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Comparison of thyroxine and 3,3′,5′-triiodothyronine metabolism in rat kidney and liver homogenates
Comparison
Michael The
effects
treatment, rates liver
of agents
of Thyroxine and 3,3’,5’-Triiodothyronine Metabolism in Rat Kidney and Liver Homogenates M. Kaplan,
added
Jeffrey
in vitro,
in vivo
and fasting for 72 hr on T,-T,
and rT,
degradation
homogenates
mogenates.
rates
were
5 mM
DTT
B. Tatro, PTU
conversion
in rat kidney
compared.
and
In kidney
stimulated
both
ho-
reactions,
whereas
0.3 mM diamide, 0.1 PM iopanoic acid, 17 PM PTU and 1 mM 2,Cdinitrophenol inhibited both reactions; 25 WM methimazole had no effect. DTT also
stimulated
homogenates. liver than showed for
of
these
reactions
in
liver
in kidney
homogenates.
Kinetic
in
analysis
that the k, for T, in kidney and liver homo-
genates k,
both
Diamide was a less potent inhibitor
were similar, but not identical, and that the rT,
in kidney
again similar. particulate
and
fraction
employed,
liver
homogenates
but not precisely of
the
the same.
were
When
homogenates
a
was
the k, for Td in two kidney preparations
was 0.8 and 1 .O PM, and in two liver preparations was
2.9
and
5.5
reduced the T,-T,
/AM. PTU conversion
administered
rates and rT, degrada-
tion rates in kidney and liver homogenates of control,
reduced
the mean
tion to < 33% of control, concentration the
mean
T,-T,
serum
to < 20%
T, concentra-
raised the mean serum rT,
to nine times control, but did not alter
serum
glutathione
it
in vivo
T,
content.
conversion
concentration A 72-hr
or the
hepatic
fast had no effect on
or rT, degradation
rates in kidney
homogenates
and had no effect on renal glutathione
content,
fasting
but
effect on TI-TJ
had
conversion
lowered
the hepatic
control.
These results,
from that
this and other there
dinase
expected
glutathione
inhibitory
content
strongly
iodothyronine
that
thesis that the iodothyronine
findings suggest
5’-monodeio-
metabolizes
rT,. The results are also compatible
and
to 79% of
along with previous
laboratories,
is a single
in rat kidney
the
in liver homogenates
both
T, and
with the hypo-
5’-monodeiodinases
in
rat kidney and liver are the same enzyme.
XTRATHYROIDAL 5’-monodeiodination of thyroxine (T,) to 3,5,3’-triiodothyronine (TJ accounts for most of the daily T, production in the rat,’ as in man.’ in the rat, liver, kidney. and pituitary have the highest T,-T, converting activity in vitro,3m5 although the pituitary contribution to the circulating T, pool is undoubtedly negligible. Conversion of T, to T, by liver and kidney tissue in vitro has been demonstrated in slice, and homogenate stutissue perfusion, dies.6-” In both liver and kidney, T,-T, converting activity resides in a particulate fraction8.‘2.” and copurifies with plasma membrane marker
E
Metabolism, Vol. 28, No. 11, (November), 1979
Roger Breitbart,
and P. Reed Larsen
preparations from enzymes. *.13.14In particulate both tissues, cytosol or thiol-reducing agents such as reduced glutathione (GSH) and dithiothreitol (DTT) are required for T,-T3 conversion.‘0,“x’6 Agents that block or oxidize sulfhydry1 groups inhibit hepatic T,-T, conversion.“,‘5 The S-monodeiodination of T, in both liver and kidney may involve transfer of the 5’-iodine from T, to an enzyme sulfhydryl group, forming a sulfenyl-iodide intermediate species.‘6,‘7 Liver and kidney are also active in degrading 3,3’,5’-triiodothyronine (rT,) in vitro.4.‘8-‘0 The major or sole degradation product of rT3 is 3,3’-diiodothyronine (Tz);i8~~20therefore, rT, degradation also represents 5’-monodeiodination of an iodothyronine. Under some reaction conditions, >90% of the rT, added to liver homogenate is converted to T,.‘8m’0 Kidney homogenate has about 75% of the rT3-T2 converting activity of liver homogenate.‘8 Hepatic T,-T, converting activity and rT,-T, converting activity are reported to copurify,14 and both are inhibited by iodoacetic acid, by 2,4-dinitrophenol and by 6-propyl-2-thiouracil (PTU).3.4.‘0.‘X PTU also inhibits T,-T, conversion in kidney homogenates.4,8.‘6 Both T,-T, conversion and rT, degradation in liver tissue in vitro are inhibited in the fasted and hypothyroid states.“.“.“m24 This information suggests that, in the rat, the hepatic enzyme that converts T4 to T, converts rT, to Tz, and that the same enzyme may be present in rat kidney. A few studies go against these hypotheses. According to one report in abstract form, renal conversion of T, to
From the Thvroid Unit, Peter Bent Brigham Hospital and Howard Hughes Medical Institute Laboratory. Harvard Medical School, Boston. Mass. Received for publication January 9, 1979. Supported in part by USPHS Grants 5-SO&RR05489-16 and I F32-AM05826. A portion of this work appeared in abstract form in Clinical Research, 27t254A. 1979. Address reprint requests to Michael M. Kaplan, Thyroid Unit, Peter Bent Brigham Hospital. Boston, Mass. 02115. tr)I979 bv Prune & Stratton, Inc. 0026-04~5/79/28/1~0010$01.00/0
1139
1140
KAPLAN ET AL.
T, is inhibited in fasting, while renal rT,-T, conversion is accelerated,” but Balsam and Ingbar,” using kidney slices, found no such inhibition of T,-T, conversion in fasting. We have reported that in hyperthyroidism, T,-T, conversion and rT, degradation rates are greater in liver homogenates but are not significantly increased in kidney homogenates.*’ Larson et a1.,6 however, found T,-T, conversion to be accelerated in kidney slices from hyperthyroid rats. The present studies were undertaken to test the hypotheses that one iodothyronine S-monodeiodinase exists in kidney that can use T4 or rT, as its substrate, and that this enzyme is the same as the hepatic iodothyronine S-monodeiodinase. We have compared the responses of T,-T, conversion and rT, degradation in rat kidney homogenates to a variety of in vitro and in vivo manipulations and compared these responses with those of the corresponding reactions in rat liver homogenates. MATERIALS
AND
METHODS
Preparation of Homogenates Male Sprague Dawley rats (Zivic Miller Laboratories, Allison Park, Pa.) were used in all experiments. In the fasting studies, the initial weights were 175-250 g and the fasted animals were deprived of food for 72 hr with free access to tap water. In two experiments, PTU was injected i.p. in a dose of 1 mg/ 100 g body weight at 0900 hr and 1700 hr for 1 or 2 days with the animals sacrificed 16 hr after the last injection; control animals were injected with vehicle in the same schedule. The animals were decapitated, trunk blood was collected, the kidneys minced and homogenized in 3 volumes (w/v) 0.05 M Tris-0.15 M NaCI, pH 7.6, using a Potter-Elvehjem homogenizer with motor driven Teflon@ pestle. Liver homogenates were prepared by the same method. Supernatants of the homogenates from centrifugation at 2000 g were used in all incubations. This preparation accurately reflects whole homogenate T, deiodination in liver, as a particulate described previously. *6 In some experiments subcellular fraction (P,) was prepared by centrifuging the 2000-g supernatant from liver or kidney homogenates at 160,000 g for 45 min. resuspending the pellet to the original volume in Tris-NaCl by homogenization, centrifuging again at 160,000 g for 30 min. and again resuspending the pellet to the original volume in Tris-NaCI.
