THEJOURNALOF BIOLOGICAL

CHEMISTRY Vol. 268, No. 20, Issue of July 15,pp. 14850-14860,1993

B 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

The Quantitative Contributionsof Mitochondrial Proton Leak and

ATP Turnover Reactions to the Changed Respiration Ratesof
Hepatocytes from Ratsof Different Thyroid Status*
(Received for publication, September 21, 1992, and in revised form, March 3, 1993)

Mary-Ellen Harper$ and Martin

D. Brand
1Q W, United Kingdom
From the Departmentof Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2

Hepatocytesfromhypothyroid rats respire more in state 3l respiration, and the other is dominant in state 4
slowly thanthosefromcontrolanimals; cells from respiration (Hafner et al., 1988,1990a). In state 3 control over
hyperthyroid rats respire faster. We have identified the respiration rate is shared approximatelyequally between
the blocks of reactions whose kinetics are significantly the reactions thatproduce protonmotive force and those that
affected by thyroid hormonesand have quantified the consume it to make ATP (Hafneret al., 1990b); most of the
contribution of flux through different blocks of en- control is in substrate transport and in the adenine nucleotide
ergy-dissipating reactions to the altered oxygen con- carrier (Groen et al., 1982). The major effect of decreases in
sumption. In cells fromhypothyroid rats compared thyroid hormones is via changes in the reactionsinvolved in
with littermate euthyroid controls, there were signifi-the phosphorylation of ADP and the exportof ATP (Hafner
cant kinetic effectson non-mitochondrial oxygen con- et al., 1990a),probably attheadenine nucleotide carrier
sumption reactions and on mitochondrial proton leak (Hoppner et al., 1987; Sterling, 1986). In state4 the respiration
but not on ATP turnover. Approximately 50% of the rate is controlled very largely by the proton leak across the
decrease in cellular oxygen consumption of hypothy- mitochondrial inner membrane (Groen et al., 1982; Brand et
roid cells was accounted for by a decrease inmitochon- al., 1988; Hafner et al., 1990b), and thyroid hormones act
drial proton leak rate and approximately50% by de- predominantly by altering this proton leak; the leak is de-
creased non-mitochondrial oxygen consumption. Met- pressed in hypothyroid mitochondria and stimulated in hy-
abolic control analysis showedthat the distributionof perthyroid mitochondria (Hafner et al., 1988, 1989; Horrum
control over oxidative phosphorylationand mitochon- et al., 1990).
drial potential in hepatocytes from hypothyroid ani- In isolated hepatocytes thathave not been presented with
mals was broadly similar to its distribution in euthy- substrates for anabolic reactions the respiration rate is inter-
roid controls. In cells from hyperthyroid rats comparedmediate between state 3 respiration and state 4 respiration.
with littermate euthyroid controls, there were signifi-The control over this “resting” respiration rate in rat hepa-
cant kinetic effects on mitochondrial proton leak and tocytes is shared amongseveral blocks of reactions. The most
on the reactions involved in ATP synthesis and con- important determinant of the respiration rate in hepatocytes
sumption but not on non-mitochondrial oxygen con- incubated in a basal medium was found to be that block of
sumption. Approximately 50% of the increase in cel- reactions responsiblefor the synthesis, export, and use of
lular oxygen consumption in hyperthyroid cells was ATP (Brown et al., 1990a) with a flux control coefficient of
accounted for by an increased mitochondrial proton 0.5; that is, the resting respiration ratewas controlled mainly
leak rate and the remainder by increased ATP turn- by the activity the “phosphorylating system” (Fig. l ) , which
over; there were no changes in non-mitochondrial oxy- includes the ATP synthase, Pi transporter, adeninenucleotide
gen consumption. carrier,andextramitochondrialATP-consuming reactions.
However, there was also significant control(C = 0.3) over the
respiration rate by the reactions that lead to production of
One of the most widely recognized effects of thyroid hor- the mitochondrial membrane potential, A$,,, (cellular cata-
mones in mammals is a n increase in the standard metabolic bolic reactions, the citric acid cycle, and the electron transport
rate. Thyroid hormone-induced increases oxygen
in consump- chain), and by the leak of protons across the mitochondrial
inner membrane (C = 0.2) (substrate oxidation and proton
tion and heat production are consistent properties of many
leak, respectively, in Fig. I) (Brown et al., 1990a). In intact
mammalian tissues including theliver. Hepatocytes andliver
cells comparatively little is known about the mechanisms or
mitochondria of hyperthyroidratshaveincreased oxygen targets that mediate theeffects of thyroid hormones on OXY-
consumption rates compared with those of euthyroid controls. gen consumption. However, Nobes et al. (1990a) showed that
Conversely, in hypothyroidismoxygen consumption rates are
slower than euthyroid rates. The abbreviations used are: state 3, respiratory state in which
In isolated mitochondria there are two separate effects of ATP synthesis is at maximal rate; state 4, respiratory state in which
thyroid hormones on oxygen consumption: one is dominant there is no ATP synthesis; C, control coefficient; e, elasticity; A$.,,
mitochondrial membrane potential; FCCP, carbonyl cyanide p-tri-
* The costs of publication of this article were defrayed in part by fluoromethoxyphenylhydrazone;TPMP+, triphenylmethylphosphon-
the payment of page charges. This article must therefore be hereby ium; subscripts e, c, m, and tot refer to extracellular, cytoplasmic +
marked “advertisement” in accordance with 18 U.S.C. Section 1734 nuclear, mitochondrial, and total intracellular pools, respectively; J ,
solely to indicate this fact. flux; subscripts S, P, and L refer to substrate oxidation, phosphoryl-
$Supported by aNatural Sciences and Engineering Research ating system, and proton leak, respectively; Js, JP,and JL refer to the
Council of Canada postdoctoral fellowship. To whom correspondence flux through the substrate oxidation, phosphorylating system, and
should be addressed. Tel.: 44-223-333-649;Fax: 44-223-333-345. the proton leak pathways, respectively.

14850

This is an Open Access article under the CC BY license.

 in Rat Hepatocytes
Effects of Thyroid Hormones on Respiration 14851

"proton
proton leak,
oxidatlon"
"substrate cycles cation
glucose, lactate,
pyruvate -ATm

\
endogenous substrates
glYCObSiS. other
cytosolic catabolic reactions, ATP synthesis
citric acid cycle,
electron
transport
chain 'phosphorylating
system"
ATPATPconsumption
k
heat
FIG. 1. The branched system of All., production and consumption. A$, is produced by substrate oxidation, which consists of all of
the steps from added glucose lactate, pyruvate, and endogenous substrates to A$,,,. Then A$,,, is consumed by the phosphorylating system,
which includes ATP production using A$, and all cellular ATP-consuming reactions, or consumed by the proton leak, which includes the
leak of protons and any cationcycles across the mitochondrial inner membrane.

