Biosci. Rep. (2010) / 30 / 413–423 (Printed in Great Britain) / doi 10.1042/BSR20090128
Characterization of non-cytosolic hexokinase
activity in white skeletal muscle from goldfish
(Carassius auratus L.) and the effect of cold
acclimation
Reinaldo Sousa DOS SANTOS*, Luan Pereira DINIZ*, Antonio GALINA† and Wagner Seixas DA-SILVA*1
*Laboratory of Bioenergetics, Institute of Medical Biochemistry, Program of Biochemistry and Cellular Biophysics, Federal University of Rio
de Janeiro, Cidade Universitária, Rio de Janeiro, Rio de Janeiro 21941-902, Brazil, and †Laboratory of Bioenergetics and Mitochondrial
Physiology, Institute of Medical Biochemistry, Program of Biochemistry and Cellular Biophysics, Federal University of Rio de Janeiro, Cidade
Universitária, Rio de Janeiro, Rio de Janeiro 21941-902, Brazil
'
$
Synopsis
HK (hexokinase) is an enzyme involved in the first step in the glucose metabolism pathway, converting glucose
into G6P (glucose 6-phosphate). Owing to the importance of skeletal muscle for fish swimming and acclimation
processes, we used goldfish (Carassius auratus L.) white muscle in order to investigate subcellular distribution and
kinetics of HK. In this study, we report that HK activity is predominantly localized in the mitochondrial fraction [NC-HK
(non-cytosolic HK)] in goldfish white muscle. Studies of the kinetic parameters revealed that the Km (Michaelis–
Menten constant) for glucose was 0.41 +
− 0.03 mM and that for mannose was 3-fold lower, whereas the affinity
for fructose was too low to be measured. The Km for ATP was 0.88 +
− 0.05 mM, whereas no activity was observed
when either GTP or ITP was used as a phosphate donor. A moderate inhibition (20–40 %) was found for ADP and
AMP. Similar to mammalian HK, G6P and glucose analogues were able to promote an inhibition of between 85
and 100 % of activity. Here, we found that acclimation of goldfish at 5 ◦ C promoted a 2.5-fold increase in NC-HK
compared with its counterpart acclimated at 25 ◦ C. However, cytosolic HK activity was not altered after thermal
acclimation. In summary, our results suggest that the goldfish has a constitutive NC-HK that shows some similarities
to mammalian HK-II and, curiously, may play a role in the broad metabolic changes required during the cold acclimation
process.
Key words: glycolysis, goldfish, hexokinase (HK), mitochondria, skeletal muscle, thermal acclimation
&
INTRODUCTION
HK (hexokinase) is a ubiquitous enzyme found in all eukaryotic
cells and is also widespread in prokaryotic organisms. This enzyme is responsible for the conversion of glucose into G6P (glucose 6-phosphate) using ATP as a phosphate donor.
Almost all vertebrates possess multiple isoforms of HK. The
major class of HK isoforms is represented in mammals, but several studies have been performed to check whether the same pattern is preserved in other vertebrates. HK-IV (or glucokinase), for
example, is absent in several animals, such as horse, cow, sheep,
llama and goat; HK-III is absent in domestic dog and many
%
rodents [1]. A study performed by Ureta [2] evaluated 97 vertebrate species from all classes and showed that HK-I and -II
isoforms found in some reptile species differ significantly from
those of the other vertebrates, showing low affinity for glucose
and a higher V max for fructose. HK-III was observed only in
some amphibians that lacked HK-I and maybe also in many teleosts. HK-III and -IV isoforms were not seen in 13 avian or 13
reptile species (except for the turtles). For many years, the presence of HK-IV was a matter for debate in fish; however, during
the last decade various studies characterized and identified some
physiological functions of the enzyme, such as regulation by feeding and dietary carbohydrates [3–6]. These observations suggest
that in the course of evolution the HK system was modified in
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Abbreviations used: ψm , mitochondrial membrane potential; CA goldfish, cold-acclimated goldfish; CS, citrate synthase; G6P, glucose 6-phosphate; HK, hexokinase; LDH, lactate
dehydrogenase; MHP, mannoheptulose; mt-HK, mitochondria-bound HK; NAG, N-acetylglucosamine; NC-HK, non-cytosolic HK; PGM, phosphoglucomutase; PK, pyruvate kinase; ROS,
reactive oxygen species; TH, total homogenate; VDAC, voltage-dependent anion channel; WA goldfish, warm-acclimated goldfish.
1 To whom correspondence should be addressed (email [email protected]).
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R.S. dos Santos and others
order to play different and specific roles depending on vertebrate
species.
