor may compliment the increase in HDAC activity to
promote the deacetylation of certain histone residues, and thereby
promote transcriptional suppression. This may also be true for GCN5L2,
the protein levels of which were also significantly reduced during late
torpor in muscle. GCN5L2 targets H3K23 for acetylation (Grant et al.,
1999), so downregulation of GCN5L2 may contribute to the reduced
acetylation of histone H3. Like CBP, GCN5L2 is also a major tran-
scriptional co-activator in a non-epigenetic sense (Nagy and Tora,
2007), so its decreased expression in muscle and liver may also gen-
erally reflect the global suppression of transcription during torpor. In
contrast, the enhanced expression of GCN5L2 at the same time in BAT
could suggest an increase in the transcriptional activation of GCN5L2
target-genes, which could be functional in this tissue given its potential
role in regulating thermogenesis when Tb drops below acceptable limits
during torpor (Boyer and Barnes, 1999), or to initiate arousal (Nizielski
et al., 1989). While the actual functions of these and other observed
changes in KAT protein expression cannot be conclusively determined
by the current study, the fact that such changes do occur is evidence to
suggest that these enzymes likely serve roles in the regulation of the
various processes implicated during hibernation.
3.2. Histone acetyltransferase activity through the torpor-arousal cycle
To further characterize the possible function of KATs in the context
of the hibernator, total HAT activity (HAT enzymatic activity is carried
out by KATs) in the four tissues was compared between euthermic (EC)
and late torpor (LT) stages (Fig. 5). While no change in HAT activity
occurred in liver or muscle between these stages, the adipose tissues
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Journal of Thermal Biology 74 (2018) 71–76
demonstrated significant fluctuations in HAT activity. In BAT, HAT
activity levels doubled during torpor as compared to control, thereby
further supporting a role for increased HAT function in this tissue
during hypometabolism. As mentioned, increased HAT activity in BAT
might reflect the need for this tissue to maintain the expression of
certain pathways involved in the thermoregulatory response, perhaps
via acetylation-induced increases in the transcriptional activation of
specific genes. In contrast, HAT activity in WAT was reduced by half
during torpor, suggesting that a reduction in the function of these en-
zymes is required in this tissue during metabolic depression. Interest-
ingly, the activity fluctuations in both tissues seem to correlate with
some of the observed protein data – in BAT, GCN5L2 levels increase
with total HAT activity during torpor, while levels of HAT1 decrease
with total activity in WAT, possibly supporting the notion that these
increases/decreases in protein levels make an actual contribution to the
total measurable acetyltransferase activity within the tissues. However,
given that these measurements only accounted for total HAT activity –
and not the activity of specific KATs – no conclusions can be made
regarding the relationship between relative changes in the protein ex-
pression of one or two enzymes, and total HAT activity. This concept
also applies to the lack of change in activity observed in muscle and
liver – although protein fluctuations occurred in both tissues, ob-
servable changes in total activity will not necessarily follow, even if the
activities of specific KATs do change. Regardless of the specificity of the
measurements, however, the current data support a role for overall
enhanced HAT activity in BAT and decreased HAT activity in WAT
during torpor, thereby providing further evidence to implicate these
enzymes in the regulation of hibernation.
3.3. Hibernation responsive acetyl-histone levels
Transcriptional control through KAT-mediated acetylation of his-
tone residues has been shown to be integral to the activation of
countless genes and the regulation of crucial cellular processes
(Carrozza et al., 2003; Jenuwein and Allis, 2001; Verdone et al., 2006).
Thus, to more fully characterize KATs in the context of hibernation, we
measured the acetylation levels of KAT-targeted histone H3 lysine 9
(H3K9ac) across the torpor-arousal cycle (Fig. 6). Acetylation of H3K9
is generally associated with active transcription, and multiple KATs
measured in this study (PCAF, GCN5L2 and CBP) target this residue
(Karmodiya et al., 2012). Similar to the measured KAT protein levels
and HAT activities, protein levels of H3K9ac showed tissue-specific
changes, suggesting that transcriptional activation/deactivation of
certain processes by differential histone acetylation may occur at var-
ious points throughout the torpor-arousal cycle. Notably, H3K9ac was
significantly elevated in BAT during torpor, which is consistent with the
transcriptional activation of gene programs regulated by this residue
being a part of this tissue's response to torpor. In fact, Evidence exists to
suggest that the regulation of uncoupling protein-1 (UCP1, the main
protein responsible for uncoupled respiration and non-shivering ther-
mogenesis in the mitochondria of BAT) is controlled by histone H3
acetylation, whereby decreased levels of the modification are asso-
ciated with reductions in ucp1 gene expression (Kiskinis et al., 2007).