mitochondrial proton leakage in intact hepatocytes from hy- effects of thyroid hormones and to quantify the relative im-
pothyroid rats was decreased compared with euthyroid con- portance of these reactions accounting for the changed resting
trols and that the decrease in oxygen consumption attribut- oxygen consumption rate.
able todecreased proton leakage accounted for approximately Top-downelasticityanalysis involvesdividinga system
30% of the difference in the respiration rate. It was assumed conceptually intoblocks of reactions which produce and con-
(incorrectly, as our data here show) that the other two-thirds sume a common intermediate (A$m in Fig. 1). The overall
were a result of differences in ATP turnover. Increased ATP kinetic response of each block to the concentration of the
turnover is thought tobe responsible for at least some of the intermediate is measured in the presence and absence of an
increased respiration in hepatocytes from hyperthyroid rats effector understudy (i.e. in cellsfrom controlortreated
(Guernsey and Edelman,1983; Nobes et al., 1989; Clausen et animals in this study). Only those blocks that have a changed
al., 1991). We have recently calculated the control coefficients kinetic response (i.e. a changed elasticity) to the intermediate
over effective P/O ratios in liver mitochondria and hepato- contain sitesof effector action which are significant in chang-
cytesand have shownthattheproton leak has negative ing the flux through the intermediate. Thus the approach
control, ATP turnover haspositive control, and the substrate allows the identification of blocks of reactions whose overall
oxidation reactions have little control (Brand et al., 1993). kinetic response to a common intermediate (such as A$,, as
Moreover, agents including thyroid hormones whichaffect in this study) has been significantly altered by treatment with
the kinetics of the proton leak tend to alter effective P/O an effector (suchasthyroidhormone).Top-downcontrol
ratios but have little effect on ATP production rate, although analysis, upon which top-down elasticity analysis is based,
this depends on any othereffects they may have. allows the calculationof the quantitative contributionof each
The mechanism by which thyroid hormones stimulate the block of reactions to the control of a rate (such as cellular
proton leak acrossthemitochondrialinnermembranein oxygen consumption) or a concentration (such as the value of
isolated mitochondria andin intact hepatocytes is unknown. A&). These approaches have been used previously to inves-
However it is likely to be indirect, presumably throughgene tigate the mechanisms by which thyroid hormone status alters
expression, as theeffects of thyroid hormones on the proton the respiration rate of the subsequently isolated liver mito-
leak in liver mitochondria in situ require at least 24 h (Hafner chondria (Hafner et al., 1988, 1989, 1990a). They have been
et al., 1988). Thyroid hormones change both the mitochondrial used to show that the thyroid hormone statusof rats affects
inner membrane surface area (Jacovcic et al., 1978) and the the state 4 respiration rate of isolated mitochondria by alter-
intrinsic permeability of vesicles formed from mitochondrial ing the kinetics of the leak of protons across the mitochondrial
innermembranephospholipids(Brand et al., 1992); these inner membrane (Hafner et al., 1988) and alters the state 3
changes explain much of the observed differences in proton respiration rate by altering the kineticsof the reactions that
leak which are induced by hypothyroidism and hyperthyroid- synthesize and export ATP (Hafneret al., 1990a). Top-down
ism. elasticity analysis has also been used successfully to investi-
Despite many studies indicating effects of thyroid hormones gate the sitesof action of glucagon (Brand et al., 1990) and of
on various component enzymes and pathways involved in a pleiotypic inhibitor, butylated hydroxyanisole (Fusi et al.,
cellular energy metabolism andoxygen consumption (for re- 1992). Glucagon treatment of rats was shown to increase the
views see Oppenheimer et al., 1987; Brand and Murphy,1987; respiration rate of isolated liver mitochondria respiring on
and Dauncey, 1991) and on A$, (Gregory and Berry, 1991; succinate solely by a stimulation of electron flow from succi-
Soboll et al., 1992) there is as yet no clear picture of the nate to ubiquinone (Brand et al., 1990). Nobes et a1 (1990b)
relative quantitative importance of such changes to overall used the method to show that fatty acids stimulate respiration
energy metabolism and oxygen consumption. Our objectives in isolated hepatocytesby increasing the substrate supply and
here were 2-fold 1)to identify the significant subcellular sites also by increasing ATP turnover, but not by causing uncou-
of action of thyroid hormone treatmentof rats on the rateof pling.
oxygen consumption by hepatocytes by applying top-down In the present paperwe use top-down elasticity analysis to
elasticity analysis, an extensionof top down control analysis identify the important sitesof action of thyroid hormones in
(Brand, 1990); and 2) to establish which energy-dissipating rats on subsequently isolated hepatocytes. We show that in
reactions change their rates asa consequence of the primary both the hypothyroid-euthyroid and the euthyroid-hyperthy-
14852 Effects of Thyroid Hormones on Respiration in Hepatocytes
roid transitions, changes in the mitochondrial proton leak
account for half of the changes in respiration rate; the re-
mainder of the changes is due to changes in non-mitochon-
drial oxygen consumption (hypothyroid) or in ATP turnover
(hyperthyroid).
EXPERIMENTALPROCEDURES
Treatment of Animals-Hypothyroidism was induced in male Wis-
tar rats through treatment with 0.05% (w/v) 6-n-propyl-2-thiouracil
in the drinking water. 6-n-Propyl-2-thiouracil treatment was begun
1-2 weeks after weaning when body weights were in the range of 140-
190 g. Rats were used 6-8 weeks later when hypothyroid animals
weighed 170-230 g and paired littermate controls weighed 380-450 g.
This treatment has been shown to decrease serum T3 and T4levels
(Carr et al., 1984; Hafner et al., 1988). Hyperthyroidism was induced
in 9-week-old male Wistar rats by 10 daily injections of 15 pgof
triiodo-L-thyronine (T3)/100 gof body weight; paired littermate con-
trols were injected with vehicle (5 mM NaOH) (Hafner et al., 1988).
All rats were allowed ad libitum access to a chow diet and were caged
in groups of two or three a t 23 "C with light 07:OO-19:OO. Rats were
killed between 09:OO and 10:00, approximately half an hour after
being anesthetized with 6 mg of sodium pentobarbitone/lOO g of body
weight.
The results described here were compiled from four separate series
of experiments. The hypothyroid series included two sets of five or
six 6-n-propyl-2-thiouracil-treated and six littermate-paired control
rats; the hyperthyroid series included two sets of six T3-injected and
six vehicle-injected littermate-paired rats. As the results from each
of the sets were indistinguishable, the results from the two sets from
the hypothyroid or hyperthyroid rats were pooled. This allowed the
comparison of data from 11 hypothyroid and 12 euthyroid rats and
from 12 hyperthyroid and 12 euthyroid rats.
Preparation and Incubation of Cells-Hepatocytes were prepared
from fed rats by a modification (Berry, 1974) of the method of Berry
and Friend (1969). The viability of cells was greater than 90% as
determined by the exclusion of 0.3% (w/v) trypan blue. An additional
indication of the high viability of the isolated cells is that all cell
preparations exhibited large responses to oligomycin, myxothiazol,
and FCCP. Dry cell weight was determined by drying a known volume
of cell suspension to a constant weight at 70 "C (usually over 24 h);
from this value we then subtracted the dry weight of an equal volume
of isolation medium (148 mM NaC1, 5 mM KC1,0.81 mM MgSO,.
7H20, 0.83 mM Na,HPO,, 0.14 mM KHzPO,, 1 mM CaCl,,25 mM
NaHC03 and 15 mM glucose). The cells were suspended in isolation