Four HK isoenzymes are found in mammalian tissues (HKs
I–IV); the isoenzymes differ in kinetic properties as well as in
subcellular and tissue distribution [7–10]. Mammalian HK-I and
-II are monomers of 100 kDa, which have high affinity for glucose
and are strongly modulated by the reaction product, G6P. In addition, Pi , glucose analogues [such as NAG (N-acetylglucosamine)
and MHP (mannoheptulose)], ADP and AMP are able to inhibit
the enzyme [11–13]. Whereas HK-I is ubiquitously and highly
expressed in brain, HK-II is mainly expressed in insulin-sensitive
tissues, such as muscle, mammary gland and adipocytes, and in
many highly glycolytic cancers [10,14,15]. In extracts prepared
from tissues, HK-I and -II activities can be found in soluble and
particulate subcellular fractions [16,17].
Similar to HK-I, HK-II is able to bind to VDACs (voltagedependent anion channels; also known as mitochondrial porins),
which are located in the outer mitochondrial membrane [18,19],
and is therefore also called mt-HK (mitochondria-bound HK).
This interaction has been shown to be physical and functional
[20] and seems to be able to positively modulate HK enzyme
activity [21] by decreasing its sensitivity to G6P [22], protecting it against proteolytic degradation [23] and providing preferred access to mitochondrially generated ATP [24]. Previously,
the association of HK with mitochondria has been suggested
to play an important role in the control of mammalian cell apoptosis, either by the inhibition of Bax-induced cytochrome c
release and apoptosis in the cell line derived from cervical cancer (HeLa) or by the inhibition of early apoptotic events mediated by Akt (also called protein kinase B) activation [25,26].
In addition, our group has shown that brain HK-I performs
a key role in preventive antioxidant defence against oxidative
stress by reducing the generation of mitochondrial ROS (reactive oxygen species) through an ADP-recycling mechanism
[27,28].
Fish HK has been more extensively studied concerning kinetic parameters, expression and tissue distribution. Its activity
has been found and characterized in many fish species and tissues, such as liver, skeletal and cardiac muscles, brain and gills
from rainbow trout, horse mackerel, gilthead sea bream and carp
[3,6,29–34]. In these studies, HK is generally used as a model
for changes induced by a wide range of stresses, such as hypoxia,
exercise, carbohydrate tolerance and temperature.
Cyprinids constitute an important model of study for fish metabolism because of their ability to overcome several kinds of stress,
such as cold acclimation and hypoxia [35–40]. Despite its classic role in glucose metabolism, little is known about other roles
attributed to HK in cyprinids. Previously, the cloning of low-K m
(Michaelis–Menten constant), HK-like cDNAs was reported in
common carp [41]. In a subsequent study, Blin et al. [42] showed
that, as in mammals, the expression of the HK-I gene is ubiquitous
(brain, liver, heart, kidney and muscle), whereas the HK-IV gene
is expressed specifically in the hepatopancreas of common carp
[43]. González-Alvarez et al. [44] confirmed that six DNA sequences denoted as possible HK in the NCBI GenBank® database
are transcribed in zebrafish (DrGLK, DrADPGK1, DrADPGK2,
DrHXK1, DrHXK2 and DrSHXK1). Furthermore, they observed that their tissue distribution is quite similar in zebrafish
and in mammals: DrGLK and DrSHXK1 are expressed in the
fish liver, DrHXK1 in brain and heart and DrHXK2 in muscle.
They also determined that the DrGLK gene is responsive to
fasting and glucose injection. In cyprinid muscle tissue, the presence of HK activity has been reported previously [45–47], but
its subcellular distribution and kinetic characterization were not
investigated.
Freshwater fish are found in environments in which the water
temperature may range from approx. 0–4 ◦ C in winter to 25–30 ◦ C
in summer. Carassius auratus, in particular, exhibits tolerance to
a wide range of temperatures. These animals frequently exhibit a
remarkable maintenance of locomotory activity patterns, which
may be related to thermal responses. The effects of cold acclimation have been investigated in goldfish and carp, mainly
with respect to muscle oxidative capacity [35–38]. Moreover,
modifications were also observed in the activity of HK, but not
in the activity of other glycolytic enzymes [39,40,46]. In these
studies, both red and white muscles from goldfish and carp that
were acclimated to low temperatures showed a compensatory
increase in HK activity. This cold-induced increase in HK activity varied from 1.8- to 4.7-fold depending on the species and
tissue. The experiment was performed using TH (total homogenate), particulate fractions and cytosolic fractions, suggesting a non-specific increase in HK activity at different subcellular sites. Furthermore, a cold-induced increase in HK activity has been observed in various fish species, such as flounder
and striped bass [48, 49]. Similarly to fish, Drosophila, rats
and mice have shown an augmented HK activity in response
to cold stress. However, in Drosophila, which is another ectotherm, HK activity increased only approx. 1.3-fold after the
acclimation period [50]. In rats exposed to low temperatures,
HK activity was 1.15- and 1.17-fold higher in cardiac and gastrocnemius muscles respectively [51]. The exception in mammals
was observed in the thermogenic brown adipose tissue of mice,
where the increase in HK activity was between 1.9- and 2.5-fold
[52,53].