Given that UCP1 expression is absolutely integral to the thermo-
regulatory function of BAT (Golozoubova et al., 2001), and is therefore
indispensable for the survival of the hibernating mammal, the tran-
scriptional activation of the ucp1 gene by H3K9 acetylation in BAT
during torpor would be unsurprising. Similarly, the significant fluc-
tuation in H3K9 acetylation between early torpor and interbout arousal
in WAT may reflect general changes in transcriptional activity that
occur over this period (i.e. suppression during the initial metabolic
decline, followed by strong reactivation during arousal). Interestingly,
the results in BAT correlate with the observed increases in HAT activity
and GCN5L2 protein levels that also occur in BAT at this time, possibly
reflecting the expected change in downstream substrate acetylation that
should occur with fluctuations in HAT activity/expression. While the
A.N. Rouble et al.
exact functions of the observed changes in H3K9 acetylation remain
unknown, the current data support the idea that epigenetic mechanisms
likely contribute to the adaptation involved in the torpor-arousal cycle.
3.4. Conclusion
This study serves as the first known investigation to provide sig-
nificant evidence to suggest that the differential expression of KATs
may be characteristic of the hibernation phenotype. Indeed, the results
discussed herein identify fluctuations in the protein levels of four of the
best-studied KATs in the literature, changes in HAT activity, and dif-
ferential acetylation of a downstream KAT histone target, at various
points throughout the torpor-arousal cycle and in a tissue-specific
manner. Some of these changes also appear to correlate. For example,
during torpor in BAT, the increased expression of the major transcrip-
tional co-activator GCN5L2 occurs concurrently with increases in HAT
activity and H3K9 acetylation, all of which are changes that point to-
wards enhanced transcriptional activation via KAT-mediated regula-
tion. While specific impacts on the hibernation phenotype by these
proteins will need to be explored further, these data likely represent
further examples of the widespread function of reversible protein
acetylation in the regulation of diverse cellular processes, and suggests
its importance to hibernator biology.
4. Materials and methods
4.1. Animal care and treatment
Animal experiments were performed as previously described
(McMullen and Hallenbeck, 2010; Rouble et al., 2013) by Dr. J.M.
Hallenbeck and were approved by the Animal Care and Use Committee
of the National Institute of Neurological Disorders and Stroke (NIH;
animal protocol no. ASP 1223– 05). Ictidomys tridecemlineatus were used
in this study and are small mammalian winter hibernators that enter
bouts of torpor lasting days to weeks with periods of rewarming and
arousal that can last ~ 24 h. Animals were sacrificed throughout the
torpor-arousal cycle, and liver, skeletal muscle, BAT, and WAT were
quickly excised and frozen in liquid nitrogen. Animals were sampled at
the following time points: (1) euthermic in the cold room (EC) main-
tained at 4 °C, as previously described (McMullen and Hallenbeck,
2010) – these animals had not entered torpor for at least 72 h and had
Tb of 36–37 °C. (2) Entrance into torpor (EN) – these animals have
shown a decline in Tb (18–31 °C) and have begun to enter torpor. (3)
Early torpor (ET) – animals that are in torpor for 24 h with a constant Tb
of 5–8 °C. (4) Late torpor (LT) – these animals had been in torpor for at
least 5 days with a constant Tb of 5–8 °C. (5) Early arousal (EA) – these
animals show a rising Tb and are sampled when Tb was 9–12 °C. (6)
Interbout arousal (IA) – these animals have arisen from torpor and their
Tb has return to euthermic levels (~ 37 °C) for ~ 18 h. Tissues were
transported on dry ice to Carleton University and stored at − 80 °C until
use.