medium and kept at 4 "C on ice before being diluted 6.6-fold into the
incubation medium described below.As it required up to 5h to
complete a series of titrations this meant that some cells were kept
on icefor up to 5 h. Repeated determinations of resting cellular
oxygen consumption (i.e. that in the absence of anyinhibitors)
showed that there was no significant effect on the A$,,, or resting
oxygen consumption over this time.
Incubations of cell suspensions were routinely carried out using
approximately 3 ml of suspension (6-9 mg, dry weight/ml) in 20-ml
stoppered glass vials at 37 "C in a shaking water bath (100 cycles/
min). The incubation medium contained 106 mM NaCl, 5 mM KC1,
25 mM NaHCOa, 0.41 mM MgSO,, 10 mM Na2HP04,2.5 mM CaCl,,
10 mM glucose, 10 mM lactate, 1 mM pyruvate, and 2.25% (w/v)
defatted bovine serum albumin. Stock 9% bovine serum albumin was
defatted by the method of Chen (1967) and dialyzed against 153 mM
NaCl and 11mM KC1. The gas phase above the cell suspension during
incubations was 95% air and 5% CO, to allow equilibration of the
medium to a pH of 7.4.
Measurement of Respiration Rate-To allow cells to establish ion
gradients after being stored on ice (Baur et al., 1975) cells were
preincubated at 37 "C in the shaking water bath for 10 min before
the addition of any inhibitors or isotopes. After a further incubation
period of 20 min oxygen consumption was measured in duplicate 2-
ml aliquots of cell suspension using two Clark-type oxygen electrodes.
The cell suspensions in the Perspex incubation chambers of the
electrodes were magnetically stirred and thermostatically maintained
at 37 "C. Respiration rates were determined approximately 3-5 min
after the addition of the cell suspension to the chambers, i.e. once
stablelinearrates were obtained. To determine the rate of non-
mitochondrial oxygen consumption, cells were incubated with maxi-
mal concentrations of oligomycin (1.0 pg/ml) and myxothiazol (1.0
p ~ and) with valinomycin (0.1 p M ) and FCCP (20 pM). The A$,
Rat
under these conditions was assumed to be zero.
Measurement of Mitochondrial Membrane Potential (A$J-The
A$,,, was measured simultaneously with oxygen consumption using
the distributionofthe lipophilic cation, triphenylmethylphosphonium
(TPMP+).Appropriate corrections were applied for plasma mem-
brane potential, cytoplasmic and mitochondrial binding of TPMP+
and other factors, as described by Nobes et al. (1990a) and asoutlined
below. The relationship between mitochondrial membrane potential
(mV) and TPMP+distribution at 37 "C is
(Eq. 1)
where V = volume, Q = apparent TPMP+ activity coefficient, and
subscripts c, m,e, and tot represent cytoplasmic + nuclear, mito-
chondrial, extracellular, and total, respectively. Thus mitochondrial
membrane potential can be calculated knowing the proportion of
cytoplasmic volume which is occupied by the mitochondrial matrix
(V,,,/VJ, the apparent activity coefficient of TPMP+ in each com-
partment (ae, a,, and a,,,), and the extent of the accumulation of
TPMP+ into the wholecell ([TPMP+]t,,/[TPMP+]e) and into the
cytoplasm in relation to the external medium ([Cl-],/[Cl-],,). The
values for these correction factors and the sources of the data are
given in Table I. The distribution of 36Cl- was also used to calculate
the plasma membrane potential (Nobes and Brand, 1989). No differ-
ences in plasma membrane potential were observed between cells
from hypothyroid and paired euthyroid controls and from hyperthy-
roid and paired euthyroid controls; values were 32 mV f 3 (S.E., n =
4), 34 mV f 3 ( n = 5), 35 mV f 3 ( n = 5), and 34 mV f 3 (n = IO),
respectively.
Hepatocytes were incubated at 37 "C in the presence of 2.5 pCi of
3H20/ml and 0.1 pCi of ['4C]methoxyinulin/ml for measurement of
cell volume; 1 pCiof [3H]TPMP+/mlfor measurement of TPMP+
accumulation; or0.1pCi of 36Cl-/ml and 2.5pCiof 3H20/ml for
measurement of 36Cl-distribution. Incubation with 3H,0 allowed the
total pellet volume to be determined while [14C]methoxyinulinallowed
the calculation of extracellular volume in pellets; the cellular volume
was then calculated as thedifference between the totalpellet volume
and the extracellular volume. Carrier inulin (0.1 mg/ml) and TPMP
bromide (0.1 p ~ were ) added to all incubations. All radiolabels and
inhibitors were added after the 10-min preincubation period, and the
incubation was continued for another 20 min. The time for equilibra-
tion of TPMP+ into hepatocytes has been shown to be 10-15 min
(Hoek et d . , 1980).
At the end of each incubation triplicate aliquots (0.70 ml) were
removed and pipetted into 1.5-ml microcentrifuge tubes and imme-
diately centrifuged in an Eppendorf microcentrifuge for 2 min. For
more accurate determinations of36Cl- distributions aliquots were
centrifuged for 2 min through 350pl of oil (42% (v/v) dinonyl
phthalate and 58% silicon fluid D.C. 550) which was layered over 100
plof 2% (v/v) Triton X-100, 0.25 M sucrose in the microcentrifuge
tube. In either case 200-p1 aliquots of the supernatant were removed
and pipetted into scintillation vials and immediately mixed with
scintillant. The residual supernatant was aspirated; the sides of each
tube were wipeddry, and 40 p1 of 20% (v/v) Triton X-100 was added;
if oil was used then theresidual supernatant andmost of the oil were
aspirated. Following suspension of the pellet by vortex mixing, the
bottom of the tube was cut off into a scintillationvial, and thepellet
was resuspended in 3.0 ml of scintillant. The radioactivities of the
supernatant andpellet were determined by dual-channel scintillation
counting for 3H and "C, 3H and =Rb, and for 3H and 36Cl,using the
appropriate quench and crossover corrections.
The apparent volume of pellet available to each isotope (its space
in pl) was calculated as dpm in total pellet divided bydpm/wlof
supernatant sample. The t3H]TPMP+accumulation ratio ([TPMP+]
,/[TPMP+],) was calculated as ([3H]TPMP+space - ["Clmethox-
~ O - ['4C]methoxyinulin space). For greater
yinulin s p a ~ e ) / ( ~ Hspace
precision correction for total pellet volume was made in each pellet
containing 36Cl-; thus, 36Cl- distribution, [Cl-],/[Cl-]., was calcu-
-
lated as [(3H,0 space ['4C]methoxyinulin space) - (3H20space -
36Cl-~pace)/(~H,O space - ['4C]methoxyinulin space)].
Quantitatiue Morphometric Cytology-To determine the proportion
of cell volume which represents mitochondrial matrix space hepato-
cytes were pelleted for electron microscopy in isolation medium
containing 2.5% (w/v) glutaraldehyde. The glutaraldehyde-fixed cells
were washed twice in 0.05% (w/v) sodium cacodylate buffer. Electron
 Effects of Thyroid
Hormones
Respiration
on Rat
in Hepatocytes 14853
TABLE I
Correction factor values and their sources for the calculation of A*,,, in hepatocytes
Values are given as mean k (S.E., n); N/A indicates S.E. not available. V,, V,, and V J V , refer to the proportion of cellular volume that
is cytoplasmic and nuclear, the proportion of cellular volume that is mitochondrial matrix, and to theratio of cytoplasmic plus nuclear volume
to mitochondrial volume. Values were determined as described under "Experimental Procedures." a,, a,, and a, refer to the proportions of
TPMP' in the mitochondria, cytoplasm, and external medium which are free (i.e. not bound); values were determined as described by Nobes
et al. (1990a) or taken from the indicated references. Cl-,t/Cl-e represents the extent of accumulation ofC1- into the cell relative to the
external medium and was determined as described under "Experimental Procedures."
Hyperthyroid Correction
Euthyroid Hypothyroid
factor
VC 0.88 (N/A)" 0.82 (N/A)" 0.81 (N/A)' 0.83 (N/A)'
V, 0.12 (N/A)" 0.18 (N/A)" 0.19 (0.03, 8)' 0.17 (0.03, 8 ) b
Vc/ Vm 7.4 (N/A)" 4.5 (N/A)" 4.3 (N/AIb 4.9 (N/A)
a, 0.51 (0.00, 3)' 0.43 (0.00, 3)' 0.30 (0.02, 3)' 0.44 (0.01, 3)'
a, 0.20 (0.02, 4)b 0.17 (0.02, 4)' 0.21 (0.02, 9)' 0.19 (O.O2,4)b
a, 0.71 (N/A)" 0.71 (N/A)" 0.71 (N/A)" 0.71 (N/A)"
cl-tot/cl-e 0.29 (0.04, 5)b 0.27 (0.03, 5)b 0.33 (0.03, 0.31 (0.03, 5)'
a From Nobes et al. (1990a).