In the present study, we investigated the impact of cold acclimation on the subcellular distribution of HK activity in white
skeletal muscle from goldfish. This subcellular localization has
been described to perform important physiological roles related
to glycolytic flux, apoptosis and control of mitochondrial ROS
in cell metabolism [27,54,55]. Herein, HK activity was found
both in soluble and particulate fractions, and most of the HK
activity was measured in the particulate fraction, which is mainly
associated with mitochondria. Owing to its relevance to metabolism, we focused on the partial kinetic characterization of NC-HK
(non-cytosolic HK), which showed some similarities to mammalian HK. Interestingly, we found that the regulation of goldfish
NC-HK is similar to that of mammalian HK-II, despite some differences. Furthermore, we described, for the first time, the presence of NC-HK in goldfish white muscle with kinetic parameters
similar to mammalian HK-II and its regulation by cold acclimation. This phenomenon was not observed in the soluble form of
the enzyme.
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Non-cytosolic hexokinase in goldfish
MATERIALS AND METHODS
Reagents and materials
were collected for enzyme activity measurements and stored at
–80 ◦ C until assayed.
To obtain mitochondrial-enriched fractions (P100 00) from rat
brain, we used the same protocol as described above.
Unless otherwise specified, all reagents were purchased from
Sigma Chemical Co. (St. Louis, MO, U.S.A.).
Protein determination
Animals
Goldfish (C. auratus L.) were purchased from a local breeder
(Rio de Janeiro, RJ, Brazil), maintained in a glass aquarium with
aerated and dechlorinated water, and fed two to three times a day
with commercial goldfish food pellets. Fish (22.41 +
− 1.26 g and
◦
C
on
a
12
h light/12 h
6.24 +
0.13
cm)
were
maintained
at
25
−
dark cycle and were generally used within one to one-and-a-half
months after arrival. All experimental and holding procedures
were approved by the Ethics Committee of the Carlos Chagas
Filho Institute of Biophysics, Health Sciences Centre, Federal
University of Rio de Janeiro (Protocol No. IBQM028).
Temperature acclimation and rates of opercular
movements
Goldfish were kept at least for 3 days in the laboratory before
starting the experiment in order to minimize the potential stress
caused by transportation. Subsequently, they were divided into
two groups of five to six fish each and transferred to a new
aquarium. One group [WA (warm-acclimated)] was kept at 25 ◦ C
◦
(+
− 0.5 C), and the other group [CA (cold-acclimated)] was subjected to an acclimation process using a refrigeration unit (347
CDG; Fanem, São Paulo, Brazil). For the CA group, the temperature of the refrigeration unit was gradually reduced 3 ◦ C per
◦
day for 1 week until it reached 5 ◦ C ( +
− 0.5 C), and the animals
were kept at this condition for four additional weeks. To measure the opercular movements, 12 individuals were placed in an
aquarium immersed in a thermostatically controlled bath at each
experimental temperature and visual recordings were made for
1 min (opercular movement was measured in beats/min).
Isolation of skeletal muscle subcellular fractions
After the acclimation period, animals were killed by decapitation.
Skeletal muscle (2–3 g) was removed immediately, cut into small
pieces and then minced six times (10 s each) in a IKA-Ultra Turrax T25 homogenizer (IKA-Works, Wilmington, NC, U.S.A.) on
ice at a 1:10 (w/v) dilution in an ice-cold isolation buffer containing 10 mM Tris/HCl buffer (pH 7.4), 250 mM sucrose, 1 mM
EDTA, 4 mM 2-mercaptoethanol, 1.5 mM KCl and 0.5 mM
PMSF. Tissue was homogenized in a Teflon glass Potter homogenizer, and TH was centrifuged at 1000 g for 15 min. The
supernatant (S1000) was carefully removed, strained through
four layers of cheesecloth and then centrifuged at 10 000 g for
15 min at 4 ◦ C. The supernatant (S100 00) was centrifuged at
100 000 g for 30 min, and the resultant supernatant (S1000 00)
was collected. The pellets obtained at each centrifugation step
were resuspended in isolation buffer and named P1000, P100 00
and P1000 00 respectively. Aliquots from all centrifugation steps
The protein concentrations of the subcellular fractions and isolated mitochondria were determined by the Folin–Lowry method
using BSA as a standard [56].
Enzyme activity assays
All enzyme activity assays were conducted at 25 ◦ C, except for
that of rat brain HK activity, which was measured at 37 ◦ C. HK
activity was determined by following NADH formation at 340 nm
according to a previously described protocol [57] with slight
modifications. The reaction medium contained 75 mM Tris/HCl
(pH 7.5), 7.5 mM MgCl2 , 0.8 mM EDTA, 1.5 mM KCl, 2.5 mM
ATP, 4 mM 2-mercaptoethanol, 0.1 % Triton X-100, 0.5 mM βNAD+ , 5 mM glucose and 0.5 unit/ml glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides. The reaction was
started by adding protein sample. For the measurement of kinetic
parameters, the substrate concentrations were varied from 0 to
100 mM for glucose, fructose or mannose and from 0 to 3 mM
for ATP, GTP or ITP. Inhibition kinetics were performed, varying
the concentration of ADP or AMP (0–3 mM), G6P (0–5 mM),
Pi (0–10 mM), NAG (0–100 mM) and MHP (0–100 mM). When
fructose was used as a substrate, 1 unit/ml phosphoglucose isomerase were used, and when mannose was used as a substrate,
2 units/ml phosphomannose isomerase plus 1 unit/ml phosphoglucose isomerase was used. For NTP kinetic measurements,
10 mM phosphocreatine and 1 unit/ml creatine kinase were added to the reaction medium as an NTP regenerator system in
order to maintain the concentrations of the triphosphate nucleotides used as substrates.
