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l0l5-8987/9l/0014-0226$2.75/0
Cell Physiol Biochem 1991;1:226-236
Physiological Changes due to Cold Adaptation in Rat Liver
Susanna Iossa, Giovanna Liverini, Antonio Barletta
Department of General and Environmental Physiology, University of Naples, Italy
Key Words. Hepatocytes • Mitochondria • Respiration • Oxygen consumption •
Cold adaptation
Introduction
When mammals are exposed to cold, they
maintain their body temperature by faculta­
tive heat production, due partly to muscle
activity of shivering and partly to other met­
abolic processes [1,2] which gradually re­
place shivering [3. 4], Several works have
been carried out to establish both the tissues
and the mechanisms involved in these pro­
cesses. It is well known that brown adipose
tissue (BAT) is involved in cold adaptation
processes by direct heat production [5-10],
and that the ability of BAT mitochondria to
produce heat depends on an uncoupling pro­
tein uniquely found in BAT: thermogenin [5,
11]. Recent estimate of the contribution by
BAT to increased metabolic rate is of about
40-50% [12], when the tissue is fully acti­
vated by exposure of the animal to cold [13],
It follows, of course, that if BAT accounts for
the above percentage in cold acclimated rats,
other tissues must be involved in the in­
creased metabolic rate.
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Abstract. The study shows that in rats acclimated to 4 °C for 15 days the following param­
eters related to gram wet liver significantly changed in respect to control rats (24 °C). The
respiration rates of isolated hepatocytes without added substrates, or with hexanoate, or
hexanoate + lactate, or hexanoate + oligomycin as substrates increased (+75, +73, +55, and
+ 44%, respectively). Similarly, an increase in mitochondrial mass (+44%), as well as in state
3 oxidative rates in isolated mitochondria using glutamate, or pyruvate + malate, or palmitoyl carnitine + malate, or succinate as substrates (+200, + 112, + 109. and + 33%, respective­
ly), was observed. Moreover, our results show that Na pumping activity significantly
increased in hepatocytes from cold acclimated rats. Since total liver mass increased in cold
acclimated rats, all the above increments gained greater importance. These findings clarify
the importance of the liver in the metabolic adjustments in the cold acclimated state.
Cold Adaptation in Rat Liver
Material and Methods
Animals. Male Wistar rats of about 200 g were
divided into two groups. One group of rats was placed
into a cold room (4 ± 1 °C) for 15 days (denoted
cold-acclimated rats). The rats in the other group
were kept at a temperature of 24 °C (denoted control
rats). All rats were kept 1 per cage under an artificial
circadian 12:12 light-dark cycle, and were fed ad libi­
tum.
Mitochondrial Isolation and Incubation. Rats were
killed by decapitation between 08.30 and 09.30 h,
without any previous food deprivation, to avoid a fur­
ther stress to cold exposed rats [23], Livers were
quickly removed and weighed. All subsequent opera­
tions were done at ice-melting temperature and using
sterilized solutions and glassware. The livers were
finely minced and washed with a medium containing
220 mM mannitol, 70 mM sucrose, 20 m M Tris, pH
7.4, 1 m M EDTA, and 1% fatty acid free bovine
serum albumin. Tissue fragments were gently homog­
enized with the same medium (1:10. w/v), in a Potter
Elvehjem homogenizer set at 500 rpm (4 strokes/
min). The homogenate was filtered through sterile
gauze and freed of debris and nuclei by centrifugation
at ! ,000 gav for 10 min; the resulting supernatant was
then centrifuged at 3,000 gav for 10 min, the mito­
chondrial pellet was washed twice and finally resus­
pended in a medium containing 80 mM KC1. 50 mM
Hepes. pH 7.0, 5 mM KH;POj. Enzymic and electron
microscopy characterization of isolated mitochondria
has shown that they are virtually pure [24], The mito­