Data collected herein. ac values were determined as described by Nobes et al. (1990a). a, and Cl-tot/Cl-evalues were not significantly
different between hypothyroid, hyperthyroid, and control hepatocytes; therefore the overall means from the four groups of data for these two
correction factors (0.19 and 0.30, respectively) were used in the calculation of A*,,, in hepatocytes from hypothyroid hyperthyroid, and control
hepatocytes.
. . Because V, and V J V , values are derived from the V, values, S.E. values are not given for these derived values.
From Hafner et al. (1988).

micrographs of osmium-stained pellet sections were prepared at a
final magnification of 6,100-fold. Mitochondrial volume was deter-
mined from the number of intersections of a 1-cm grid that overlay
mitochondria as described by Weibel (1969). Mitochondrial volume
was calculated as the total number of intersections in mitochondria
divided by the total number of intersections in cells (less the total
number of intersections in cellular lipid droplets). The cellular volume
was corrected for the volume of intracellular lipid droplets as TPMP'
is not taken up into fat (Davis et al., 1981). Mitochondrial matrix
volume was calculated as 56.5% of total mitochondrial volume based
on the work of Loud (1968), who calculated this weighted mean from
the percentage matrix volume of midzonal, peripheral, and central
liver cells and thepercentage of these cell types in the whole liver.
Measurement of ATP Content-Total ATP content of cells was
determined using cell extracts preparedby precipitating 200 plof cell
suspension (0.8-1.2mg, dry weight/ml) with 500 p1 of 3% (w/v)
HClO, at 0 "C. Determinations were carried out in parallel to deter-
minations of the A$, and oxygen consumption. After the cell extracts
were prepared they were kept on ice for up to 5 h before the ATP
content was assayed. Control experiments using known amounts of
ATP standard and repeated determinations of resting cellular ATP
conczntrations over the 5-h period showed that ATPin these extracts
was stable for this period of time. Five min before the ATP assay the
cell extracts were neutralized with 250 p1 of 1 M KOH in 1 M Hepes.
The ATP content was determined in triplicate using firefly lantern
extract (10 mg/ml H20)in a Du Pont Luminescence Biometer (Lakin-
Thomas and Brand, 1987).
Experimental Approach t o Top-down Elasticity Analysis-The
overall elasticities to changes in the A$, of the reactions that produce
the A$, (cellular catabolic reactions, the citric acid cycle and the
electron transport chain) and those that consume it (ATP synthesis
and turnover, and the proton leak pathway) were then studied. The
response of the A$, producers to the A$, was measured by titrating
the A$,,, consumers wlth oligomycin (0.05-1.0 pg/ml). The elasticity
of the proton leak pathway to the A$, was measured from titrations
with myxothiazol (0.025-0.50 p ~ in) the presence of saturating
amounts of oligomycin (1.0 pg/ml). Preliminary experiments showed
that theaddition of greater concentrations of oligomycin resulted in
no further decrease in cellular oxygen consumption. Thus it was
assumed that thephosphorylating system (oxidative ATP synthesis)
was completely inhibited at this concentration. With the subsequent
additions of myxothiazol (to inhibit complex 111 of the respiratory
chain) thekinetic response of the leak t o the A$, can then be assessed
(Brand et al., 1988). The elasticity of the phosphorylating system to
the A& was measured from titrations with myxothiazol alone (0.05-
0.20 pM), and corrections were later made for the amount of oxygen
required to balance the rate of proton leakage at each A$, measured.
Elasticity coefficients and flux control coefficients from the A$, and
hepatocyte oxygen consumption data were calculated using the equa-
tions previously published (Hafner etal., 1990b;Brown et al., 1990b).
Statistical Analysis-Data were analyzed using unpaired Student's
t tests. Any differences between linear regression lines were assessed
by analysis of covariance using the computer program COMPREG
(Wiggans et al., 1983). A p value of less than 0.05was considered
statistically significant.
Materials-Oligomycin, valinomycin, bovine serum albumin (frac-
tion V), collagenase (type IV), firefly lanternextract, inulin, and
trypan blue were from Sigma. Myxothiazol and disodium ATP were
from Boehringer Mannheim.FCCPand TPMP iodidewerefrom
Aldrich. Dinonyl phthalate andsilicon fluid D.C. 550 were from BDH
Chemicals. Na3'C1 and 3H20 were from Amersham. ['4C]Methoxyi-
nulin and [3H]TPMP bromide werefromDu Pont-New England
Nuclear. Water-insoluble compounds were dissolved in dimethyl sulf-
oxide.
RESULTSANDDISCUSSION
Resting Oxygen ConsumptionRates of Hepatocytes-In
these studies the hepatocytes were from fed animals andwere
incubatedin a minimal medium withouttheaddition of
substrates for anabolic reactions.However when incubated in
this medium thehepatocytes from hypothyroid,euthyroid
and hyperthyroid rats were most likely using different sub-
strates as energy sources. As glucose, even at 15 mM, is a
minor substrate for hepatocytes and as glycogen and fatty
acids are excellent substrates, it is probable that the hepato-
cytes from hypothyroid and euthyroid rats were using the
latter as their major substrates. In hepatocytes from hyper-
thyroid rats, where glycogen and lipid droplets are substan-
tially depleted, the major substrates used were likely lactate,
pyruvate, and, t o a lesser extent, glucose. Thus under these
incubation conditions, hepatocytes isolated from hypothyroid
and euthyroid rats arelikely to be "glycolytic," whereas those
isolated from hyperthyroid rats are"gluconeogenic." For sim-
plicity, we will describe all of these cells, incubated without
inhibitors, as "resting" cells.
Mean oxygen consumption rates were 5.35 f 0.25 (S.E., n
= 11) and 6.15 f 0.22 ( n = 12) nmol of Oz/min/mg dry weight
for hepatocytes from hypothyroid rats and paired euthyroid
control rats, respectively. Both values are similar to those
values reported previously (Nobes et aL, 1990a). With cells
from hyperthyroid and paired euthyroid control rats oxygen
consumption rates were 9.88 f 0.48 ( n = 12) and 5.98 f 0.27
( n= 12) nmolof Oz/min/mg dryweight, of cells. These values
from hyperthyroid and euthyroid rats areslightly lower than
those reported by Gregory and Berry(1992) (after accounting
for their dry to wet weight conversion factor); their higher
14854 Effects of Thyroid Hormones on Respiration in Rat Hepatocytes

A 200 ratesare likely due, at leastinpart,tothe provision of

180 E palmitate in the incubationmedium. Rates were significantly
lower ( p = 0.03) with cells from hypothyroid rats andsignif-
icantly higher ( p < 0.001) with cells from hyperthyroid rats
than with the corresponding euthyroid cells.
Top-down Elasticity Analysis of the Kinetics of Substrate
Oxidation, Proton Leak, and the Phosphorylating System to
the A+,-Our initial purpose in each set of studies was to
determine which of the many known targets of thyroid hor-
mone actionwere responsible for the difference in respiration
rate of the isolated cells under these incubation conditions.
This was done by measuring the kineticresponse to the A+,
of the different subsystems of energy metabolism shown in
0 1 2 3 4 Fig. 1 to identify those subsystems that were significantly
J (nmol Vmlnlmg dry collr) affected by altering thyroid hormone status (i.e. by applying
L top-down elasticity analysis). For the analysisof the kinetics
of the three blocks of reactions depicted in Fig. 1 the non-
mitochondrial membrane portionof oxygen consumption was
B subtracted from the total cellular oxygen consumption.