The G6P effect was assessed by a radiometric assay using
[14 C1 ]mannose as a substrate as previously described [58] with
slight modifications. The reaction medium used contained 75 mM
Tris/HCl (pH 7.5), 7.5 mM MgCl2 , 0.8 mM EDTA, 1.5 mM
KCl, 2.5 mM ATP, 4 mM 2-mercaptoethanol, 0.1 % Triton
X-100, 5 mM NaF, 5 mM NaN3 and 2 mM [14 C1 ]mannose (18.5
Bq/nmol; Amersham Pharmacia Biotech). The reaction was started by the addition of the mitochondrial fraction derived from
goldfish white muscle and was incubated for 30 min at 25 ◦ C in
a final volume of 30 μl. The reaction was then stopped by the
addition of 30 μl of cold ethanol. An aliquot of 25 μl of the final
sample was spotted on a 2.4 cm Whatman DE 81 ion-exchange
disc (Whatman, Maidstone, Kent, U.K.). The discs were allowed
to dry and were then mounted in a vacuum manifold and washed
eight times with 15 ml of distilled water. The amount of phosphorylated product adsorbed on the discs was then determined
by liquid-scintillation counting. A preparation of rat brain mitochondria was used as a positive control for the measurement of
G6P inhibition, but in this case, the reaction was incubated at
37 ◦ C.
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R.S. dos Santos and others
Kinetic constants were calculated by nonlinear regression analysis applied to the Michaelis–Menten equation using PRISM
software (GraphPad Software, San Diego, CA, U.S.A.). According to previous work [40], goldfish HK is very sensitive, and its
activity is lost after a 24 h storage period. Thus all HK assays
were measured within 12 h of fraction preparation.
LDH (lactate dehydrogenase), PGM (phosphoglucomutase)
and PK (pyruvate kinase) activities were measured as described
previously [34]. CS (citrate synthase) activity was measured by
using a previously described method [59]. Mitochondrial ATPase
activity was measured in the absence and presence of 5 mM NaN3
in a reaction medium containing 50 mM Mops/Tris (pH 7.0),
4 mM MgCl2 and 1 mM ATP. The difference between these activities is referred to as ATPase azide-sensitive and is related to
mitochondrial Fo F1 -ATPase. The amount of Pi derived from ATP
hydrolysis was determined by a colorimetric method [60].
Statistical analysis
All data were analysed using PRISM software (GraphPad Software) and are expressed as means +
− S.E.M. The Student’s t test
was used to compare the differences between two groups, whereas
the one-way ANOVA test, with the Newman–Keuls test as a posttest, was used to compare the differences between more than two
groups. P < 0.05 was used to reject the null hypothesis.
RESULTS
Variation of opercular movement rate during
temperature acclimation
In order to validate our experimental protocol to acclimation,
we measured the effect of temperature over the opercular movement rate (or ventilation rate), which was performed by counting
operculum opening during a specific period of time. Despite
an indirect measurement, this rate is a useful tool to determine whether our experimental protocol is efficient. We observed
that ventilation rate decreased during temperature lowering and
reached its lowest level at approximately the 10th day, when water temperature was 5 ◦ C. However, this rate slightly rose after
some days and remained the same until the end of the acclimation period, suggesting a partial compensation. It is noteworthy
that fish maintained at 25 ◦ C had no changes in ventilation rate
during the acclimation period (Figure 1). Thus we suggest that
our experimental protocol is useful to determine changes caused
by cold acclimation.
Intracellular distribution of HK activity in WA
goldfish white skeletal muscle
To address the question of the subcellular distribution of HK
activity in white skeletal muscle from goldfish, we submitted
the tissue homogenate to differential centrifugation in order
to obtain distinct subcellular fractions. The total HK activity
Figure 1 Rates of opercular movements at various acclimation
temperatures
The control group (䊉) was evaluated only at 25 ◦ C during all the acclimation period and the group submitted to cold acclimation (䊊) was
evaluated at different temperatures (25, 22, 19, 16, 13, 10, 7 and
5 ◦ C) during the first week. After that, the temperature was constant
at 5 ◦ C and measurements were made at 3 day intervals. The broken
line indicates when the temperature was constant at 5 ◦ C. Values are
the means +
− S.E.M. for 12 fish per experimental group. **P < 0.001
compared with the same group on the 10th and 13th days.
of goldfish white muscle was found to be equally distributed
between the cytosolic- and mitochondrial-enriched fractions,
with only a minor contribution from the microsomal fraction (Figure 2A). Even though total HK activity was basically the same
in all subcellular fractions, higher specific activities were observed in the mitochondrial- and microsomal-enriched fractions
(Figure 2B).