chondrial protein content was determined by the
method of Hartree [25], using bovine serum albumin
as the protein standard, while succinic dehydrogenase
activity was measured at 30 °C by the method of Lee
and Lardy [26], Mitochondrial oxygen consumption
was measured polarographically, with a Clark type
electrode (Yellow Springs Instruments. Yellow
Springs, Ohio, USA), maintained in a chamber at
30 °C, using the above suspension medium supple­
mented with 1% fatty acid free bovine serum albumin
as the incubation medium. Measurements were made
within 2 h. following the isolation of the mitochon­
dria. The mitochondria were allowed to oxidize their
endogenous substrates for a few minutes. Substrate
was then added to determine state 4 oxygen consump­
tion rate. Six minutes later, ADP (at a final concentra­
tion of 0.3 m M ) was added and state 3 rate was mea­
sured. The ratio between state 3 and 4 (RCR) and
ADP/O ratios were calculated according to Estabrook
[27] , Concentrations of the various substrates used
are noted in the tables. ATP production rate during
state 3 respiration was measured monitoring the dis­
appearance of inorganic phosphorus in the above
incubation medium in the presence of 5 mM ADP
[ 28] ,
Preparation and Incubation o f Liver Cells. Rat
liver cells were prepared as described by Seglen [29],
except that the rat was anesthetized by the intraperi­
toneal administration of chloral hydrate (40 mg/100 g
body weight). The hepatocytes were washed and sus­
pended in a medium containing 120 mMNaCI, 5 mM
KC1, 50 mM Hepes. pH 7.4, 1 mM KH: P 0 4, 2 mM
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Although there are some reports [14-17],
little is known on the role played by other
tissues in the adaptation to cold, and the
molecular mechanism by which this role
could be carried out is also poorly under­
stood. An involvement of the rat liver in cold
adaptation has been previously suggested by
Stoner [18], Maekubo et al. [19] and Guern­
sey and Stevens [20], Moreover, our pre­
vious results have shown that notable mor­
phological [21] and functional [22] modifica­
tions occur in the liver mitochondrial com­
partment from rats exposed to cold. Since
the mitochondria are the principal cellular
sites of metabolic energy production, we
thought it would be interesting to investigate
their involvement in the acclimation to cold.
To this purpose we have measured, in cold
acclimated rats, the respiration of isolated
hepatocytes together with the oxygen con­
sumption and ATP production of isolated
mitochondria under different conditions of
stimulation of mitochondrial respiration;
moreover, we have correlated these parame­
ters to the liver mitochondrial protein con­
tent. The results obtained are clearly indica­
tive of the involvement of the liver in the
cold acclimation processes.
227
228
Iossa/Liverini/Barletta
Table 1. Body mass and hepatic parameters in control and colc-adapted rats
Body mass, g
Liver, g wet weight/anima!
Liver, g wet weight/100 g body weight
Number of hepatocytes per g wet weight
Cold-adapted rats
247+18
8.4±0.6
3.4 + 0.2
150± 5X 106
251+20
11.0 ±0.9*
4.4 + 0.3*
I48± 4X 106
The values are the means ± SEM of 10 different experiments.
p < 0.05 versus 0.
CaCL, 1.2 mM MgS04, 2% fatty acid free bovine
serum albumin. The hepatocytes were routinely 90%
viable, as determined by trypan blue exclusion.
Hepatocyte oxygen consumption was measured
polarographically, with a Clark type electrode main­
tained in a chamber at 37 °C. Aliquots corresponding
to about 106 viable cells were incubated in the above
suspension buffer with different suostrates. at the
concentrations reported in the tables.
Materials. Collagénase (type IV), ADP, rotenone,
succinate, malate, pyruvate, palmitoyl carnitine, glu­
tamate, ATP, oligomycin, ouabain, lactate, hexanoate, were purchased from Sigma Chemical Co., St.
Louis, USA. All other reagents used were of the high­
est purity commercially available.