Incubation Procedure Incubations were carried out immediately after preparation of the homogenates. In the in vivo experiments (fasting and PTU administration) homogenates from each animal were incubated separately. In the in vitro studies, homogenates from several animals were pooled. Incubations were carried out with 1 ml homogenate or PZ per incubation tube
at 37°C. L-T,, 1.3 pM (this and all other concentration values refer to final concentration in homogenate) was added, and aliquots were taken at 0 (immediately after T, addition) and 15 min. or D,L-rT,, 15.4 nM, was added and aliquots taken at 0 and I, 2, or 4 min. The optimal durations for the rT, incubations were determined by preliminary experiments so that the rate of rT, degradation would be both measurable and constant during the incubation period. T,, rT,, and other agents were added in IO-40 ~1 volumes. Incubations using P, were carried out in closed vessels under nitrogen to minimize oxidation of added GSH. The timed aliquots of the incubation mixtures were immediately mixed with 2 ~0195% ethanol and stored at -4OC for at least 18 hr. Other details of the incubation procedure have been described previously.4
Assay Methods The ethanol extracts were assayed for T, and rT, by radioimmunoassay. The T, assay has been described.26 The rT, assay was modified from that used previously,4 although the same antibody was used. Each assay tube contained (1) 25 r.d blank ethanolic extract of homogenate (no T, or rT, added) in the standards or 25 r.d unknowns, (2) 25 ~1 rT, standards in glycine-acetate buffer, pH 8.6 (GAB), 0.1% ovalbumin, or 25 ~1 GAB, 0.1% ovalbumin (unknowns), (3) 950 ~1 GAB, 0.01% ovalbumin with anti-rT,, 1:70000, and (4) [12’1] rT,. Free and bound tracer were separated with dextran-coated charcoal. The sensitivity of the assay was 2 pg/tube. T, and rT, concentrations were corrected for recovery, and reaction rates were calculated as described previousIY.~ Rates were expressed as fmol T, produced or fmol rT, degraded/min/mg protein. In the kinetic analyses of the rate of rT, degradation as a function of rT, concentration, the mean rT, concentration during the course of the incubation, determined as described previously,4 was used in calculations as suggested by Segel. *’ Lineweaver-Burk analysis was employed. Serum T,. T,, rT,, and TSH concentrations were measured by radioimmunoassay.4~2*~“’ The rT, serum assay was modified by use of 100 ~1 serum, 0.05% sodium salicylate as blocker, and dextran-coated charcoal separation of bound and free label. Cross reactivity of T, in rT, assay, 0.04%. was subtracted for each serum, as described! Protein was measured in duplicate by the Lowry method” with BSA as standard. Glutathione content (reduced plus oxidized forms) of liver and kidney tissue was estimated as described by Owens and Belcherr’ in metaphosphoric acid extracts of tissue. Extracts were mixed with Ellman’s reagent, and AOD,,,/min was measured after addition of glutathione reductase and NADPH. Glutathione determinations were done in duplicate. All results are presented as mean + SE. The t test for unpaired values was used to compare means. Equations for the regression lines in kinetic analyses were calculated by the least squares method.
Reagents Iopanoic acid was generously provided by Dr. F. C. Nachod of the Sterling-Winthrop Research Institute, Rensselaer, N.Y. Methimazole (2-mercapto-I-methylimidazole)
TI AND rT, METABOLISM
1141
IN KIDNEY AND LIVER
Eflects on Reaction Rates of Agents Added to Homogenates In Vitro
from the Aldrich Chemical Co., Milwaukee, and 6-propyl-2-thiouracil (PTU) from General Biochemicals, Chagrin Falls, Ohio. T,, T,, dithiothreitol, diamide (diazene-dicarboxylic acid bis (N, N-dimethylamide)), 2,4-dinitrophenol, and the reagents for the glutawas obtained
Wis.,
thione assay were obtained St. Louis, MO. D,L-rT,
from the Sigma Chemical
was generously
supplied
Results of several studies are shown in Table 1, and are expressed as percent of control rates to facilitate comparison of effects on the two reactions and comparison of experiments on different days. Two sulfhydryl-active agents were tested: DTT, a thiol-reducing agent,33 and diamide, a thiol-oxidizing agent.34 DTT increased the rates of T,-T, conversion and rT, degradation in kidney homogenates to 2-3 times the control values. The reaction rates with 20 mM DTT were not greater than those at 5 mM DTT. Diamide significantly inhibited T,-T, conversion and rT, degradation in kidney homogenates at 0.3 mM and 0.6 mM. lopanoic acid inhibited T,-T, conversion and rT, degradation in kidney homogenates. The extent of inhibition of the two reactions was quite similar at the three iopanoic acid concentrations tested. Seventeen PM PTU reduced the T,-T, conversion rate and rT, degradation rate in kidney homogenates to onethird of control, whereas 25 FM methimazole had no effect. Dinitrophenol at 1 mM almost
Co..
by Dr. Hans
Cahnmann.
RESULTS
Time, Temperature, and pH Effects on T,T, Conversion in Kidney Homogenates Production of T, from T, proceeded at a constant rate for 20 min and then slowed; continued production was measured up to 60 min but little or none occurred from 60 to 150 min. Degradation of rT, in homogenates of normal kidneys occurred at a constant rate for approximately 2 min, after which the rate decreased appreciably. Temperature and pH dependencies of T,-T, conversion were very similar to those reported by Chiraseveenuprapund et al.’ There was no measurable T, degradation (< lo?%) when 15.4 nM T, was added to kidney homogenates and incubations were carried out for 40 min. Table 1. Effect of Agents Added In Vitro on T.-T,
Conversion and rT, Degradation in Rat Tissue Homogenates
T,-T, ConversionRate (% Control ? SE) Agent
Kldnev
rT, begradat,onRate (% Control ? SE) LlVW
Kldney
LPJer
Dithiothreitol 5mM 20 mM
313 * 15’
183 + 35t
190 t 34t
191 + 45t
217 + 5’
189 + 13t
194 f 19t
192 f 15t
Diamide 0.3 mM
42 ? 8‘
91?6NS
19 * 7”
83 _+6 NS
0.6 mM
22 & 3’
74 + 4 NS
18 * 7’
45 * lot (73 + 7t)
lopanoic acid 0.1 PM
34 + 3*
(58 i 5’)
39 * 3’
l.OpM
16 + 5*
I10 + 2’)
13 + 8”
(43 + 6’)
4 + 2’
(8 + 3’)
5 t 34
(19 * 41)
10.0
pM
Propylthiouracil 17 PM
31 *9”
33 + 3’
Methimazole 25 PM
108 r 18NS
88 t 4 NS
2,4-dinitrophenol 1 .O mM
5 * 2”
(48 ? 16t)
10 ? 2t
(21 & 21)
rT, 19 nM
48 + 9t
(31 t 8’)
39 nM
16 t 7’
(3 ? l*)
Incubations were performed in triplicate or quadruplicate simultaneously with 3-6
control incubations. Experiments with the different
agents and different tissues were done at different times. Homogenates from livers or kidneys of 4-6 rats were pooled and incubated with 1.3 gM L-T, or 15.4 nM D.L-rT,. Values in parentheses have been reported previously4 and are included for comparison.
‘p -c0.01 tp -c0.05
compared to simultaneous control. compared to simultaneous control.
NS. Not significant.
1142
completely inhibited renal T,-T, conversion and rT, degradation, and rT, reduced the renal T,T, conversion rate in a dose-dependent manner. In these in vitro studies, therefore, the quantitative and qualitative responses of T,-T, conversion and rT, degradation in kidney homogenates were quite similar. Results of corresponding experiments in liver homogenates are also shown in Table I. DTT increased hepatic T,-T, conversion and rT, degradation rates to twice the control rates. Diamide, at 0.6 mM, partially inhibited hepatic rT, degradation but had no other significant effect in the hepatic incubations. In an earlier study, higher concentrations of diamide inhibited T,-T, conversion in identically prepared liver homogenates in a dose-dependent manner.26 The effects of iopanoic acid, PTU, and methimazole on hepatic T,-T, conversion and rT, degradation, previously reported,4 were quite similar in liver and kidney homogenates, as were the inhibitory effects of rT, on T,-T, conversion. Dinitrophenol was a somewhat more potent inhibitor in kidney homogenates than in liver homogenates.