Since the association between HK and mitochondria has been
proposed as a physiological mechanism to regulate metabolism
and biological processes, we decided to characterize the kinetic properties of only the NC-HK in a mitochondrial-enriched
fraction, since it was the fraction with the highest specific activity. Because the lability described for this enzyme prohibited us
from purifying it, we then decided to work with a mitochondrialenriched fraction, a system that has been validated by other authors [16,17,34].
Kinetic properties of goldfish NC-HK
We measured the kinetic constants for hexose substrates and
NTP in NC-HK from goldfish white muscle (Figure 3 and
Table 1). As expected, a higher V max for NC-HK was found
with glucose as a substrate, whereas mannose showed a lower
−1
−1
V max (glucose: 34 +
− 1 nmol · mg · min ; mannose: 22 +
−1
−1
−1
nmol · mg · min ; P = 0.0001). Curiously, fructose was not
an efficient substrate for NC-HK up to 50 mM (Figure 3A), which
differs from the HK activity in mammalian tissues [7,11,61]. The
analysis of kinetic parameters for NTPs showed that the V max obtained for ATP was similar to that found for glucose as a hexose
−1
−1
substrate (34 +
− 1 nmol · mg · min , Figure 3B and Table 1),
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Figure 2
Subcellular distribution of HK activity in goldfish white muscle
(A) Total and (B) specific activities in different subcellular fractions from goldfish. White muscle subcellular fractions are:
TH; pellet (P1000) and supernatant (S1000) from centrifugation at 1000 g; pellet (P10000) and supernatant (S100 00)
from centrifugation at 100 00 g; pellet (P1000 00) and supernatant (S1000 00) from centrifugation at 1000 00 g. Values
are means +
− S.E.M. for nine measurements (three different preparations).
Table 1 Kinetic constants for goldfish NC-HK activity
The Km values for hexoses were measured with 2.5 mM ATP, whereas
the Km values for NTPs were measured with 5 mM glucose. For assay
conditions, see the Materials and methods section. Values are the
means +
− S.E.M. for four to five measurements (four different preparations). ***P < 0.001 compared with glucose. In this Table, V max is
given in units of nmol of hexose 6-phosphate/mg per min. Km is given
in units of mM. Abbreviations: ND, not determined; NA, no activity
detected.
Substrates
V max
Km
V max /K m
Glucose
34 +
−1
0.43 +
− 0.03
79
Fructose
ND
ND
ND
Mannose
22 +
− 1***
39 +
−3
0.14 +
− 0.02
0.88 +
− 0.05
GTP
NA
NA
NA
ITP
NA
NA
NA
ATP
157
44
but no HK activity was detected when GTP and ITP were tested
as phosphate donors. Thus it seems that NC-HK utilizes only
ATP, but not GTP or ITP, as a phosphate donor (Figure 3B).
The apparent K m values for glucose, mannose and ATP were
calculated and are shown in Table 1. Because NC-HK did not
reach maximal activity with fructose as a substrate and had no
activity with GTP and ITP as phosphate donors, the apparent
K m values for these substrates were not determined. As shown
in Table 1, NC-HK from goldfish white muscle has a higher affinity for mannose than for glucose (glucose: 0.43 +
− 0.03 mM;
mannose: 0.14 +
0.02
mM;
P
<
0.001),
indicating
that
this isoen−
zyme preferentially phosphorylates mannose. This fact may also
be observed through analysis of the V max /K m ratio, which indicates catalytic efficiency. Thus it was found that NC-HK exhibited
a catalytic efficiency for mannose that was 2-fold higher than that
for glucose (Table 1).
For ATP, which was found to be the unique phosphate donor
utilized by NC-HK in goldfish, the K m was similar to that
found for mammalian HK-II (NC-HK goldfish white muscle:
0.88 +
− 0.05 mM; mammalian HK-II: 0.7 mM; [14]).
Effect of ADP, AMP and Pi on goldfish NC-HK
activity
We next tested the effects of ADP, AMP and Pi on NC-HK
activity. These molecules are known to have an inhibitory effect on mammalian HK-II activity in the millimolar range
[10,13,14,62,63]. However, the effects of these inhibitors were
less pronounced on goldfish NC-HK activity (Table 2). We found
that ADP and AMP (both at 3 mM) inhibited NC-HK activity by
23 and 41 % respectively (Table 2). In addition, the highest Pi
concentration tested (10 mM) was only able to inhibit 12 % of
the NC-HK activity (Table 2). These results suggest that NC-HK
from goldfish white muscle is less sensitive to possible allosteric
inhibition by ADP, AMP and Pi when compared with HK-II from
mammals.