Statistics. Data are given as means ± SEM. Anal­
ysis of variance was used to determine significant dif­
ferences among means, while differences between in­
dividual means were examined by the two-tailed Stu­
dent’s t test, when a significant F value was obtained.
For derived parameters, the Gaussian law of error
propagation was applied.
Results
Body Weight and Some Hepatic
Parameters in Control and
Cold-Acclimated Rats
Table 1 presents the body and liver mass
and hepatocyte number per gram wet weight
in control and cold-acclimated animals. No
difference in body weight was observed at
the end of the experimental period between
control and 15-day cold-exposed rats, indi­
cating that the latter thrived in the cold, and
could be considered cold-acclimated. We
also observed that cold exposure doubled rat
food intake, in agreement with previous sim­
ilar observations [8, 30]. On the other hand,
significant variations in liver weight oc­
curred; in fact, liver weight, expressed per
animal or per 100 gram body weight, was sig­
nificantly higher (+31 and + 29%, respective­
ly) in cold-acclimated rats than in control
rats. No significant variations in the hepato­
cyte number per gram wet weight were ob­
served in rats living at the two different envi­
ronmental temperatures (table 1). As the
percent contribution of parenchymal cells
(about 83%) to the total hepatic volume was
unaffected by cold exposure [21], it follows,
of course, that the number of parenchymal
liver cells per gram wet liver is approxi­
mately 125 X 106 in both groups.
Respiration Rates o f Isolated
Hepatocytes in Control and
Cold-Acclimated Rats
The oxygen consumption in isolated he­
patocytes from control and cold acclimated
rats was measured under different condi-
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*
Control rats
Cold Adaptation in Rat Liver
229
Additions
Control
rats
Cold-adapted
rats
1 None
1.6 ± 0.2
(5.4 ±0.4)
2.8 ±0.3*
( 12.3 ± 1.2)*
2
Hexanoate
2.2 ±0.2’
(7.5 ± 0.5)*
3.8 ± 0.4* •
( 16.7 ± 1.4)* *
3 Hexanoate
+ lactate
2.7 ±0.2*
(9.2 ±0.8)*
4.2 ±0.4**
( 18.5 ± 1.5)*-*
4
0.9 ± 0.1
(3.1 ±0.3)'
1.3±0. I*-*
(5.7 ±0.4)**
Hexanoate
+ oligomycin
The values are the means ± SEM of 10 different
experiments and are expressed as pmol CT/min X g
wet liver. The values in parentheses are expressed as
pmol CT/min X liver/100 g body weight.
* Significant effect of cold exposure (p < 0.05).
* Significant effect of additions (p < 0.05) compar­
ing: value 2 with 1, or value 3 with 2, or value 4 with 2.
Isolated liver cells were incubated at 37 °C as
described in Materials and Methods. Final substrate
concentrations were: hexanoate 4 mA/; lactate 10
mM \ oligomycin 5 pg/ml.
tions of stimulation of mitochondrial respi­
ration (table 2).
Liver cells from cold-acclimated rats, in­
cubated without added substrate (basal
state), exhibited an oxygen consumption sig­
nificantly higher (+75%) than those from
control rats. When the cells were provided
with additional substrate, namely hexa­
noate, an increase in oxygen consumption
for both animal groups occurred in compari­
son to respective basal states. Moreover,
with added hexanoate, hepatocytes from
cold-acclimated rats exhibited an increase of
73% compared with those from control rats.
A further increase in the respiration rate of
the cells from both animal groups was
achieved by adding lactate to the incubation
medium containing hexanoate. since lactate
is a gluconeogenic substrate which increases
the ADP availability. Under these condi­
tions, too. the oxygen consumption was sig­
nificantly higher (+55%) in the cells from
cold-acclimated animals than in those from
control rats.