Reaction Kinetics The effect of substrate concentration on the T,-T, conversion rate and the rT, degradation rate was studied. Eight T, concentrations between 0.65 and 10.5 &4. and seven rT, concentrations between 1.5 and 30.8 nA4 were used. Table 2 lists the resulting kinetic parameters with the corresponding parameters for the hepatic T,-T, conversion and rT, degradation reactions reported previously.4 To obtain a clearer comparison of hepatic and renal T,-T, conversion, P, preparations from liver and kidney were incubated in the presence of 8 mM GSH. Preliminary studies using P, from both liver and kidney showed that the T,-T, conversion rate was very low in the absence of added GSH and increased progressively as GSH was added to the incubation mixtures in concentrations from 2 to 20 mM. The 8 mM GSH concentration was slightly higher than the measured endogenous hepatic GSH content in the fed state, 6.1 pmol/g (2 pg/mg). T, was added in 7 concentrations between 0.13 and 13 FM in two experiments each for kidney and liver. The
KAPLAN ET AL.
Table 2.
Kinetic Parameters
Degradation
Parameter T.-T,
Conversion
and rT,
Kidney Homogenate*
Liver Homogenate*t
0.9
7.7
85
130
30
7.5
Kidney
LlWX
P, Fractiont
P* Fraction$
Conversion
k, for T, (*Mt V,,
for T.-T,
in Liver and Kidney Homogenates
0.8,
1.0
2.9, 5.5
(fmol T,/min/mg protein)
149, 161
438,
187
rT, Degradation k, for rf, (nM) V,
(fmol rT,/min/mg protein)
‘Separate T,-T,
926 homogenate
360 preparations were used to assess
conversion and rT, degradation.
tData reported previously.4 SResults of two separate experiments for each tissue. P, is the 2000-
160,000-g
pellet resuspended to the original volume
in Tris-NaCI. Incubations of this fraction were performed with 8 mM GSH added.
kinetic parameters calculated tions are shown in Table 2.
from these incuba-
Effects of PTU In Vivo Reaction rates and serum hormone concentrations in rats treated with PTU for I or 2 days and in control rats are shown in Table 3. Both PTU treatment schedules caused marked inhibition of T,-T, conversion and rT, degradation in liver and kidney homogenates to less than 20% of control values. The mean serum T, concentrations in the PTU-treated groups and the control groups were not significantly different. In the PTU-treatment groups, the mean serum T, concentrations were less than 32% of the control values. In the l-day experiment, the PTUtreated rats had a much higher serum rT, concentration of 7.2 ng/dl compared to the control value of 0.8 ng/dl. PTU treatment did not alter hepatic glutathione content. Eflects of 72-hr Fast The results of these studies are shown in Table 4. There was no significant inhibition of T,-T, conversion or rT, degradation in renal homogenates from 72-hr fasted rats as compared to fed rats. This was true whether the rates were calculated per gram tissue (not shown) or per mg protein. The protein content was similar in kidney homogenates from the fed rats, 21.3 *
T, AND rT, METABDLISM
1143
IN KIDNEY AND LIVER
Table 3. Effects of In Vivo PTU Administration
on lodothyronine
Days of PTU Treatment
Measurement
Metabolism
in the Rat
ControlGroup (mean + SE)
PTU-TreatedGroup (mean + SE)
Kidney T.-T,
Conversion rate
(fmol T,/min/mg
protein)
2
26.4
t 1.1
4.4 + 1.7”
rT, Degradation rate (fmol rT,/min/mg
2
protein)
98 + 16
<7*
Liver T.-T,
Conversion rate
(fmol T,/min/mg
protein)
GSH content (&mg
40.5
+ 2.0
3.0 + l.B*
42.0
? 5.4
4.5 f 3.1*
1
163 + 23
protein)
2
330 + 49
tissue)
1
rT, Degradation rate (fmol rT,/min/mg
1 2
T, Concentration (pg/dl) T, Concentration (ng/dl) rT, Concentration (ng/dl)
2.00
t6’ <6’
+ 0.26
1.90 + 0.29
1
3.2 k 0.4
3.7 + 0.1
2
3.4 & 0.2
3.2 + 0.2
1
32 + 3
10 * 2*
2
43 + 9
10 i- 2T
1
0.8 ? 0.2
7.2 t 1.9$
Rats were injected with PTU. 1 mg/lOO g body weight i.p., or with vehicle, twice daily for 1 or 2 days and sacrificed 16 hr after the last injection. Four PTU-treated
rats and four control rats were used in the l-day experiment and in the 2-day experiment, and all the
measurements in the two experiments were made on tissues from the same rats. Kidney and liver homogenates from each rat were incubated separately with 1.3 M L-T, or 15.4 nM D,L-rT,.
lp
< 0.001
compared to control.
Tp < 0.01 compared to control. $p < 0.02 compared to control.
Table 4.
Effects of a 72-N
Fast on lodothyronine
Metabolism
in Tissue Homogenates,
and Serum Thyroid Hormone
Measurement
”
and on Tissue Glutathione
Content
Concentrations
Fed tmean + SE)
Fasted (mean + SE)
P
Kidney T,-T,
Conversion rate
fmol T,/min/mg
16
protein
pmol T,/min/lOO g body wt rT, Degradation rate fmol rT,/min/mg
8 4
protein
GSH content (pg/mg tissue)
35.8
? 4.1
1.90 * 0.40 186 + 29
4
0.68
12 8 4
28.6
* 4.9
NS
1.79 f 0.27
NS
194 * 32
NS
t 0.06
NS
+ 0.02
0.68
38.4
+ 4.0
18.0 + 1.8
< 0.001
23.4
? 1.9
8.3 ? 0.9
< 0.001
1.98 + 0.09
1.57 * 0.13
< 0.05
Liver T,-T,
Conversion rate
fmol T,/min/mg
protein
pmol T,/min/ 100 g body wt GSH content (pg/mg tissue)
T, Concentration @g/dl)
16
4.1 + 0.2
1.6 + 0.1
< 0.001
T, Concentration fng/dl)
16
53 * 4
20 + 2
< 0.001
Abbreviation: NS. not significant. In each of four experiments, T,-T,
conversion was measured in kidney homogenates from 4 fed and 4 fasted rats, In some of the
experiments other measurements were made as well. Comparisons between fed and fasted rats were made on tissues processed simultaneously. Results of the experiments have been pooled: “n”
is the number of fed and fasted rats for which the particular measurements are available. Tissue homogenates from each rat were incubated separately with 1.3 ELML-T, or 15.4 nM D,L-rT,. The
T,-T,
conversion rates expressed as pmol/min/lOO
g body wt are indices of total renal and hepatic enzyme activity.
1144
KAPLAN
0.8 mg/ml, n = 16, and kidney homogenates from the fasted rats, 21.4 k 1.O mg/ml, n = 16. An index of total renal T,-T, converting activity was calculated using the known homogenate dilution and the weight of both kidneys, normalizing to 100 g body weight. Since neither the T, nor the endogenous cofactor concentrations were saturating and since some activity may have been lost in the 2000-g sediment, this index is not an absolute measure of maximal tissue activity, but should reflect the presence or absence of a change in the overall T,-producing capacity of the tissue. There was no significant difference between the fed and fasted groups in this index of renal T,-T, conversion, In liver homogenates, the expected decrease in the T4-T, conversion rate was found. The rate per mg protein in the fasted group was 47% of the rate in the fed group. The index of total hepatic activity in the fasted group was 35% of the index in the fed group. Renal GSH content was similar in the fed and fasted rats, whereas hepatic GSH content in the fasted group was 79% of the hepatic GSH content in the fed group. DISCUSSION
These studies add to previously reported characteristics of T,-T, conversion in rat kidney homogenate by demonstrating inhibition by iopanoic acid, 2,4-dinitrophenol, and diamide, and showing no change in the fasted state. Balsam and Ingbar” have recently reported no change in Td-T, conversion rates in kidney slices from fasted rats. There is no clear explanation for the contradicting results of Gavin et al.,” who found a decrease in renal T4-T3 conversion. Degradation of rT, in kidney homogenate is shown here to share these properties of TI-Tj conversion and also share properties of renal T4-T, conversion previously reported: inhibition by PTU in vivo16and in vitro,4,8*‘6stimulation by DTT,16 and absence of inhibition by methimazo1e.8y’6Renal rT, degradation and renal T4-T, conversion are inhibited to a similar degree in hypothyroidism.2’ As noted earlier, rT, degradation proceeds mainly or entirely by 5’-monodeiodination,‘s-20 as does T,-T3 conversion. In sum, T4-Tj conversion and rT, degradation in kidney homogenates respond in the same way to all in vitro and in vivo influences tested to date; therefore, the hypothesis that a single enzyme catalyzes these two reactions has strong support.