Effect of G6P on goldfish NC-HK activity
Since strong inhibition by their product, G6P (K i = 2.0 × 10−5
M), is often said to be a striking property of the glycolytic mammalian HK-I and -II, we investigated whether G6P would also
be able to inhibit goldfish NC-HK. Because HK activity is usually measured by an enzyme-coupled assay, which detects the
G6P formed, addition of exogenous G6P prohibits the use of this
approach. Thus, in order to address this question, we used an alternative methodology (a radiometric assay) and exploited the capacity of goldfish NC-HK to also phosphorylate [14 C1 ]mannose.
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Figure 3
Kinetic parameters of NC-HK activity in the mitochondrial fraction
(A) Hexose substrates: glucose (䊉), mannose (䊊) and fructose (䉱). (B) NTP: ATP (䊏), GTP (䊐) and ITP (). Four different
preparations (4–5 measurements) were evaluated. (A, B) Only the mean values are shown.
Table 2 Inhibitory profile of goldfish NC-HK activity
For ADP, AMP, Pi and G6P, the inhibitory effect was measured 4–9
times (3 or 4 different preparations). For NAG and MHP, the inhibitory effect was measured three times (one preparation). The inhibition
was the maximal inhibitory effect observed. *P < 0.05 compared with
100 % activity (nmol of G6P/mg per min, measured in the absence of
inhibitor).
Inhibitors
dependent manner (Figure 4B). At 100 mM of glucose analogues,
NC-HK activity was inhibited by 88 % (NAG) and 100 % (MHP)
respectively (Table 2). Thus, like mammalian HK, goldfish NCHK activity is inhibited by glucose analogues, such as NAG and
MHP.
Inhibition (%)
ADP
23
AMP
41*
Pi
12
MHP
100*
NAG
88*
G6P
86*
Goldfish NC-HK was greatly affected by the addition of G6P
(Figure 4A, closed symbols). The IC50 calculated for the reaction was estimated to be 38 μM. This result was similar to that
observed for rat brain mitochondrial HK, which is known to be
inhibited by G6P (Figure 4A, open symbols). Similar concentrations of G6P (up to 5 mM) were able to almost completely
inhibit both HK activities (see Table 2 for goldfish data). The
result observed with rat brain HK is in accordance with results from other previously described studies (for reviews, see
[14,64]).
Effect of glucose analogues NAG and MHP on
goldfish NC-HK activity
In mammals, it is well known that glucose analogues competitively inhibit HK activity with respect to glucose [11,12]. Here,
we tested the effect of two glucose-analogue HK inhibitors, NAG
and MHP, on NC-HK activity. With a fixed amount of glucose
(5 mM), NAG and MHP inhibited NC-HK activity in a dose-
Effect of cold acclimation on HK activity in goldfish
white skeletal muscle
Cold acclimation induces several changes in goldfish white
muscle metabolism. Previously, it has been shown that HK activity increases after thermal acclimation [40]. However, this activity
was investigated using a supernatant obtained from centrifugation at 1000 g, which contains both soluble and particulate fractions; therefore the specificity of the subcellular effect was not
examined. Thus we investigated the subcellular distribution of
HK activity after 1 month of cold acclimation at 5 ◦ C. After the
acclimation period, subcellular fractions from CA and WA goldfish skeletal muscle were prepared and assayed for HK activity
(Figure 5). CA goldfish showed a subcellular localization profile
similar to that found for WA in both total and specific HK activities. Nevertheless, as previously seen [40,46], cold acclimation
promoted an increase in HK activity in both the TH (2.6-fold) and
the supernatant obtained from centrifugation at 1000 g (3.8-fold).
Interestingly, we found that this increase in HK activity occurred
only in the particulate fraction, i.e. mitochondria and microsome,
with no changes in the cytosolic fraction (Figure 5A). Curiously,
the highest increase was observed in the microsomal fraction
(7.6-fold) in CA goldfish compared with WA goldfish, whereas
the mitochondrial fraction was 2.8 times higher. Likewise, specific activities were higher in CA goldfish, following the same
profile exhibited in total HK activity (Figure 5B). Mitochondrial
and microsomal fractions increased by 2- and 3.8-fold respectively. Once again, the specific activity of HK in the cytosolic
fraction did not change in CA goldfish.
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Figure 4
Effect of G6P and glucose analogues on HK activity
(A) Effect of G6P on goldfish white muscle NC-HK (䊉) and rat brain HK-I (䊊). (B) Glucose analogues: NAG (䉱) and
MHP (). Values are means +
− S.E.M. for goldfish G6P experiments (four measurements in three different preparations),
+
means +
− S.D. for rat brain G6P (three measurements in one rat preparation) and means − S.D. for NAG and MHP goldfish
experiments (three measurements in one fish preparation).
Figure 5
Effect of cold acclimation on the subcellular distribution of HK activity in goldfish white skeletal muscle
(A) Total and (B) specific activities in different subcellular fractions from goldfish. Fractions are the same as in Figure 2.