In the presence of oligomycin. an inhibi­
tor of ATP synthase, the minimal rate of
oxygen consumption necessary to balance
proton leak and maintain a high ApH+ was
determined. Obviously, a decrease in oxygen
consumption occurred in the cells from both
groups of animals. This decrease was not
attributable to a fall in cellular ATP levels or
to a resulting interference with fatty acid
activation, as the concentration of oligomy­
cin employed by us has been shown not to
cause a marked depletion of cellular ATP,
probably by a compensatory increase in the
rate of glycolysis [31]. Under these condi­
tions, too, the cells from cold-acclimated rats
exhibited a respiration rate significantly
higher (+44%) than cells from control rats.
Due to an increase in the relative contri­
bution of the liver weight to the total body
mass in cold-acclimated rats, the increases in
respiratory rates after 15 days of cold expo­
sure were much more marked when the re­
sults were expressed on a 100-gram body
weight basis (table 2).
Rat Liver Mitochondrial Mass in
Control and Cold-Acclimated Rats
Rat liver mitochondrial mass was deter­
mined by assaying the succinic dehydroge­
nase activity in liver homogenates and in
isolated liver mitochondria (table 3). The
specific activity of this enzyme was not af­
fected by cold exposure but when the activity
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Table 2. Hepatocyte respiration in cold-accli­
mated rats
230
Iossa/Liverini/Barletta
Table 3. Rat liver mitochondrial mass in cold-acclimated rats
Control rats
Succinic dehydrogenase activity in the homogenate
pmol/min X g wet liver
Succinic dehydrogenase activity in isolated mitochondria
pmol/min X mg protein
Mitochondrial proteins, mg/g wet liver
Mitochondrial proteins, mg/liver/100 g body weight
9.5±0.5
13.7 + 0.8*
0.28 ±0.02
34 + 2
116± 12
0.28 ±0.03
49 ±4*
216 ± 21 *
The values are the means ± SEM of 10 different experiments.
p < 0.05 versus 0.
was assayed in the liver homogenates and
expressed per gram wet liver, significant
changes occurred, namely, an increase of
about 44% in cold-acclimated rats in respect
to control rats. Thus, the increased total suc­
cinic dehydrogenase activity reflected an in­
crease (+44%) in the mitochondrial mass per
gram wet liver in cold-acclimated rats. When
the data are expressed per liver, this increase
was much more marked (+88%).
ADP-Stimulated Respiration o f Isolated
Rat Liver Mitochondria from Control
and Cold-Acclimated Rats
The effect of 15 days of cold exposure on
ADP-stimulated (state 3) respiration of rat
liver mitochondria was studied using lipid
and nonlipid substrates to involve different
modes of transportation into the mitochon­
dria, different dehydrogenases and different
sites of entry of reducing equivalents into the
mitochondrial respiratory chain. The high
values of RCR (8.3) and ADP/O (2.6) ratios
obtained with glutamate, and also with all
the other substrates utilized [data not
shown], indicated the high quality of the
mitochondrial preparation. Moreover, no
significant difference was observed in the
RCR and ADP/O values of mitochondria
from rats living at the two different environ­
mental temperatures [data not shown]. Liver
mitochondria from cold-acclimated rats had
higher rates of state 3 respiration than con­
trol animals with all the substrates utilized,
except when the succinate was the substrate
(fig. la). In fact, state 3 respiration of mito­
chondria from cold-acclimated animals in­
creased by 110, 47 and 45% using glutamate,
or pyruvate + malate, or palmitoyl-camitine
+ malate as the substrate, respectively. Due
to the increase in the liver mitochondrial
mass in cold-acclimated rats, the increases in
respiratory rates in these animals were much
more marked when the results were ex­
pressed per gram wet liver (+200, +112 and
+ 109%, respectively) (fig. lb); moreover, a
significant increase (+33%) in state 3 respi­
ration occurred also with the succinate as the
substrate (fig. lb).
As shown in figure 2, mitochondria from
cold-acclimated rats had higher rates of ATP
production per gram wet liver than those
from control animals, whatever the substrate
utilized.