4.2. Protein extraction and immunoblotting
Total protein extraction and immunoblotting was performed as
previously described (Rouble et al., 2013). Membranes were blocked
with milk (2.5–5%, 20–30 min) or polyvinyl alcohol (1 mg/mL,
30–70 kDa PVA, 45–60 s) in tris-buffered saline with Tween-20 (TBST).
Targets were probed with antibodies for HAT1 (Genetex, GTX110643),
GCN5L2 (Cell Signaling, #3305), PCAF (Cell Signaling, #3378), CBP
(Cell Signaling, #7389), or H3K9ac (Cell Signaling, #9649)
(1:1000–12000 v/v in TBST) overnight at 4 °C. Membranes were then
probed with HRP-conjugated anti-rabbit secondary antibodies (1:1500-
8000 v/v in TBST, 30–60 min) at room temperature and visualized with
chemiluminescence using the ChemiGenius Bio Imaging System (Syn-
gene, Frederick, MD, USA). Membranes were then stained with
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Journal of Thermal Biology 74 (2018) 71–76
Coomassie blue (0.25% w/v Coomassie brilliant blue, 7.5% v/v acetic
acid, 50% methanol) for loading standardization.
4.3. Lysine acetyltransferase activity assay
Total histone acetyltransferase (HAT) activity during EC and LT was
assayed in liver, skeletal muscle, BAT, and WAT total protein extracts
using the EpiQuick HAT Activity/Inhibition Assay Kit (Epigentek, P-
4003) as per the manufacturer's instructions. Samples were prepared as
above with the exception that extracts were not mixed with 2 × SDS
loading buffer. Briefly, in each assay well, 50 µL of 1:50 HAT substrate
(supplied by the manufacturer) was incubated at room temperature for
45 min, which was then aspirated and each well was washed with
150 µL of wash buffer (supplied by the manufacturer) three times.
Then, added to each well was 2 µL of protein extracts, 26 µL of HAT
assay buffer (supplied by the manufacturer), and 2 µL of acetyl CoA
(1:20 v/v from 30 mM stock in HAT assay buffer), which were then
incubated for 60 min at 37 °C. The wells were then washed three times
as above and 50 µL of capture antibody (supplied by the manufacturer)
was added and incubated for 60 min at room temperature on an orbital
shaker. The wells were wash four times, and 50 µL of detection anti-
body (1:1000, supplied by the manufacturer) was added and incubated
for 30 min at room temperature on an orbital shaker. Each well was
then washed five times and 100 µL of developer solution was added to
each well and incubated for 10 min at room temperature on an orbital
shaker in the dark. 50 µL of stop solution was then added to each well
and the absorbance of each well was read at 450 nm using a Powerwave
HT spectrophotometer (BioTek). Three wells with additional assay
buffer instead of protein extracts were run during the assay to act as
negative controls as per the manufacturer's instructions.
4.4. Quantification and statistics
Band densities on chemiluminescent immunoblots were quantified
using GeneTools (Syngene, Frederick, MD). Band densities were stan-
dardized against the summed intensity of Coomassie stained protein
bands in the same lane and then normalized to their respective EC
condition. Data are expressed as mean ± SEM, n = 4. Statistical ana-
lysis of the data was performed by a one-way ANOVA with a Dunnett's
post-hoc test (p < 0.05) to correct for multiple comparisons using
SigmaPlot 12 statistical package software (Systat Software Inc., San
Jose, CA, USA). HAT activity assays were corrected using negative
control wells, data are expressed as mean ± SEM, n = 4 and normal-
ized to the EC samples. Statistical analysis of HAT activity assay results
was performed by Student's t-tests (p < 0.05).
Acknowledgments
The authors thank Dr. J.M. Hallenbeck and Dr. D.C. McMullen
(NINDS, NIH, Bethesda) for providing the tissue samples for this study
and Jan Storey for assistance in the editing of the manuscript.
Funding
This work was supported by a Discovery grant (Grant # 6793) from
the Natural Sciences and Engineering Research Council (NSERC) of
Canada. ANR held a NSERC CGSM Scholarship, and KBS holds the
Canada Research Chair in Molecular Physiology.
Conflicts of interest
The authors report no conflicts of interest.
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