White bars represent WA goldfish, and grey bars represent CA goldfish. Values are means +
− S.E.M. for nine measurements
(three different preparations per group). # P < 0.05, *P < 0.01, **P < 0.001 compared with the WA group.
Effect of cold acclimation on the activity of other
enzymes
As described above, cold acclimation is able to alter the activity
of several enzymes involved in goldfish muscle metabolism, and
these effects are mainly associated with mitochondrial oxidative
metabolism, whereas glycolysis remains unchanged [39,40]. Because HK activity was increased in the particulate fraction but
not in the cytosolic fraction, we tested whether this subcellular
distribution occurred only for HK or whether it could also be
observed for other enzymes associated with specific metabolic
pathways. In order to investigate these points, we measured the
activity of other cytosolic and mitochondrial marker enzymes:
PGM, LDH, PK, Fo F1 -ATPase and CS. As shown in Table 3,
cold acclimation had no effect on the specific activities of PGM,
LDH and PK in both the mitochondrial-enriched and cytosolic
fractions. In contrast, as seen before, mitochondria-associated
HK activity was increased 2-fold in CA fish compared with WA
fish, whereas cytosolic HK activity was the same under both conditions. Moreover, a similar increase to that observed for HK was
also found for the mitochondria-marker enzymes, CS and Fo F1 ATPase, in the mitochondrial fraction, but not in the cytosolic
fraction. It is worth noting that cytosolic-marker enzymes had
low specific activities in the mitochondrial fraction, indicating a
low level of contamination. Low specific activities were observed
for CS and Fo F1 -ATPase in the cytosolic fraction because they
are typical enzymes from mitochondria.
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R.S. dos Santos and others
Table 3 Specific activities of mitochondrial and cytosolic
enzymes in WA and CA goldfish
Specific activities were measured as described in the Materials and
methods section and are expressed as nmol of product/mg per
min. Mitochondrial and cytosolic fractions correspond to P10000 and
S100000 respectively. Values are the means +
− S.E.M. for 4–5 measurements (one to three different preparations per group). *P < 0.05;
†P < 0.01 compared with the WA group. ND, not detected.
Subcellular fractions
Enzymes
Conditions
HK
WA
CA
PGM
WA
LDH
WA
PK
WA
CA
CA
CA
Fo F1 -ATPase
WA
CS
WA
CA
CA
Mitochondrial
fraction
Cytosolic
fraction
41 +
− 10
83 +
− 6*
58 +
− 29
100 +
− 39
289 +
− 80
390 +
− 80
319 +
− 55
410 +
− 111
148 +
− 23
365 +
− 54†
22 +
−6
55 +
− 5†
2+
−1
3+
−1
1030 +
− 70
1100 +
− 70
4652 +
− 404
4773 +
− 345
2328 +
− 88
2422 +
− 316
33 +
− 14
21 +
− 14
ND
ND
DISCUSSION
It was suggested that the glycolytic flux in muscle in goldfish
is much lower than in other fish species owing to lower enzyme
activities, including HK, phosphofructokinase I and PK, in this
tissue [40]. In this same study, the researchers demonstrated that
HK activity from goldfish acclimated to 15 ◦ C compensated positively when compared with goldfish acclimated to 30 ◦ C. The
same effect was observed previously [46] using a TH from white
muscle.
In the literature, HK activity in goldfish muscle is usually
measured using either a TH or a supernatant from centrifugation
at 1000 g, which contains several subcellular fractions such as
the mitochondria, microsome and cytosol. This is not a problem
since there is a classical concept of the subcellular distribution
of glycolytic enzymes, and many authors assume that they have
a soluble localization. Thus the analysis of the subcellular distribution of HK becomes important since this enzyme can be
found to be either associated with different organelles or soluble in the cytosol [10]. More recently, several new functions
have been attributed to this enzyme, such as mediation of apoptosis [25,26], ROS production [27,28] and plant sugar sensing
[65].
Herein, we describe for the first time some characteristics
of the subcellular localization of HK in goldfish white muscle.
Moreover, kinetics and inhibition parameters for NC-HK were
measured as well as the thermal acclimation effect on HK subcellular distribution. We found that HK activity is mainly located in
particulate fractions, and it seems to play a role in the biochemical
changes that occur during cold acclimation.
Subcellular distribution analysis showed that, although total
activity is well distributed over all subcellular compartments,
HK specific activity is higher in fractions in which it may be
anchored, such as in mitochondria and microsome fractions (Figure 2). These results are interesting since HK is classically known
as a ‘cytosolic enzyme’ and, unlike this title, HK specific activity
in the cytosolic fraction was very low. Because the association
between HK and mitochondria may play a role in cell metabolism, we decided to investigate the HK activity found in the
mitochondrially-enriched fraction, which was defined here as
NC-HK.