E Fig. 2 shows the effects of hypothyroidism on the kinetics

2oo
180

160

140
1- of the three blocks of reactions of Fig. 1. There is clearly a
difference in the rateof the proton leak pathway at anygiven
mitochondrial membrane potential between the hypothyroid
and euthyroid states (Fig. 2 4 ) ; the proton leak rate is up to
twice as greatin the euthyroidcells as it is in the hypothyroid
120 - cells. This confirms and expands the results of Nobes et al.
100 - (1990a). The effect was also seen with isolated liver mito-
chondria (Hafner et al., 1989).
80 - In contrast to the kinetics of mitochondrial proton leak,
Fig. 2B shows that there was no significant difference in the
60 I . 1 . 1 . 1 . 1 rate of the A+,-producing reactions at the same values of A+,
0 1 2 3 4 5 between the hypothyroid and euthyroid states. Thus although
J (nmol0 Imlnlmg dry collr) hypothyroidism has been reported t o result in decreased con-
S 2 centrations of the cytochromes (Horrum et al., 1985), de-
creased respiratory chain activity (Clot and Baudry, 1982),
and decreased activities of enzymes that increase the supply
of reducing equivalents to the mitochondrial matrix (Oppen-
C heimer et al., 1977), such changes cause insignificant changes

2oo
180 1 in the overall response of substrate oxidation to theA+, and
cannot cause thedifferences between the respiration rates of
hypothyroid and euthyroid hepatocytes under these experi-
mental conditions.
Similarly, Fig. 2C shows that contrary to expectation, there

t
was no significant difference in the rateof the phosphorylat-
ing system at any given value of the A+, between the hypo-
thyroid and euthyroid states. The activity and capacity of the
120 100 adenine nucleotide transporter are decreased in liver mito-
chondria during hypothyroidism (Portnay et ul., 1973; Hoch,
1977; Mak et al., 1983; Verhoeven et al., 19851, and the activity
of the M$+-stimulated ATPase is decreased by 20% in thy-
0 1 2 3 4 5 6 roidectomized rats (Clot and Baudry,1982), but such changes
causeinsignificantchangesinthe overallresponse of the
Jdnmol ~/mln/mgdry wllr)
phosphorylating system to the A+, and cannot cause the
FIG. 2. Comparison of the kinetic responses of the proton differences between the respiration rates of hypothyroid and
leak (A), substrate oxidation ( B ) ,and phosphorylating system euthyroid hepatocytes under these experimental conditions.
(C)with A$m in hepatocytes from hypothyroid and euthyroid Thus the only block of reactions to have a different kinetic
rats. Open symbols, hypothyroid cells; filled symbols, paired euthyroid response to the A+, is the mitochondrial proton leak, and
controls. A , the kinetic responseof mitochondrial proton leakto A$,,, thischangecausesall of the difference inmitochondrial
(myxothiazol titration of oligomycin-inhibited respiration) showing
that the absence of thyroid hormones decreases the rate of proton oxygen consumption between hypothyroid and euthyroidhe-
leak at any given A$m. E, the kinetic responseof substrate oxidation patocytes.
to A$,,, (oligomycin titration of resting respiration) showing that the The effects of hyperthyroidism on the kinetics of the proton
absence of thyroid hormones causes no difference in the kinetics ( p ~ ~ ~~