ET AL.
Many of the properties of T4 and rT,-5’monodeiodination in liver homogenates are quite similar to those in kidney homogenates, but differences do exist. Neither renal reaction is substantially inhibited in the fasted state, whereas both hepatic reactions are.4V”,23,24 GSH is probably the endogenous sulfhydryl-containing cofactor for iodothyronine monodeiodination,26.35so the discrepancy during fasting may be explained, at least in part, by the finding that renal GSH content is not altered in fasting, whereas hepatic GSH content falls (Table 3). Decreases in rat hepatic GSH content during fasting and protein deprivation have been described previously,36,37 whereas renal GSH content is maintained during protein deprivation.36 Both renal reactions are inhibited by lower diamide concentrations than the hepatic reactions. The degree of inhibition of T,-T, conversion in liver homogenate by diamide is probably determined by the endogenous GSH content.26 Since the GSH measurements reported here, as well as those of others,36.3sindicate that renal GSH content is 30%40% of hepatic GSH content, the finding that higher diamide concentrations are required for inhibitory effects in liver homogenates than in kidney homogenates can be explained on this basis. The kinetic studies do not resolve the question of the number of enzymes involved in these reactions. The apparent k, for T., and rT, in liver and kidney homogenates were similar, but not exactly the same. These k, values are in agreement with the data of others in both liver31’8*‘9~39 and kidney’ homogenates. In liver4 and kidney homogenates from normal rats, > 80% of 15.4 nM D,L-rT, is degraded if incubations are longer than 10 min, showing that D- and L-rT, are both degraded. A difference between the kinetic parameters for D- and L-rT, would not substantially change the marked difference in k,s for T4 and rTJ. Since liver and kidney cytosol proteins bind T4 and rT3,4’ since these proteins have different binding capacities in the two tissues,“’ and since endogenous GSH content differs in liver and kidney, the kinetic parameters in homogenates from the two tissues may not be strictly comparable. When particulate fractions were incubated with the same GSH concentration to eliminate these factors, apparent k,s for T, in the two tissues were closer, but still not precisely the same. Final assessment of
T, AND rT, METABDLISM
1145
IN KIDNEY AND LIVER
the identity or non-identity of liver and kidney iodothyronine S-monodeiodinases must await further enzyme purification. PTU, administered in vivo, inhibits extrathyroidal T,-T, conversion in rat and man, resulting in a lower serum T3/T4 ratio.4’43 The present results show that PTU given in vivo has the same inhibitory effect on hepatic T4-T, conversion as on renal T,-T, conversion.‘6 In addition, given in vivo, PTU inhibits rT, degradation in liver and kidney homogenates, and causes a significant elevation in serum rT, concentrations. The changes induced by PTU in liver and kidney metabolism of the iodothyronines can thus explain the acute alterations in serum iodothyronine concentrations. The extreme elevation of serum rT, after PTU treatment in pregnant rats reported recently44 was not found here. To the extent that homogenate results can be
applied to the intact animal, the results of fasting studies suggest that the liver is far more important than the kidney in the overall reduction in the T4-T, conversion that occurs in the fasted rat.26945If V,,, values obtained by the present method reflect relative in vivo reaction rates, then the liver is a quantitatively more important site of extrathyroidal T3 production than the kidney. The quantitative contributions of the liver and of the kidney to total in vivo extrathyroidal T, production are not established; more nearly physiologic techniques, such as organ perfusion and assessment of T, production at other sites, are needed to completely define the sources of T, in the intact animal.
ACKNOWLEDGMENT We thank Anne Duli for expert secretarial assistance.
REFERENCES 1. Abrams GM, Larsen PR: Triiodothyronine and thyroxine in the serum and thyroid glands of iodine-deficient rats. J Clin Invest 52:2522-2531, 1973 2. Schimmel M, Utiger RD: Thyroidal and peripheral production of thyroid hormones. Review of recent findings and their clinical implications. Ann Intern Med 87:760-768, 1977 3. Chopra IJ: A study of extrathyroidal conversion of thyroxine (T,) to 3,3’,5-triiodothyronine (T,) in vitro. Endocrinology 101:453-463, 1977 4. Kaplan MM, Utiger RD: Iodothyronine metabolism in rat liver homogenates. J Clin Invest 61:459-47 I, 1978 5. Silva JE, Kaplan MM, Cheron RG, et al: Thyroxine to 3,5.3’-triiodothyronine conversion by rat anterior pituitary and liver. Metabolism 27:1601ll607, 1978 6. Larson FD, Tomita K. Albright EC: Deiodination of thyroxine to triiodothyronine by kidney slices of rats with varying thyroid function. Endocrinology 57:338-344, 1955 7. Glitzer MS, Symchowicz S, Gross J: Metabolism of labeled thyroxine in perfused rabbit kidney in vitro. Fed Proc 15:76,1956 8. Chiraseveenuprapund P, Buergi U. Goswami A, et al: Conversion of L-thyroxine to triiodothyronine in rat kidney homogenate. Endocrinology 102:6 12-622, I978 9. Becker DV, Prudden JF: The metabolism of “‘Ilabeled thyroxine and triiodothyronine and diiodotyrosine by an isolated perfused rabbit liver. Endocrinology 64: 136-l 48. 1959 10. Visser TJ, Van der Does-Tobt I, Dotter R, et al: Subcellular localization of a rat liver enzyme converting thyroxine to triiodothyronine and possible involvement of essential thiol groups. Biochem J 157:479-493. 1976 1I. Balsam A, Ingbar SH: The influence of fasting, diabetes and several pharmacological agents on the pathways of thyroxine metabolism in rat liver. J Clin Invest 62:415424, 1978
12. Hesch RD, Brunner G, Soling HD: Conversion of thyroxine (T,) and triiodothyronine (T,) and the subcellular localisation of the converting enzyme. Clin Chim Acta 59:209-213, 1975 13. Leonard JL, Rosenberg IN: Subcellular distribution of thyroxine 5’-deiodinase in the rat kidney: A plasma membrane location. Endocrinology 103:274-280, 1978 14. Maciel RMB, Ozawa Y, Chopra IJ: Plasma membrane: A site for T, and reverse T, (rT,) outer ring monodeiodinase activities. Clin Res 26:309, 1978 (abstract) IS. Chopra IJ: Sulfhydryl groups and the monodeiodination of thyroxine to triiodothyronine. Science 199:904-906, 1978 16. Leonard JL, Rosenberg IN: Thyroxine S’-deiodinase activity in rat kidney: Observations on activation by thiols and inhibition by propylthiouracil. Endocrinology 103:21372144, 1978 17. Visser TJ: A tentative review of recent in vitro observations of the enzymatic deiodination of iodothyronines and its possible physiologic implications. Mol Cell Endocrinol 10:241-247. 1978 18. Chopra IJ, Wu SY, Nakamura Y, et al: Monodeiodination of 3,5,3’-triiodothyronine and 3,3’,5’-triiodothyronine to 3,3’-diiodothyronine in vitro. Endocrinology 102: 10991106. 1978 19. Hiifner M, Grussendorf M: Investigations on the deiodination of thyroxine (T+) to 3,3’-diiodothyronine (3,3’T2) in rat liver homogenate. Clin Chim Acta 85:243-251, 1978 20. Visser TJ, Fekkes D, Dotter R, et al: Sequential deiodination of thyroxine in rat liver homogenate. Biochem J 174:221-229. 1978 21. Kaplan MM, Utiger RD: Iodothyronine metabolism in liver and kidney homogenates from hyperthyroid and hypothyroid rats. Endocrinology 103:156-161, 1978 22. Balsam A, Sexton F, Ingbar SH: The effect of thy-
1146
KAPLAN
roidectomy, hypophysectomy and hormone replacement on the formation of triiodothyronine from thyroxine in rat liver and kidney. Endocrinology 103:1759-1767, 1978 23. Harris ARC, Fang SL, Vagenakis AG, et al: Effect of starvation, nutriment replacement and hypothyroidism on in vitro hepatic T, to T, conversion in the rat. Metabolism 27:1680-1690, 1978 24. Grussendorf M, Htifner M: Induction of the thyroxine (T,) to triiodothyronine (T,) converting enzyme in rat liver by thyroid hormone analogs. Clin Chim Acta 80:61-66, 1977 25. Gavin LA, McMahon FA, Cavalieri RR: Thyroxine deiodination in rat liver and kidney: The differential effects of glucose refeeding. Program of the 54th Meeting of the American Thyroid Association, T6, 1978 (abstract) 26. Kaplan MM: Subcellular alterations causing reduced hepatic thyroxine 5’-monodeiodinase activity in fasted rats. Endocrinology 104:58-64, 1979 27. Segel I: Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems. New York, Wiley, 1975, p 57 28. Larsen PR: Direct immunoassay of triiodothyronine in human serum. J Clin Invest 51:1939-1949, 1972 29. Larsen PR, Dockalova J, Sipula D, et al: Immunoassay of thyroxine in unextracted human serum. J Clin Endocrinol Metab 37:177-182, 1973 30. Silva JE, Larsen PR: Peripheral metabolism of homologous thyrotropin in euthyroid and hypothyroid rats: Acute effects of thyrotropin releasing hormone, T3 and Tq. Endocrinology 302:1783-1796, 1978 31. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with Folin phenol reagent. J Biol Chem 193:265-275, 32. Owens
1951 CWI,
Belcher
RV:
A calorimetric
micro-
method for the determination of glutathione. Biochem J 94:705-7 11, 1965 33. Cleland WW: Dithiothreitol, a new protective agent for SH groups. Biochemistry 3:480-482, 1964 34. Kosower NS, Kosower EM, Wertheim B: Diamide, a new reagent for the intracellular oxidation of glutathione to
the disulfide.