The kinetic properties investigated for this enzyme are in
agreement with those observed for HK-II from rat and human
skeletal muscles [12,66], suggesting that the isoform studied
here could be a type II-like HK. Indeed, the results obtained
by González-Alvarez et al. [44] in zebrafish suggest that HK distribution in cyprinids is similar to that in mammals, which may
support our speculation about the identity of NC-HK found in
goldfish white skeletal muscle. However, two of our results for
NC-HK were in disagreement with those for mammalian HK-II:
we found a markedly low affinity for fructose (no activity up to
50 mM) and a weaker inhibition by ADP, AMP and Pi in our
present study of NC-HK. Among mammalian HK isoenzymes, a
high K m for fructose is characteristic of HK-IV [67]. However,
until now, we did not know whether this low affinity for fructose
has any physiological role in goldfish white muscle metabolism.
Concerning ADP, AMP and Pi inhibition, the lack of regulation
indicates that goldfish NC-HK is not allosterically modulated by
these molecules.
Cold acclimation induces several metabolic responses in goldfish muscle, including an increase in mitochondrial volume density and mitochondrial enzyme activity [35,46]. As described
previously [40,46], we also found that CS and Fo F1 -ATPase
were increased in the mitochondrial fraction from CA goldfish white muscle in a similar proportion, suggesting a higher
mitochondrial abundance after cold acclimation. In addition,
HK activity followed this pattern, and cold acclimation was
able to promote a compartment-specific HK activity increase,
in which only the particulate fractions (mitochondria and microsomes) were augmented by thermal acclimation (Figure 5 and
Table 3).
A couple of questions might raise about a role for NC-HK during the cold acclimation process in goldfish muscle; the increase
in unsaturations that accompanies thermal acclimation in some
fish species renders mitochondrial membranes more susceptible
to peroxidation by ROS. In addition, the higher oxidative capacity
in CA fish could enhance ROS formation inside the mitochondrial matrix, since ROS generation is highest when the magnitude of the ψm (mitochondrial membrane potential) is also
high [68]. Therefore it is probable that ROS production might be
elevated in CA cyprinids, since these fish exhibit both high oxidative capacity and changes in phospholipid polyunsaturation after
cold acclimation [36,38]. da-Silva et al. [27] proposed a mechanism for reducing mitochondrial ROS formation based on the
association between HK and mitochondria: the mt-HK–VDAC–
ANT (adenine nucleotide translocator) complex, together with
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Non-cytosolic hexokinase in goldfish
Fo F1 -ATPase, would represent the core of an ADP recycling
system that uses glucose and ATP as fuels and whose function is
to maintain low ψm and prevent mitochondrial ROS generation.
Thus it is reasonable to think that HK activity might play a crucial
role during higher mitochondrial respiration in CA goldfish, acting as a preventive antioxidant defence, a hypothesis that needs
to be tested. This hypothesis is supported by the specific increase
in HK activity in the particulate fraction, but not in the cytosolic fraction, after cold acclimation. Furthermore, as stated above,
ROS generation by CA mitochondria is thought to be higher than
in its WA counterpart.
Although we do not know the mechanisms underlying these
processes, they may be related to an increase in HK binding to
mitochondria, following higher mitochondrial abundance, rather
than a transcriptional effect on HK expression after cold acclimation. In addition, further studies are needed to investigate whether the association between HK and mitochondria can
also be modulated by G6P or clotrimazole, as in mammals
[69,70].
In conclusion, we showed for the first time that HK activity
in goldfish white muscle is mainly found in particulate fractions rather than cytosolic fractions. The analyses of kinetic and
inhibitory parameters indicate that NC-HK activity in goldfish
white muscle, which is present in the mitochondrially-enriched
fraction, is quite similar to type II HK expressed in mammalian
skeletal muscle. Furthermore, our results suggest that cold acclimation promotes an increase in mitochondrial enzyme activity
(CS and Fo F1 -ATPase), which may be related to a higher mitochondrial density volume after cold exposure. Curiously, HK
activity was elevated only in the particulate fraction, but not in the
cytosolic compartment, indicating that cold acclimation causes
a particular subcellular effect on HK activity. We speculate that
an association between NC-HK and mitochondria, as well as its
increase in specific activity, may play an important role during
the acclimation process, but further studies are required in order
to elucidate the physiological importance of this association.
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ACKNOWLEDGMENT
We are grateful to Leopoldo de Meis for the laboratory facilities.
14
15
FUNDING
This study was supported by grants from the PRONEX [Programa de
Núcleos de Excelência; grant no. E-26/171.525/2006]; Fundação
Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de
Janeiro [grant no. E26/170.615/2007]; MCT/CNPq [Ministério
da Ciência e Tecnologia/Conselho Nacional de Desenvolvimento
Cientı́fico e Tecnológico; grant no. 485372/2007-0]; and Pew Charitable Trusts Foundation. R.S.S. is the recipient of a fellowship from
the CAPES (Fundação Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior) of Brazil. L.P.D. is the recipient of a fellowship from the FAPERJ (Fundação Carlos Chagas Filho de Amparo à
Pesquisa do Estado do Rio de Janeiro).
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Received 16 October 2009/4 January 2010; accepted 7 January 2010
Published as Immediate Publication 7 January 2010, doi 10.1042/BSR20090128
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