> 0.05 by analysis of covariance). C, the kinetic response of the
phosphorylatingsystem to A$m (myxothiazoltitration of resting
respirationintheabsence of oligomycin minusthe myxothiazol
titration in the presence of oligomycin) showing that the absence of
thyroid hormones causes no difference in the kinetics ( p > 0.05 by
analysis of covariance). Each point represents mean -C S.E. of 11
determinations for hypothyroid rats and 12 determinations for euthy-
roid control rats. Each A$, determination was carried out in tripli-
cate; each oxygen consumption determination was carried out simul-
taneously in duplicate.
 Effects of Thyroid
Hormones
leak and phosphorylating system pathways are shown in Fig.
3. Withhepatocytesfromhyperthyroidrats,the A$, de-
creased during the titration witholigomycin; thus it was not
possible to determine the kinetics of the substrate oxidation
reactions. The A&, should be higher in state 4 respiration
than in the resting state since the A$,,, is not dissipated by
the reentry of H' during the synthesisof ATP. Preliminary
results showed that in the presenceof saturating amounts of
oligomycin (1.0 pg/ml) the ATP concentration decreased by
34% to a concentration of approximately 1.8 mmol of ATP/
*
liter of cell water 0.29 (S.E., n = 5). In hepatocytes from
euthyroid rats the resting ATP concentration decreased by
24% to approximately 2.9 mmol of ATP/liter of cell water f
0.44 (S.E., n = 5) after the addition of maximal amounts of
200-
180 - significant difference in the rate of the proton leak at each
0 1 2 3
J ( m o l 0 Iminlmg dry calls)
L 2
200 r
180 - dependent oxygen
0 1 2 3 4 5 6
J ( m o l 0 /mln/mg dry calls)
P 2
FIG. 3. Comparison of the kinetic responses of the proton
leak ( A ) and the phosphorylating system ( B ) to Aqc, in
hepatocytes from hyperthyroid and euthyroid rats. Open sym-
bols, hyperthyroid cells; filkd symbols, paired euthyroid controls. A ,
the kinetic response of mitochondrial proton leak to A$,,, (myxothia-
zol titration of oligomycin-inhibited respiration) showing that the
hyperthyroid state results in an increased rate of proton leak a t any
given A$,. B , the kinetic response of the phosphorylating system to
A$,,, (myxothiazol titration of resting respiration in the absence of
oligomycin minus the myxothiazol titration in the presence of oligo-
mycin) showing that the hyperthyroid state results in an increased
rate of the phosphorylating system at any given A&,. The lines are
significantly different ( p < 0.05). Each point representsmean f S.E.
of 12 determinations. Each A$, determination was carried out in
triplicate; each oxygen consumption determination was carried out
simultaneously in duplicate.
on Respiration in Rat Hepatocytes 14855
oligomycin (1 pg/ml). Because the hepatocytes from hyper-
thyroid rats have an increased demand for ATP (see below)
it appears that, unlike cells from euthyroid and hypothyroid
animals, they are unable to maintain their phosphorylation
potential by glycolysis whenoxidative phosphorylationis
prevented by the addition of oligomycin. This confirms pre-
vious observations (Nobes et al., 1990a). Thus in hyperthyroid
cells oligomycin not only inhibits the phosphorylation reac-
tions but also lowers ATP and indirectly inhibits substrate
oxidation, so making it unsuitable for titrations designed to
measure theoverall kinetics of substrate oxidation. However,
these secondary effects should not affect the kinetics of the
proton leak, so myxothiazol titrations in the presence (or
absence) of maximal amounts of oligomycin still accurately
reflect the kinetics of the proton leak (or proton leak plus
phosphorylating
system). Fig. 3A shows
there that was a
value of the in cells from hyperthyroid animals; it was a t
least 2.5-fold greater than in controls. This confirms results
from earlier studies usingliver mitochondria from hyperthy-
roid rats (Hafner etal., 1988) and reveals that theincrease in
proton leakobserved in isolated mitochondriapersistsin
hepatocytes andis not an artifactof the isolation techniques.
There were alsolarge and significant differences in the
kinetic response of the phosphorylating system to the A$,,,
(Fig. 3B). At any given A$, the rate of the phosphorylating
system was a t least four times greater in the hyperthyroid
cells than in the euthyroid cells. These differences in the
overall kinetics of thephosphorylationsystem might be
caused by the reported alterations in the adenine nucleotide
4
carrier (Babior et al., 1973; Hoch, 1977; Mak et al., 1983) or
phosphate carrier (Clot and Baudry, 1982) or by changes in
the kineticsof ATP-consuming reactions,for example, of the
Na'/K'-ATPase. The synthesis of the Na'/K'-ATPase is
controlled by thyroid hormone (Lingrel et al., 1990). Indeed
fairly large proportions of hyperthyroid-induced increases in
cellular oxygen consumption havebeen attributed to increases
in Na+/K'-ATPase activity (Ismail-Beigi andEdelman,
1970).estimates
However
early these of Na+/K'-ATPase-
overestimates
consumption
are be- mainly
cause of the long incubation times of cells with the Na+/K'-
ATPase inhibitor, ouabain (Himms-Hagen, 1976; Nobes et
al., 1989; Clausen et al., 1991). More conservative estimates
in the rangeof 5 3 0 % for the increase inoxygen consumption
that is attributable to increased Na'/K+-ATPase activity have
been reported (Folke and Sestoft, 1977; Nobes et al., 1989;
Clausen etal.. 1991).
Thus the primary targets for the actions of thyroid hor-
mones on oxidative phosphorylation in hepatocytes are the
proton leak for the euthyroid-hypothyroid transition, and the
proton leak and the phosphorylating system for the euthyroid-
hyperthyroid transition. There are also changes in non-mi-
tochondrial oxygen consumption in thetransitiontothe
hypothyroid state but not to the hyperthyroid state. These
are the only kinetically significant targets for thyroid hor-
mone effects under the conditions we have examined; other
changes do not contribute to the changed respiration rates
that we have observed.
Top-down Control Analysis in Hepatocytes from Hypothy-
roid Ruts-Top-down elasticity analysis provides all of the
data necessary to complete a top-down control analysisof the
system being studied (seeBrown et al., 1990b). Top-down
control analysis then provides quantitative answers to ques-
tions about the importance of particular steps (or groups of
steps) in controlling theflux through a pathway (mathemati-
cally described in the form of a flux control coefficient), or
14856 Effects of Thyroid
Hormones
controlling the concentration of particularintermediates
(mathematically described in the form of aconcentration
control coefficient). A top-down control analysis was con-
ducted with the data from the hypothyroid cells and paired
controls, and the results are presented here. Because we did
not determine the kinetics of the substrateoxidation pathway
in the cells from the hyperthyroid rats, it was not possible to
carry out a top-down control analysis on these cells.
Table IIA provides the overall elasticities to theAqm of the
substrate oxidation, phosphorylating system, and proton leak
pathways. The values in both the hypothyroid and euthyroid
states aresimilar. Despite the apparently divergent elasticities
between groups, an analysis of covariance showed no signifi-
cant differences ( p > 0.05) in the elasticities to the Aqm of
the substrate oxidation and phosphorylating system pathways
between the hypothyroid and thetwo euthyroid groups. Table
IIA also provides the overall elasticities to the Aqm of the
phosphorylating system and proton leak pathways for hyper-
thyroid cells. The elasticity of the phosphorylating system in
hyperthyroid cells is significantly different (analysis of covar-
iance; p < 0.05) from those in hypothyroid cells and from
those determined for both sets of euthyroid controls. Because
the elasticity of the proton leak pathway is nonlinear an
analysis of covariance could not be used to testfor significant
differences in the elasticities.
The flux control coefficients of the three reaction blocks
over the rate of each of the blocks and their concentration
control coefficients over the Aqm were calculated from these
on Respiration Rat
in Hepatocytes
elasticities and are shown in Table 11. All of the values for
cells from hypothyroid rats aregenerally similar to those from
euthyroid controls. Because the elasticities to the A+,,, of the
substrate oxidation and phosphorylating system pathways
between hypothyroid and both setsof euthyroid controls were
not significantly different, any significant difference in flux
control coefficients (which are derived from these elasticities)
is highly unlikely. Table I1 shows that thecontrol coefficients
in hypothyroid cells were no moredifferent from their paired
controls than the two euthyroid sets were from each other,
supporting this conclusion. The results show that despite
changes in the kinetics of the proton leak reactions (Fig. 2 A )
the distribution of control between different reactions over
the three pathways and over the A$, is maintained in hepa-
tocytes from hypothyroid rats. It is also clear that the exper-
imental scatter in the datawould have to be much less before
small changes ineither elasticities or control coefficients
could be reliably observed.
The results from both hypothyroid and euthyroid cells in
the resting state indicate that the phosphorylating system
(0.4-0.5) and substrate oxidation (0.3-0.4) exert most of the
control over mitochondrial oxygen consumption ( J s ) ,whereas
the remainder of the control is through the proton leak (0.2-
0.3). These results arebroadly similar to those obtained with
hepatocytes from euthyroid rats (Brown et al., 1990a); how-
ever our results show slightly higher control by substrate
oxidation and a correspondingly lower control by the phos-
phorylating system over mitochondrial oxygen consumption,

TABLEI1
Ouerall elasticities to A*,,, and control coefficients over rates and over A*,,, in hepatocytes
from hypothyroid and euthyroid (hypothyroid-paired and hyperthyroid-paired) rats
Overall elasticities to A*,,, in hepatocytes from hyperthyroid rats are also given (the elasticity to AT,,, of substrate oxidation and the
control coefficients could not be calculated as substrate oxidation could not be titrated in these cells). A, overall elasticities to A*,,, of
substrate oxidation, phosphorylating system, and proton leak. Flux control coefficients of substrate oxidation, phosphorylating system and
proton leak pathway over B, substrate oxidation rate; C, the phosphorylating system flux; D, the proton leak pathway flux. E, concentration
control coefficient over A q m by the substrate oxidation, phosphorylating system, and proton leak. Results were calculated from the mean
values of A*, and hepatocyte mitochondrial oxygen consumption determinations from 11hypothyroid rats and 12 paired euthyroid controls
(Fig. 2) and from 12 hyperthyroid rats and12 paired euthyroid controls (Fig. 3). For the elasticities represented by straight lines (i.e. substrate
oxidation and phosphorylating system) analyses of covariance were conducted to determine if elasticities were significantly different. Identical
symbols indicate no significant difference ( p > 0.05), and different symbols indicate significant differences ( p < 0.05) between elasticity
coefficients of the same block of reactions from cells of rats having different thyroid hormone status under either resting or state 4 respiration
states. Similar values of elasticities and control coefficients were obtained if non-mitochondrial oxygen consumption was not subtracted first.
Resting State 4
Euthyroid Euthvroid Euthyroid Euthyroid
Hypothyroid (hypothyroid- (hyperthyroid- Hyperthyroid Hypothyroid (hypothyroid- (hyperthyroid-
paired) paired) paired) paired)