Biochem
Biophys
Res Commun
ET AL.
37:593-596,
1969 35. Balsam A, Ingbar SH: The mechanism of regulation of triiodothyronine (Ta) generation from thyroxine (T4) in liver of normal and fasted rats. Clin Res 26:489A, 1978 (abstract) 36. Leaf G, Neuberger A: The effect of diet on glutathione content of the liver. Biochem J 41:280-287, 1947 37. Tataeishi N, Higashi T, Shinya S, et al: Studies on the regulation of glutathione level in rat liver. J Biochem 75:93103.1974 38. Tietze F: Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal Biochem 27:502-522, 1969 39. HiIfner M, Grussendorf M, Ntokalou M: Properties of the thyroxine (T4) monodeiodinating system in rat liver homogenate. Clin Chim Acta 78:251-259, 1977 40. Dillman W. Surks MI, Oppenheimer JH: Quantitative aspects of iodothyronine binding by cytosol proteins of rat liver and kidney. Endocrinology 95:492-498, 1974 41. Larsen PR, Frumess RD: Comparison of the biological effects of thyroxine and triiodothyronine in the rat. Endocrinology 100:980-988, 1977 42. Saberi
M, Sterling
FH,
Utiger
RD: Reduction
in
extra-thyroidal triiodothyronine production by propylthiouracil in man. J Clin Invest 55:218-223, 1975 43. Geffner DL, Azukizawa M, Hershman J: Propylthiouracil blocks extrathyroidal conversion of thyroxine to triiodothyronine and augments thyrotropin secretion in man. J Clin Invest 55:224-229. 1975 44. Benson MC, Liu JP, Huang YP, et al: Differential effects of triiodothyronine and 3,5-dimethyl-3’-isopropyl-lthyronine treatment of maternal rats upon hepatic L-triiodothyronine aminotransferase activity in fetal rats. Endocrinology 102:562-567, 1978 45. Nathanielsz PW: The effect of diet and acute starvation on the deiodination of thyroxine and triiodothyronine in the thyroidectomized rat. J Physiol 206:701-710, 1970
Michael The
effects
treatment, rates liver
of agents
of Thyroxine and 3,3’,5’-Triiodothyronine Metabolism in Rat Kidney and Liver Homogenates M. Kaplan,
added
Jeffrey
in vitro,
in vivo
and fasting for 72 hr on T,-T,
and rT,
degradation
homogenates
mogenates.
rates
were
5 mM
DTT
B. Tatro, PTU
conversion
in rat kidney
compared.
and
In kidney
stimulated
both
ho-
reactions,
whereas
0.3 mM diamide, 0.1 PM iopanoic acid, 17 PM PTU and 1 mM 2,Cdinitrophenol inhibited both reactions; 25 WM methimazole had no effect. DTT also
stimulated
homogenates. liver than showed for
of
these
reactions
in
liver
in kidney
homogenates.
Kinetic
in
analysis
that the k, for T, in kidney and liver homo-
genates k,
both
Diamide was a less potent inhibitor
were similar, but not identical, and that the rT,
in kidney
again similar. particulate
and
fraction
employed,
liver
homogenates
but not precisely of
the
the same.
were
When
homogenates
a
was
the k, for Td in two kidney preparations
was 0.8 and 1 .O PM, and in two liver preparations was
2.9
and
5.5
reduced the T,-T,
/AM. PTU conversion
administered
rates and rT, degrada-
tion rates in kidney and liver homogenates of control,
reduced
the mean
tion to < 33% of control, concentration the
mean
T,-T,
serum
to < 20%
T, concentra-
raised the mean serum rT,
to nine times control, but did not alter
serum
glutathione
it
in vivo
T,
content.
conversion
concentration A 72-hr
or the
hepatic
fast had no effect on
or rT, degradation
rates in kidney
homogenates
and had no effect on renal glutathione
content,
fasting
but
effect on TI-TJ
had
conversion
lowered
the hepatic
control.
These results,
from that
this and other there
dinase
expected
glutathione
inhibitory
content
strongly
iodothyronine
that
thesis that the iodothyronine
findings suggest
5’-monodeio-
metabolizes
rT,. The results are also compatible
and
to 79% of
along with previous
laboratories,
is a single
in rat kidney
the
in liver homogenates
both
T, and
with the hypo-
5’-monodeiodinases
in
rat kidney and liver are the same enzyme.
XTRATHYROIDAL 5’-monodeiodination of thyroxine (T,) to 3,5,3’-triiodothyronine (TJ accounts for most of the daily T, production in the rat,’ as in man.’ in the rat, liver, kidney. and pituitary have the highest T,-T, converting activity in vitro,3m5 although the pituitary contribution to the circulating T, pool is undoubtedly negligible. Conversion of T, to T, by liver and kidney tissue in vitro has been demonstrated in slice, and homogenate stutissue perfusion, dies.6-” In both liver and kidney, T,-T, converting activity resides in a particulate fraction8.‘2.” and copurifies with plasma membrane marker
E
Metabolism, Vol. 28, No. 11, (November), 1979
Roger Breitbart,
and P. Reed Larsen
preparations from enzymes. *.13.14In particulate both tissues, cytosol or thiol-reducing agents such as reduced glutathione (GSH) and dithiothreitol (DTT) are required for T,-T3 conversion.‘0,“x’6 Agents that block or oxidize sulfhydry1 groups inhibit hepatic T,-T, conversion.“,‘5 The S-monodeiodination of T, in both liver and kidney may involve transfer of the 5’-iodine from T, to an enzyme sulfhydryl group, forming a sulfenyl-iodide intermediate species.‘6,‘7 Liver and kidney are also active in degrading 3,3’,5’-triiodothyronine (rT,) in vitro.4.‘8-‘0 The major or sole degradation product of rT3 is 3,3’-diiodothyronine (Tz);i8~~20therefore, rT, degradation also represents 5’-monodeiodination of an iodothyronine. Under some reaction conditions, >90% of the rT, added to liver homogenate is converted to T,.‘8m’0 Kidney homogenate has about 75% of the rT3-T2 converting activity of liver homogenate.‘8 Hepatic T,-T, converting activity and rT,-T, converting activity are reported to copurify,14 and both are inhibited by iodoacetic acid, by 2,4-dinitrophenol and by 6-propyl-2-thiouracil (PTU).3.4.‘0.‘X PTU also inhibits T,-T, conversion in kidney homogenates.4,8.‘6 Both T,-T, conversion and rT, degradation in liver tissue in vitro are inhibited in the fasted and hypothyroid states.“.“.“m24 This information suggests that, in the rat, the hepatic enzyme that converts T4 to T, converts rT, to Tz, and that the same enzyme may be present in rat kidney. A few studies go against these hypotheses. According to one report in abstract form, renal conversion of T, to
From the Thvroid Unit, Peter Bent Brigham Hospital and Howard Hughes Medical Institute Laboratory. Harvard Medical School, Boston. Mass. Received for publication January 9, 1979. Supported in part by USPHS Grants 5-SO&RR05489-16 and I F32-AM05826. A portion of this work appeared in abstract form in Clinical Research, 27t254A. 1979. Address reprint requests to Michael M. Kaplan, Thyroid Unit, Peter Bent Brigham Hospital. Boston, Mass. 02115. tr)I979 bv Prune & Stratton, Inc. 0026-04~5/79/28/1~0010$01.00/0
1139
1140
KAPLAN ET AL.