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*
Cold-adapted rats
Cold Adaptation in Rat Liver
231
0
15
Days o f cold exposure
I Succinate
H i Glutamate
Pyruvate * malate
E33Palmitoyl
carnitine * malate
I
I Succinate
■ Glutamate
•y/A Pyruvate + malate
IDS Palmitoyl
carnitine » malate
Ouabain-Suppressible Oxygen
Consumption in Isolated Hepatocytes
from Control and Cold-Acclimated Rats
We have measured oxygen consumption
in isolated liver parenchymal cells from con­
trol and cold-acclimated rats in the presence
and absence of 1 mM ouabain and the results
are shown in table 4. The specific Na/K
ATPase inhibitor, ouabain, decreased oxy­
gen consumption by 13% in hepatocytes
from control rats and by 18% in those from
cold-acclimated animals. The ouabain-suppressible oxygen consumption in hepato­
cytes from cold-acclimated rats was in­
creased by 130% compared with the value
observed in hepatocytes from control rats,
indicating that 25% of the increase in oxygen
consumption that results from cold acclima­
tion was directly attributable to increased
Na pumping activity. Moreover, it should be
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Days o f cold exposure
Fig. 1. Rat liver mitochondrial state 3 oxidative
rates in cold-acclimated rats expressed as pmol
CL/min X mg protein (a) and as pmol CL/min X g
wet liver (b). The values are the means ± SEM of 10
different experiments. * p < 0.05 versus 0. Isolated
mitochondria were incubated as described in Materi­
als and Methods. Concentrations of the various sub­
strates were: succinate 10 m M + rotenone 3.75 pM \
glutamate 10 mM: pyruvate 10 m M + malate 2.5
mA/; palmitoyl carnitine 40 pA/ + malate 2.5 mM.
Fig. 2. ATP production in rat liver mitochondria
from cold-acclimated rats expressed as pmol ATP
synthesized/min X g wet liver. The values are the
means ± SEM of 10 different experiments. * p <
0.05 versus 0. Concentrations of the various sub­
strates were the same as in figure 1.
Iossa/Liverini/Barletta
Table 4. Ouabain-suppressible oxygen consump­
tion in isolated hcpatocytes from cold-acclimated
rats
Additions
Control
rats
Cold-acclimated
rats
Hexanoate
2.2 ±0.2
3.8 ±0.3*
(7.5 ±0.5) ( 16.7 ± 1.4)*
Hexanoate
+ ouabain
1.9 ±0.1*
(6.5 ±0.3)*
Ouabain-suppressible
oxygen consumption
0.3 ±0.1
( 1.0 ± 0.3)
3.1 ±0.2**
■f
Ö
+1
N©
0.7 ±0.1*
(3.1 ±0.3)*
The values are the means ± SEM of 10 different
experiments and are expressed as pmol CWmin X g
wet liver. The values in parentheses are expressed as
pmol Cb/min X liver/100 g body weight.
* Significant effect of cold exposure (p < 0.05).
♦ Significant effect of ouabain addition (p <
0.05).
Isolated liver cells were incubated at 37 'C as
described in Materials and Methods. Final substrate
concentrations were: hexanoate 4 mM \ ouabain 1
mM. Ouabain-suppressible oxygen consumption re­
presents the contribution of Na/K. ATPase activity to
total hepatic oxygen consumption.
noted that, due to the increased liver mass in
cold-exposed rats, the total hepatic ouabainsuppressible oxygen consumption gained
greater importance (table 4).
Discussion
The present study shows that the expo­
sure of rats to cold for 15 days results in
extensive changes in the oxygen consump­
tion of isolated rat liver cells. In addition,
the analysis of the values of oxygen con­
sumption obtained under different condi­
tions of stimulation of mitochondrial respi­
ration gives us useful information about the
situation existing in rat liver cells from both
control and cold-acclimated rats (table 2).