-22.45% -8.90' -13.93' -65.28* -19.38' -31.54*

8.56' 1.96$
9.14' 5.44'
6.96 3.06 3.61 2.61 6.97 3.97 3.87

0.26 0.44 0.25 0.10 0.17 0.14

0.51 0.35 0.46
0.23 0.21 0.29 0.90 0.83 0.86

0.28 0.57 0.29

0.81 0.64 0.82
-0.09 -0.21 -0.11

0.23 0.19 0.19 0.10 0.17 0.14

-0.16 -0.12 -0.12
0.93 0.93 0.93 0.90 0.83 0.86

0.03 0.06 0.05 0.01 0.04 0.04

-0.02 -0.04 -0.03
-0.01 -0.02 -0.02 -0.01 -0.04 -0.04
 Effects of Thyroid Hormones on Respiration in Rat Hepatocytes 14857
again, perhapsbecause of the inclusion of lactate and pyruvate synthesis) as described by Brand (1990). Non-mitochondrial
in our incubation medium. The concentration control coeffi- oxygen consumption was identified as that which was insen-
cients over the Aqm are also similar to the values determined sitive to saturating amounts of myxothiazol and oligomycin
by Brown etal. (1990a). The results for the oligomycin- (indicated by the shaded bar in each graph in Fig. 4). Oxygen
induced state 4 for both hypothyroid and euthyroid cells show consumption used to drive the proton leak cycle across the
that respiration rate in intact hepatocytes is predominantly mitochondrial inner membrane in the resting state was meas-
(but not completely) controlled by proton leak (0.8-0.9), very ured as the rate of the proton leak (oxygen consumption in
muchasitisinisolated liver mitochondriarespiringon the presence of oligomycin, titrated with myxothiazol) at the
succinate (Brand et al., 1988; Hafner et al., 1990b). same value of A$, as in restingcells (hatched bars in Fig. 4),
QuantitativeAnalysis of the Effectsof Altered Proton Perme- as describedpreviously (Nobes et al., 1990a; Brown etal.,
ability and Altered Non-mitochondrial Oxygen Consumption 1990a). The remaining oxygen consumption was usedto drive
on Resting Oxygen Consumption in Hepatocytes from Hypo- ATP synthesis (clear bars in Fig. 4).
thyroid Rats-Fig. 4 shows the titrations of respiration in The resting oxygen consumption rate attributable to the
hypothyroid and euthyroid cells with oligomycin and myxo- activities of each of the three pathways is given in thediagram
thiazol which were used to constructFig. 2. From the titration inserted between the elasticity curves in Fig. 4. This diagram
curves it ispossible to calculate theoxygen consumption used also shows estimates of the percentof the difference in resting
intherestingstate for energy-dissipatingreactions(non- oxygen consumption from hepatocytes of hypothyroid and
mitochondrial oxygen consumption, proton leak, and ATP euthyroid ratswhich is attributable todifferences in the rates

FIG. 4. Relationship between A$m

and respiration rate in hepatocytes

~
from hypothyroid rats ( A ) and
paired euthyroid controls (23).Titra- 1O(
tions were carried out from the resting
state (furthest point on the right). The
response of the A$,,, producers (substrate
oxidation) to A$, (0)was measured by
titrating the A$, consumers with oligo-
mycin (0.1, 0.2, 0.5, and 1.0 pglml). The 40 0
20 1 2 3 4 5 6 7 8 9 1 0 1
elasticity of the proton leak (0)to A$,,,
was measured by titrating with myxo- Oxy* con8urnptlon( m o l 9/mlnlmg dry wlh)
thiazol (0.05, 0.10, 0.20, and 0.50 phi) in
the presence of saturating oligomycin
(1.0 bg/ml). The combined elasticity of
the A$,,, consumers (ATP synthesis and
1Leak
~~

turnover, plus proton leak) (A)to A$,,, Resting cellular oxygen consumptlon(nmol4 /min/mg) Nonmitochondrbl
was measured from titrations with
myxothiazol alone (0.05, 0.10, and 0.20 0 ATP turnover
p ~ ) To. determine the non-mitochon-
drial rate of oxygen consumption, cells
Hypothyrold
s 5.35 rn 3.05 I+
were incubated in the presence of maxi-
mal concentrations of oligomycin (1.0
Euthyroid
I 6.15 m 3.02 I+
rg/ml) and myxothiazol (1.0 phi) and
with valinomycin (0.1p ~and ) FCCP (20
phi). Each point represents the mean &
S.E. of 11 determinations for hypothy-
52% Non-mitochondriai 52% Proton bek
i
roid rats and 12 determinations for eu-
% Contrlbutlon to tho difference in mating cdkriar oxygen consumption
thyroid control rats. Each A$,,, determi-
nation was carried out in triplicate; each
oxygen consumption determination was
carried out simultaneously in duplicate.
The bar graphs in the top left corner of
A and B show the oxygen consumption
calculated to be due to non-mitochon-
drial reactions, proton leak, and ATP
turnover. The centralpanel compares B
these bar graphs and shows how the dif-
ference in respiration rate between the
two conditions is distributed. S.E. (for a
1
non-mitochondrial oxygen consump-
tion) and pooled S.E. (for proton leak-
dependent and ATPturnover-dependent
oxygen consumption) are indicated; the
S.E. for the point on each proton leak
curve at the resting A$,,, was estimated
as themean of the S.E. for the two data
points adjacent to it.
"1
f
0 1 2 3 4 5 6 7 8 9 1 0 1 1
9
O w n con8umpkn ( m o l lmlnhng dry d b )
14858 Effects of Thyroid Hormones on Respiration
of the three pathways. Of the difference in resting oxygen
consumption between hepatocytes from hypothyroid and eu-
thyroid rats (p< 0.03),approximately 52% could be attributed
to differences in non-mitochondrial oxygen consumption
processes. The difference in the amount of non-mitochondrial
oxygen consumption between hypothyroid and euthyroid he-
patocytes was statistically significant (p < 0.01). The remain-
der of the difference in resting oxygen consumption between
hepatocytes from hypothyroid and euthyroid rats, about48%,
was dueto a difference in mitochondrial oxygen consumption.
Allof the difference in mitochondrial oxygen consumption
could be attributed to differences in the rate of proton leak.
The rateof proton leak was significantly lower in hepatocytes
from hypothyroid rats than those from euthyroid controls ( p
< 0.05).
The observed differences in non-mitochondrial oxygen con-
sumption were unexpected. A recent report from this labora-
tory (Nobes et al., 1990a) does not describe differences in
non-mitochondrial oxygen consumption between hepatocytes
from hypothyroid and euthyroid rats. The apparent discrep-
ancy between the previous results and the present work is
because non-mitochondrial oxygen was assessed by Nobes et
al. (1990a) for only one hypothyroid rat and one euthyroid
rat, and thiscomparison yielded no significant difference (0.34
uersus 0.36 nmol of 02/min/mg, wet weight, of liver, respec-
tively). Using techniques similar to those described above,
Nobes et al. (1990a) concluded that approximately one-third
of the change in resting oxygen consumption rate in hepato-
cytes from hypothyroid rats was due to differences in proton
leakage and assumed that the other two-thirds were due to
differences in ATP turnover. The present results show that
this assumption was incorrect.
The mechanisms involved in the changes in non-mitochon-
drial oxygen consumption are unknown but mayinvolve
altered microsomal and/or peroxisomal oxidative pathways.
Changes in microsomal oxygen consumption could involve
altered activities of the P450-dependent reactions. In light of
the recent report of Ram and Waxman (1992) that hypothy-
roidism in rats led to a 75-85% depletion of hepatic micro-
somal P450 reductase activity and protein, it is likely that at
least some of the decrease in non-mitochondrial oxygen con-
sumption in the hepatocytes from our hypothyroid rats is as
a result of decreased P450 reductase activity. P450 reductase
is reported to be an obligatory, and often “rate-limiting’’
component of microsomal P450-dependent reactions (Miwa
et al., 1978; Kaminsky and Guengerich, 1985); it participates
in monooxygenation reactions catalyzed by a large number of
individual P450 enzymes. Moreover if one assumes that there
is approximately 15 mg of microsomal protein/g, wet weight,
of liver (Sato and Omura, 1978) to estimate the change in
oxygen consumption that would correspond to thechanges in
the activity of P450 reductase as described by Ram and
Waxman (1992), it is evident that the changes in oxygen
consumption wouldbe in the same order of magnitude as
those that we have experimentally observed here. However,
this is only a rough estimate which is based on V,,, values;
further evidence is needed to generate any quantitative re-
sults. Furthermore, there are also likely to be decreases in
oxygen consumption occurring as a resultof changes in other
oxygen-dependent systems, such as those in peroxisomal OX-
idative pathways in which oxygen is used asa hydrogen
acceptor to produce hydrogen peroxide or those involved in
desaturation of fatty acids.
All of the difference in mitochondrial oxygen consumption
between cells from hypothyroid and euthyroid rats could be
accounted for by a difference in the rate of mitochondrial
Rat
Hepatocytes
in
proton leakage. There was a very small increase in the resting
oxygen consumption required to balance the activity of the
phosphorylating system in the hypothyroid cells; thus ATP
synthesis is not decreased in hypothyroid cells as might have
been expected, and thedecreased respiration rate of hypothy-
roid cells is not due to a decrease in ATP demand by cyto-
plasmic pathways. The results clearly demonstrate that the
decrease in resting oxygen consumption which is observed in
hepatocytes from hypothyroid rats is due half to a decrease
in non-mitochondrial oxygen consumption and half to a de-
crease in proton cycling across the mitochondrial inner mem-
brane. They also indicate that anychanges that may occur in
the phosphorylating system (e.g. the phosphate transporter,
the adenine nucleotide carrier) are insignificant in terms of
the altered resting oxygen consumption rate in these cells.
There were no significant differences in the A+m between
cells from the hypothyroid and euthyroid rats either at the
resting state where the values were 162mV k 5 (S.E., n = 11)
and 155 mV f 5 ( n = 121, respectively, or at state 4 where
the values were 167 mV k 4 ( n = 11) and 166 mV k 5 ( n =
12), respectively.
Quantitative Analysis of the Effects of the Altered Kinetics
of the A+,-consuming Reactions on Resting Oxygen Comump-
tion inHepatocytes from HyperthyroidRats-Fig. 5 shows the
titrations of respiration in euthyroid and hyperthyroid cells
with oligomycin and myxothiazol which were used to con-
struct Fig. 3. As before, the oxygen consumption used in the
resting state for energy-dissipating reactions can be calcu-
lated. In this case, because of the secondary effects of oligo-
mycin via the ATP concentration, the titration with myxo-
thiazol in the presence of saturating oligomycin had to be
extrapolated back a small distance to estimate the oxygen
consumption rate used to drive the proton leak at theresting
value of A+,.
All ofthe difference in resting oxygen consumption between
hepatocytes from hyperthyroid and euthyroid rats ( p 0.001)
could be attributed to differences in mitochondrial oxygen
consumption. There wasno significant difference innon-
mitochondrial oxygen consumption between hyperthyroid and
euthyroid cells. Of the difference in mitochondrial oxygen
consumption, approximately 48% could be accounted for by
differences in the rate of proton leak, and about 55% could
be accounted for by differences in the rate of ATP turnover.
Thus increased proton leak rate ( p < 0.001) and increased
ATP synthesisandturnover ( p c 0.01) contribute about
equally to the increased respiration rate of hepatocytes from
hyperthyroid rats as compared with euthyroid controls. To
check that theconclusions were not substantially affected by
the small extrapolation involved in the calculation above, we
also conducted an analysis at the highest potential actually
measured in the hyperthyroid leak titration (128 mV). At this
potential there was still a clear stimulation of both proton
leak flux and ATP turnover flux in hyperthyroid cells com-
pared with euthyroid controls.
Resting state Aqm was significantly lower ( p = 0.002) in
hyperthyroid cells (142 mV f 4; n = 12) than in euthyroid
cells (162 mV k 4; n = 12) as expected if the A+,,, consumption
for ATP synthesis and by the proton leak is increased more
than the A+, production. It was not possible to determine
state4 in hyperthyroid hepatocytes because of the sec-
ondary effects of oligomycin; state 4 A$= in cells from the
euthyroid controls was 166 mV (k 5; n = 12).
CONCLUSIONS
The differences in oxygen consumption rate between he-
patocytes from hypothyroid and euthyroid rats are entirely
 Effects of Thyroid Hormoneson Respiration in RatHepatocytes 14859