T, is inhibited in fasting, while renal rT,-T, conversion is accelerated,” but Balsam and Ingbar,” using kidney slices, found no such inhibition of T,-T, conversion in fasting. We have reported that in hyperthyroidism, T,-T, conversion and rT, degradation rates are greater in liver homogenates but are not significantly increased in kidney homogenates.*’ Larson et a1.,6 however, found T,-T, conversion to be accelerated in kidney slices from hyperthyroid rats. The present studies were undertaken to test the hypotheses that one iodothyronine S-monodeiodinase exists in kidney that can use T4 or rT, as its substrate, and that this enzyme is the same as the hepatic iodothyronine S-monodeiodinase. We have compared the responses of T,-T, conversion and rT, degradation in rat kidney homogenates to a variety of in vitro and in vivo manipulations and compared these responses with those of the corresponding reactions in rat liver homogenates. MATERIALS
AND
METHODS
Preparation of Homogenates Male Sprague Dawley rats (Zivic Miller Laboratories, Allison Park, Pa.) were used in all experiments. In the fasting studies, the initial weights were 175-250 g and the fasted animals were deprived of food for 72 hr with free access to tap water. In two experiments, PTU was injected i.p. in a dose of 1 mg/ 100 g body weight at 0900 hr and 1700 hr for 1 or 2 days with the animals sacrificed 16 hr after the last injection; control animals were injected with vehicle in the same schedule. The animals were decapitated, trunk blood was collected, the kidneys minced and homogenized in 3 volumes (w/v) 0.05 M Tris-0.15 M NaCI, pH 7.6, using a Potter-Elvehjem homogenizer with motor driven Teflon@ pestle. Liver homogenates were prepared by the same method. Supernatants of the homogenates from centrifugation at 2000 g were used in all incubations. This preparation accurately reflects whole homogenate T, deiodination in liver, as a particulate described previously. *6 In some experiments subcellular fraction (P,) was prepared by centrifuging the 2000-g supernatant from liver or kidney homogenates at 160,000 g for 45 min. resuspending the pellet to the original volume in Tris-NaCl by homogenization, centrifuging again at 160,000 g for 30 min. and again resuspending the pellet to the original volume in Tris-NaCI.
Incubation Procedure Incubations were carried out immediately after preparation of the homogenates. In the in vivo experiments (fasting and PTU administration) homogenates from each animal were incubated separately. In the in vitro studies, homogenates from several animals were pooled. Incubations were carried out with 1 ml homogenate or PZ per incubation tube
at 37°C. L-T,, 1.3 pM (this and all other concentration values refer to final concentration in homogenate) was added, and aliquots were taken at 0 (immediately after T, addition) and 15 min. or D,L-rT,, 15.4 nM, was added and aliquots taken at 0 and I, 2, or 4 min. The optimal durations for the rT, incubations were determined by preliminary experiments so that the rate of rT, degradation would be both measurable and constant during the incubation period. T,, rT,, and other agents were added in IO-40 ~1 volumes. Incubations using P, were carried out in closed vessels under nitrogen to minimize oxidation of added GSH. The timed aliquots of the incubation mixtures were immediately mixed with 2 ~0195% ethanol and stored at -4OC for at least 18 hr. Other details of the incubation procedure have been described previously.4
Assay Methods The ethanol extracts were assayed for T, and rT, by radioimmunoassay. The T, assay has been described.26 The rT, assay was modified from that used previously,4 although the same antibody was used. Each assay tube contained (1) 25 r.d blank ethanolic extract of homogenate (no T, or rT, added) in the standards or 25 r.d unknowns, (2) 25 ~1 rT, standards in glycine-acetate buffer, pH 8.6 (GAB), 0.1% ovalbumin, or 25 ~1 GAB, 0.1% ovalbumin (unknowns), (3) 950 ~1 GAB, 0.01% ovalbumin with anti-rT,, 1:70000, and (4) [12’1] rT,. Free and bound tracer were separated with dextran-coated charcoal. The sensitivity of the assay was 2 pg/tube. T, and rT, concentrations were corrected for recovery, and reaction rates were calculated as described previousIY.~ Rates were expressed as fmol T, produced or fmol rT, degraded/min/mg protein. In the kinetic analyses of the rate of rT, degradation as a function of rT, concentration, the mean rT, concentration during the course of the incubation, determined as described previously,4 was used in calculations as suggested by Segel. *’ Lineweaver-Burk analysis was employed. Serum T,. T,, rT,, and TSH concentrations were measured by radioimmunoassay.4~2*~“’ The rT, serum assay was modified by use of 100 ~1 serum, 0.05% sodium salicylate as blocker, and dextran-coated charcoal separation of bound and free label. Cross reactivity of T, in rT, assay, 0.04%. was subtracted for each serum, as described! Protein was measured in duplicate by the Lowry method” with BSA as standard. Glutathione content (reduced plus oxidized forms) of liver and kidney tissue was estimated as described by Owens and Belcherr’ in metaphosphoric acid extracts of tissue. Extracts were mixed with Ellman’s reagent, and AOD,,,/min was measured after addition of glutathione reductase and NADPH. Glutathione determinations were done in duplicate. All results are presented as mean + SE. The t test for unpaired values was used to compare means. Equations for the regression lines in kinetic analyses were calculated by the least squares method.
Reagents Iopanoic acid was generously provided by Dr. F. C. Nachod of the Sterling-Winthrop Research Institute, Rensselaer, N.Y. Methimazole (2-mercapto-I-methylimidazole)
TI AND rT, METABOLISM
1141
IN KIDNEY AND LIVER
Eflects on Reaction Rates of Agents Added to Homogenates In Vitro
from the Aldrich Chemical Co., Milwaukee, and 6-propyl-2-thiouracil (PTU) from General Biochemicals, Chagrin Falls, Ohio. T,, T,, dithiothreitol, diamide (diazene-dicarboxylic acid bis (N, N-dimethylamide)), 2,4-dinitrophenol, and the reagents for the glutawas obtained
Wis.,
thione assay were obtained St. Louis, MO. D,L-rT,
from the Sigma Chemical
was generously
supplied
Results of several studies are shown in Table 1, and are expressed as percent of control rates to facilitate comparison of effects on the two reactions and comparison of experiments on different days. Two sulfhydryl-active agents were tested: DTT, a thiol-reducing agent,33 and diamide, a thiol-oxidizing agent.34 DTT increased the rates of T,-T, conversion and rT, degradation in kidney homogenates to 2-3 times the control values. The reaction rates with 20 mM DTT were not greater than those at 5 mM DTT. Diamide significantly inhibited T,-T, conversion and rT, degradation in kidney homogenates at 0.3 mM and 0.6 mM. lopanoic acid inhibited T,-T, conversion and rT, degradation in kidney homogenates. The extent of inhibition of the two reactions was quite similar at the three iopanoic acid concentrations tested. Seventeen PM PTU reduced the T,-T, conversion rate and rT, degradation rate in kidney homogenates to onethird of control, whereas 25 FM methimazole had no effect. Dinitrophenol at 1 mM almost
Co..
by Dr. Hans
Cahnmann.