When we examine the respiration values
of isolated rat liver cells from control rats,
we see that the oxygen consumption mea­
sured without added exogenous substrate
(basal respiration) depends not only on the
ATP/ADP ratio, as it is generally believed
[32, 33], but also on substrate supply to the
electron transport chain [34], In fact, the
addition of an external substrate, such as
hexanoate, stimulates endogenous respira­
tion of about 40%. This stimulation is much
greater than would be anticipated on the
basis of the potential ATP demands arising
from the metabolic interactions induced by
substrate addition [35] and is due to im­
proved substrate supply to the electron
transport chain, as shown by Brand and
Nobes [34], On the other hand, the oxygen
consumption rate of isolated rat liver cells,
with hexanoate added, reflects the intracellu­
lar ATP/ADP ratio, as the addition of lac­
tate, which stimulates gluconeogenesis and
hence ATP hydrolysis, causes a further in­
crease in respiration. However, this increase
(about 20%) is less marked than that ob­
tained by others [35], using isolated hepatocytes from 24-hour starved rats: in fact, it is
well known that starvation induces an acti­
vation of hepatic gluconeogenic pathway
[36], while well fed rats (like the ones used by
us) show a partial inactivation of liver gluco­
neogenesis [36].
The analysis of the respiration rates ob­
tained with isolated hcpatocytes from coldacclimated rats shows that oxygen consump­
tion increased significantly compared with
control rats (table 2). With regard to basal
respiration, if it is limited in control cells by
substrate supply, then the increase observed
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232
in cold-acclimated rats (+75%) is due to an
improvement in substrate supply in isolated
hepatocytes from cold-acclimated rats, in
agreement with the observed increase in sub­
strate availability which takes place during
cold exposure [37], In addition, it should be
noted that the basal respiration in hepato­
cytes from cold-acclimated rats is similar to
that measured in hepatocytes from control
rats with hexanoate and lactate added, sug­
gesting that cold exposure elicits an increase
in ADP production. However, the observa­
tion that hexanoate addition causes an in­
crease in oxygen consumption in cold-accli­
mated hepatocytes leads to the conclusion
that the increased substrate supply does not
saturate the mitochondrial oxidative capaci­
ty. Lactate addition leads to stimulation of
only 10% of hexanoate-supported respira­
tion in cold-acclimated rats, supporting the
above hypothesis that in this condition not
only substrate supply but also ADP produc­
tion is enhanced, probably because of an
activation of ATP-consuming pathways.
We also measured oxygen consumption
in isolated hepatocytes from both control
and cold-acclimated rats in the presence of
oligomycin, an inhibitor of ATP synthase,
obtaining the minimal rate of oxygen con­
sumption necessary to balance proton leak
and maintain an elevated ApH+ [38]. This
value depends both on proton leak and on
mitochondrial mass: assuming that no varia­
tion in passive permeability of the mito­
chondrial inner membrane occurs during
cold exposure, the increase of 44% found in
oligomycin-limited respiration of hepato­
cytes from cold-acclimated rats (table 2)
could be explained by an increase in the
mitochondrial mass per gram wet liver. This
observation is confirmed by the determina­
tion of the mitochondrial protein content
233
per gram wet liver, calculated by assaying the
succinic dehydrogenase activity in liver ho­
mogenates and in isolated liver mitochon­
dria (table 3). In fact, after 15 days of cold
exposure, the mitochondrial protein content
per gram wet liver increased by 44%; more­
over, due to the concomitant increase in the
liver mass in cold-exposed rats (table 1), the
total hepatic mitochondrial protein content
increased by about 90%.
Our results obtained from rat liver iso­
lated mitochondria also show that mitochon­
drial capacities to oxidize lipid and nonlipid
substrates, except succinate, increased after
15 days of cold exposure (fig. la). Moreover,
state 3 respiratory rates obtained with succi­
nate were always higher, both in control and
cold-acclimated rats, than those obtained us­
ing the other substrates.