-
FIG. 5. Relationship between A$,,,
and respiration rate in hepatocytes
from euthyroid control rats ( A ) and
Oxygon conaumptlon (nmol02hln/mg dry collr)
hyperthyroid rats ( B ) . As in Fig. 4
except thatthedeterminations were
made on hepatocytes from hyperthyroid
rats and paired euthyroid controls. The
response of substrate oxidation to A$,,,
could not be measured in hyperthyroid
cellsbecause oligomycin caused a de-
crease in A$,,,; theresponse of theproton
leak to A$= was extrapolated by eye to
Euthyroid
* I5.98 lzz2zzP 2.00

thesame value of A$,,, as inresting cells

sorespiration
the
that attributable
to
proton leak and ATP synthesis in the
restingstate could be calculated. The
S.E. associatedwiththisextrapolated
Hyperlhyroid
* = 9.88
-
t
5.03

-
t
point was taken to be the mean S.E. of 4
8% Proton bak 55% ATP turnover
the other points on the myxothiazol ti-
tration curve. Each data point represents % Contribution to tho dlfference in resting cellular oxygen consumption
mean f S.E. of 12 determinations for
hyperthyroid rats and12 determinations
for euthyroid control rats. Each A$,,, de-
termination was carried out in triplicate;
each oxygen consumption determination
was carried out simultaneously in dupli-
cate. S.E. (for non-mitochondrial oxygen
consumption) and pooled S.E. (for pro-
ton leak-dependent and ATP turnover-
dependent oxygen consumption) are in-
dicated
the S.E. for the
point on each B 200
proton leak curve at theresting A$,,, was 180
estimated as the mean of t,he S.E. forthe 160
two data points adjacentto it.
e

I I
0 0
0 1 2 3 4 5 6 7 8 9 1 0

Oxygon conoumptlon(nmol 02/rnlWmg dry atlo)

due to changes in the propertiesof the mitochondrial proton hypothyroidism and hyperthyroidism alterations in the rate
leak andinnon-mitochondrial oxygen consumption.The of mitochondrialproton leak are responsiblefor approxi-
mechanisms involved in the changes in non-mitochondrial mately 50% of the differences in resting oxygen consumption
oxygen consumption are unknown but are likely to involve in intact hepatocytes from hypothyroid or hyperthyroid rats
altered microsomal and/or peroxisomal oxidative pathways as compared with euthyroid rats. The remaining50% of the
and warrant further investigation. These results show that differences are due to decreased non-mitochondrial oxygen
during hypothyroidism any changes that may occur in the consumption in hypothyroid cells and to increased ATP turn-
catabolic pathways, the citricacid cycle, the electron transport over in hyperthyroid cells.
chain, or the cellular ATP-consumingreactions,haveno Acknowledgments-We thank Mark Leachfor experttechnical
significant role in causing changes in respiration rate in rat assistanceincludingthepreparation of the hepatocytes. We also
hepatocytes, at least, under our experimental conditions. The thank Gina Briggs and John Richardson for assistance with the rats.
differences in resting oxygen consumption rate between he- We are grateful to Dan Hill for preparing the electron micrographs,
to Richie Porter for advice on experimental techniques, and to Ber-
patocytes from hyperthyroid and euthyroid rats are caused by nard Korzeniewski for criticism of the manuscript.
changes in the kinetics of the proton leak and of ATP turn-
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