RESULTS
Time, Temperature, and pH Effects on T,T, Conversion in Kidney Homogenates Production of T, from T, proceeded at a constant rate for 20 min and then slowed; continued production was measured up to 60 min but little or none occurred from 60 to 150 min. Degradation of rT, in homogenates of normal kidneys occurred at a constant rate for approximately 2 min, after which the rate decreased appreciably. Temperature and pH dependencies of T,-T, conversion were very similar to those reported by Chiraseveenuprapund et al.’ There was no measurable T, degradation (< lo?%) when 15.4 nM T, was added to kidney homogenates and incubations were carried out for 40 min. Table 1. Effect of Agents Added In Vitro on T.-T,
Conversion and rT, Degradation in Rat Tissue Homogenates
T,-T, ConversionRate (% Control ? SE) Agent
Kldnev
rT, begradat,onRate (% Control ? SE) LlVW
Kldney
LPJer
Dithiothreitol 5mM 20 mM
313 * 15’
183 + 35t
190 t 34t
191 + 45t
217 + 5’
189 + 13t
194 f 19t
192 f 15t
Diamide 0.3 mM
42 ? 8‘
91?6NS
19 * 7”
83 _+6 NS
0.6 mM
22 & 3’
74 + 4 NS
18 * 7’
45 * lot (73 + 7t)
lopanoic acid 0.1 PM
34 + 3*
(58 i 5’)
39 * 3’
l.OpM
16 + 5*
I10 + 2’)
13 + 8”
(43 + 6’)
4 + 2’
(8 + 3’)
5 t 34
(19 * 41)
10.0
pM
Propylthiouracil 17 PM
31 *9”
33 + 3’
Methimazole 25 PM
108 r 18NS
88 t 4 NS
2,4-dinitrophenol 1 .O mM
5 * 2”
(48 ? 16t)
10 ? 2t
(21 & 21)
rT, 19 nM
48 + 9t
(31 t 8’)
39 nM
16 t 7’
(3 ? l*)
Incubations were performed in triplicate or quadruplicate simultaneously with 3-6
control incubations. Experiments with the different
agents and different tissues were done at different times. Homogenates from livers or kidneys of 4-6 rats were pooled and incubated with 1.3 gM L-T, or 15.4 nM D.L-rT,. Values in parentheses have been reported previously4 and are included for comparison.
‘p -c0.01 tp -c0.05
compared to simultaneous control. compared to simultaneous control.
NS. Not significant.
1142
completely inhibited renal T,-T, conversion and rT, degradation, and rT, reduced the renal T,T, conversion rate in a dose-dependent manner. In these in vitro studies, therefore, the quantitative and qualitative responses of T,-T, conversion and rT, degradation in kidney homogenates were quite similar. Results of corresponding experiments in liver homogenates are also shown in Table I. DTT increased hepatic T,-T, conversion and rT, degradation rates to twice the control rates. Diamide, at 0.6 mM, partially inhibited hepatic rT, degradation but had no other significant effect in the hepatic incubations. In an earlier study, higher concentrations of diamide inhibited T,-T, conversion in identically prepared liver homogenates in a dose-dependent manner.26 The effects of iopanoic acid, PTU, and methimazole on hepatic T,-T, conversion and rT, degradation, previously reported,4 were quite similar in liver and kidney homogenates, as were the inhibitory effects of rT, on T,-T, conversion. Dinitrophenol was a somewhat more potent inhibitor in kidney homogenates than in liver homogenates.
Reaction Kinetics The effect of substrate concentration on the T,-T, conversion rate and the rT, degradation rate was studied. Eight T, concentrations between 0.65 and 10.5 &4. and seven rT, concentrations between 1.5 and 30.8 nA4 were used. Table 2 lists the resulting kinetic parameters with the corresponding parameters for the hepatic T,-T, conversion and rT, degradation reactions reported previously.4 To obtain a clearer comparison of hepatic and renal T,-T, conversion, P, preparations from liver and kidney were incubated in the presence of 8 mM GSH. Preliminary studies using P, from both liver and kidney showed that the T,-T, conversion rate was very low in the absence of added GSH and increased progressively as GSH was added to the incubation mixtures in concentrations from 2 to 20 mM. The 8 mM GSH concentration was slightly higher than the measured endogenous hepatic GSH content in the fed state, 6.1 pmol/g (2 pg/mg). T, was added in 7 concentrations between 0.13 and 13 FM in two experiments each for kidney and liver. The
KAPLAN ET AL.
Table 2.
Kinetic Parameters
Degradation
Parameter T.-T,
Conversion
and rT,
Kidney Homogenate*
Liver Homogenate*t
0.9
7.7
85
130
30
7.5
Kidney
LlWX
P, Fractiont
P* Fraction$
Conversion
k, for T, (*Mt V,,
for T.-T,
in Liver and Kidney Homogenates
0.8,
1.0
2.9, 5.5
(fmol T,/min/mg protein)
149, 161
438,
187
rT, Degradation k, for rf, (nM) V,
(fmol rT,/min/mg protein)
‘Separate T,-T,
926 homogenate
360 preparations were used to assess
conversion and rT, degradation.
tData reported previously.4 SResults of two separate experiments for each tissue. P, is the 2000-
160,000-g
pellet resuspended to the original volume
in Tris-NaCI. Incubations of this fraction were performed with 8 mM GSH added.
kinetic parameters calculated tions are shown in Table 2.
from these incuba-
Effects of PTU In Vivo Reaction rates and serum hormone concentrations in rats treated with PTU for I or 2 days and in control rats are shown in Table 3. Both PTU treatment schedules caused marked inhibition of T,-T, conversion and rT, degradation in liver and kidney homogenates to less than 20% of control values. The mean serum T, concentrations in the PTU-treated groups and the control groups were not significantly different. In the PTU-treatment groups, the mean serum T, concentrations were less than 32% of the control values. In the l-day experiment, the PTUtreated rats had a much higher serum rT, concentration of 7.2 ng/dl compared to the control value of 0.8 ng/dl. PTU treatment did not alter hepatic glutathione content. Eflects of 72-hr Fast The results of these studies are shown in Table 4. There was no significant inhibition of T,-T, conversion or rT, degradation in renal homogenates from 72-hr fasted rats as compared to fed rats. This was true whether the rates were calculated per gram tissue (not shown) or per mg protein. The protein content was similar in kidney homogenates from the fed rats, 21.3 *
T, AND rT, METABDLISM
1143
IN KIDNEY AND LIVER
Table 3. Effects of In Vivo PTU Administration
on lodothyronine
Days of PTU Treatment
Measurement
Metabolism
in the Rat
ControlGroup (mean + SE)
PTU-TreatedGroup (mean + SE)
Kidney T.-T,
Conversion rate
(fmol T,/min/mg
protein)
2
26.4
t 1.1
4.4 + 1.7”
rT, Degradation rate (fmol rT,/min/mg
2
protein)
98 + 16
<7*
Liver T.-T,
Conversion rate
(fmol T,/min/mg
protein)
GSH content (&mg
40.5
+ 2.0
3.0 + l.B*
42.0
? 5.4
4.5 f 3.1*
1
163 + 23
protein)
2
330 + 49
tissue)
1
rT, Degradation rate (fmol rT,/min/mg
1 2
T, Concentration (pg/dl) T, Concentration (ng/dl) rT, Concentration (ng/dl)
2.00
t6’ <6’
+ 0.26
1.90 + 0.29
1
3.2 k 0.4
3.7 + 0.1
2
3.4 & 0.2
3.2 + 0.2
1
32 + 3
10 * 2*
2
43 + 9
10 i- 2T
1
0.8 ? 0.2
7.2 t 1.9$
Rats were injected with PTU. 1 mg/lOO g body weight i.p., or with vehicle, twice daily for 1 or 2 days and sacrificed 16 hr after the last injection. Four PTU-treated
rats and four control rats were used in the l-day experiment and in the 2-day experiment, and all the
measurements in the two experiments were made on tissues from the same rats. Kidney and liver homogenates from each rat were incubated separately with 1.3 M L-T, or 15.4 nM D,L-rT,.
lp
< 0.001
compared to control.