In general, the rates of oxygen uptake by
mitochondria may reflect either respiratory
chain or adenine nucleotide translocase or
ATP synthase or substrate permeation or
substrate dehydrogenation activity, depend­
ing on the substrate used [39-41], Therefore,
various substrates were used to represent a
variety of entries into the citric acid cycle
and electron transport chain: glutamate en­
ters the citric acid cycle at a-chetoglutarate
and pyruvate can be converted to acetylCoA; both the substrates are NAD-linked
and electrons enter at site 1 of the electron
transport chain. Succinate is FAD-linked
and enters the electron transport chain at
site 2, while palmitoyl-carnitine, through the
beta-oxidative pathway, produces both
NADH and FADH.
Using succinate as the substrate, the value
of state 3 of respiration is limited by the ade­
nine nucleotide system (transmembrane
ATP/ADP exchange and intramitochondrial
ATP synthesis) [42], as shown by the fact
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Cold Adaptation in Rat Liver
234
oxidative capacity. The elevated ATP pro­
duction rate so obtained (fig. 2) is in good
agreement with the enhanced metabolic rate
of isolated hepatocytes from cold-acclimated
rats (table 2) and with the increased capacity
for hepatic gluconeogenesis which is associ­
ated with cold acclimation [44]. In addition,
our results show that about 25% of the in­
creased hepatic metabolic rate in cold-accli­
mated rats is directly attributable to in­
creased Na pumping activity (table 4), which
in turn elicits an increased liberation of met­
abolic heat.
All our present results emphasize the role
of the liver in the metabolic adjustments in
the cold-acclimated state and outline the
mechanism by which this role is carried
out.
Acknowledgement
The authors would like to thank Prof. M. Orunesu
for his kind hospitality and are indebted to Prof. E.
Fugassa for her advice on preparation of isolated
hepatocytes.
The work was supported by MPI.
References
1 Jansky L: Nonshivering thermogenesis and its
thermoregulatory significance. Biol Rev 1973;48:
85-132.
2 Himms-Hagen J: Cellular thermogenesis. Annu
Rev Physiol 1976:38:85-132.
3 Harl JS. Heroux O. Dcpocas F: Cold acclimation
and the electromyogram of unanesthetized rats. J
Appl Physiol 1956;9:404-408.
4 Foster DO. Frydman ML: Tissue distribution of
cold induced thermogenesis in conscious warm or
cold acclimated rats reevaluated from changes in
tissue blood flow: The dominant role of brown
adipose tissue in the replacement of shivering by
nonshivering thermogenesis. Can J Physiol Phar­
macol 1979;57:257-270.
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that with succinate, an uncoupler such as
carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which releases the respiratory
chain from the phosphate acceptor control
and thus renders it maximally active, gives a
respiratory rate which is about 40% higher
than that of state 3 [43], On the other hand,
for the other substrates here used, the above
factor cannot be rate-limiting, as it is charac­
terized by a value sufficient to account for a
more rapid oxidation of the succinate. So we
can conclude that during the oxidation of
pyruvate, glutamate and palmitoyl carnitine
only substrate permeation and dehydroge­
nases, as far as the system involved in succi­
nate oxidation, can be rate-limiting. These
considerations, together with the result that
oxygen consumption does not vary in iso­
lated mitochondria from cold adapted ani­
mals using succinate, bring us to conclude
that the observed increase in oxygen con­
sumption with the other substrates is the
result of the activation of some limiting step
as far as the complex II of the respiratory
chain. Liver mitochondria thus improve
their oxidative capacity, and consequently
ATP production, by increasing dehydroge­
nase activities and substrate permeation,
and therefore the supply of reducing equiva­
lents, rather than by increasing the respira­
tory chain capacity itself. In addition, the
improvement of the mitochondrial oxidative
capacity and of ATP production is more sub­
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Accepted: December 14, 1990
Giovanna Liverini
Dipartimento di Fisiología Generale ed
Ambiéntale
Via Mezzocannone 8
1-80134 Napoli (Italy)
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