Tp < 0.01 compared to control. $p < 0.02 compared to control.
Table 4.
Effects of a 72-N
Fast on lodothyronine
Metabolism
in Tissue Homogenates,
and Serum Thyroid Hormone
Measurement
”
and on Tissue Glutathione
Content
Concentrations
Fed tmean + SE)
Fasted (mean + SE)
P
Kidney T,-T,
Conversion rate
fmol T,/min/mg
16
protein
pmol T,/min/lOO g body wt rT, Degradation rate fmol rT,/min/mg
8 4
protein
GSH content (pg/mg tissue)
35.8
? 4.1
1.90 * 0.40 186 + 29
4
0.68
12 8 4
28.6
* 4.9
NS
1.79 f 0.27
NS
194 * 32
NS
t 0.06
NS
+ 0.02
0.68
38.4
+ 4.0
18.0 + 1.8
< 0.001
23.4
? 1.9
8.3 ? 0.9
< 0.001
1.98 + 0.09
1.57 * 0.13
< 0.05
Liver T,-T,
Conversion rate
fmol T,/min/mg
protein
pmol T,/min/ 100 g body wt GSH content (pg/mg tissue)
T, Concentration @g/dl)
16
4.1 + 0.2
1.6 + 0.1
< 0.001
T, Concentration fng/dl)
16
53 * 4
20 + 2
< 0.001
Abbreviation: NS. not significant. In each of four experiments, T,-T,
conversion was measured in kidney homogenates from 4 fed and 4 fasted rats, In some of the
experiments other measurements were made as well. Comparisons between fed and fasted rats were made on tissues processed simultaneously. Results of the experiments have been pooled: “n”
is the number of fed and fasted rats for which the particular measurements are available. Tissue homogenates from each rat were incubated separately with 1.3 ELML-T, or 15.4 nM D,L-rT,. The
T,-T,
conversion rates expressed as pmol/min/lOO
g body wt are indices of total renal and hepatic enzyme activity.
1144
KAPLAN
0.8 mg/ml, n = 16, and kidney homogenates from the fasted rats, 21.4 k 1.O mg/ml, n = 16. An index of total renal T,-T, converting activity was calculated using the known homogenate dilution and the weight of both kidneys, normalizing to 100 g body weight. Since neither the T, nor the endogenous cofactor concentrations were saturating and since some activity may have been lost in the 2000-g sediment, this index is not an absolute measure of maximal tissue activity, but should reflect the presence or absence of a change in the overall T,-producing capacity of the tissue. There was no significant difference between the fed and fasted groups in this index of renal T,-T, conversion, In liver homogenates, the expected decrease in the T4-T, conversion rate was found. The rate per mg protein in the fasted group was 47% of the rate in the fed group. The index of total hepatic activity in the fasted group was 35% of the index in the fed group. Renal GSH content was similar in the fed and fasted rats, whereas hepatic GSH content in the fasted group was 79% of the hepatic GSH content in the fed group. DISCUSSION
These studies add to previously reported characteristics of T,-T, conversion in rat kidney homogenate by demonstrating inhibition by iopanoic acid, 2,4-dinitrophenol, and diamide, and showing no change in the fasted state. Balsam and Ingbar” have recently reported no change in Td-T, conversion rates in kidney slices from fasted rats. There is no clear explanation for the contradicting results of Gavin et al.,” who found a decrease in renal T4-T3 conversion. Degradation of rT, in kidney homogenate is shown here to share these properties of TI-Tj conversion and also share properties of renal T4-T, conversion previously reported: inhibition by PTU in vivo16and in vitro,4,8*‘6stimulation by DTT,16 and absence of inhibition by methimazo1e.8y’6Renal rT, degradation and renal T4-T, conversion are inhibited to a similar degree in hypothyroidism.2’ As noted earlier, rT, degradation proceeds mainly or entirely by 5’-monodeiodination,‘s-20 as does T,-T3 conversion. In sum, T4-Tj conversion and rT, degradation in kidney homogenates respond in the same way to all in vitro and in vivo influences tested to date; therefore, the hypothesis that a single enzyme catalyzes these two reactions has strong support.
ET AL.
Many of the properties of T4 and rT,-5’monodeiodination in liver homogenates are quite similar to those in kidney homogenates, but differences do exist. Neither renal reaction is substantially inhibited in the fasted state, whereas both hepatic reactions are.4V”,23,24 GSH is probably the endogenous sulfhydryl-containing cofactor for iodothyronine monodeiodination,26.35so the discrepancy during fasting may be explained, at least in part, by the finding that renal GSH content is not altered in fasting, whereas hepatic GSH content falls (Table 3). Decreases in rat hepatic GSH content during fasting and protein deprivation have been described previously,36,37 whereas renal GSH content is maintained during protein deprivation.36 Both renal reactions are inhibited by lower diamide concentrations than the hepatic reactions. The degree of inhibition of T,-T, conversion in liver homogenate by diamide is probably determined by the endogenous GSH content.26 Since the GSH measurements reported here, as well as those of others,36.3sindicate that renal GSH content is 30%40% of hepatic GSH content, the finding that higher diamide concentrations are required for inhibitory effects in liver homogenates than in kidney homogenates can be explained on this basis. The kinetic studies do not resolve the question of the number of enzymes involved in these reactions. The apparent k, for T., and rT, in liver and kidney homogenates were similar, but not exactly the same. These k, values are in agreement with the data of others in both liver31’8*‘9~39 and kidney’ homogenates. In liver4 and kidney homogenates from normal rats, > 80% of 15.4 nM D,L-rT, is degraded if incubations are longer than 10 min, showing that D- and L-rT, are both degraded. A difference between the kinetic parameters for D- and L-rT, would not substantially change the marked difference in k,s for T4 and rTJ. Since liver and kidney cytosol proteins bind T4 and rT3,4’ since these proteins have different binding capacities in the two tissues,“’ and since endogenous GSH content differs in liver and kidney, the kinetic parameters in homogenates from the two tissues may not be strictly comparable. When particulate fractions were incubated with the same GSH concentration to eliminate these factors, apparent k,s for T, in the two tissues were closer, but still not precisely the same. Final assessment of
T, AND rT, METABDLISM
1145
IN KIDNEY AND LIVER
the identity or non-identity of liver and kidney iodothyronine S-monodeiodinases must await further enzyme purification. PTU, administered in vivo, inhibits extrathyroidal T,-T, conversion in rat and man, resulting in a lower serum T3/T4 ratio.4’43 The present results show that PTU given in vivo has the same inhibitory effect on hepatic T4-T, conversion as on renal T,-T, conversion.‘6 In addition, given in vivo, PTU inhibits rT, degradation in liver and kidney homogenates, and causes a significant elevation in serum rT, concentrations. The changes induced by PTU in liver and kidney metabolism of the iodothyronines can thus explain the acute alterations in serum iodothyronine concentrations. The extreme elevation of serum rT, after PTU treatment in pregnant rats reported recently44 was not found here. To the extent that homogenate results can be
applied to the intact animal, the results of fasting studies suggest that the liver is far more important than the kidney in the overall reduction in the T4-T, conversion that occurs in the fasted rat.26945If V,,, values obtained by the present method reflect relative in vivo reaction rates, then the liver is a quantitatively more important site of extrathyroidal T3 production than the kidney. The quantitative contributions of the liver and of the kidney to total in vivo extrathyroidal T, production are not established; more nearly physiologic techniques, such as organ perfusion and assessment of T, production at other sites, are needed to completely define the sources of T, in the intact animal.
ACKNOWLEDGMENT We thank Anne Duli for expert secretarial assistance.
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KAPLAN
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