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Dietary Modification of Thyroxine Deiodination in
Rat Liver is Not Mediated by Hepatic Sulfhydryls
LAURENCE A. GAVIN, FRANCIS A. MCMAHON, and M. MOELLER, Division of
Endocrinology/-Metabolismn, Veterans Administration Medical Center and
Department ofAledicine, Universityl ofCalifornia, San Francisco, California 94121
ABSTRACT The enzymatic deiodination of thy- INTRODUCTION
roxine (T4) is thiol dependent. Fasting (72 h) depresses Caloric intake appears to be a major physiological
hepatic T4 deiodination and lowers the hepatic content regulator of thyroid hormone activation. It has been
of nonprotein sulfhydryls (NP-SH) and reduced gluta- demonstrated that both short-term fasting (1) and long-
thione (GSH). It has been proposed that the fasting term starvation (2-4) significantly depress the circu-
effect may be mediated through these alterations in lating levels of3,5,3'-triiodothyronine (T3)' and elevate
hepatic suilflydryls. To test the importance of tissue 3,3',5'-triiodothyronine (reverse-T3; rT3) in man. In
(hepatic) thiol content in the modification of T4 de- man these dietary induced changes in thyroxine (T4)
iodination consequent to dietary manipulation, we deiodination are aconsequence ofadecrease in the daily
examined the sequential deiodination of T4 to 3,5,3'- production ofT3 and in the disposal ofrT3 (2-4). Tissue
triiodothyronine (T3) (5'-deiodination) and 3,3',5- studies in animals, particularly in the rat liver, tend to
triiodothyronine (reverse T3, rT3) (5-deiodination) support these in vivo findings (5, 6).
in liver homogenates without added thiol from groups The actual mechanisms by which fasting induces
of rats fed Purina lab chow (P) (a protein-rich diet), these changes have not been fully elucidated. Previous
glucose alone (G), or glucose plus cysteine (G,) for 72 h reports suggest that the effects of fasting result from a
or fasted (F) for the same period. The initial rate ofeach change in the concentration ofdeiodinase (7, 8) and/or
reaction was compared to the tissue concentrations of in the availability of a cofactor (9, 10).
NP-SH and GSH. T4 deiodination is thiol dependent (11) and, as the
Dietary manipulation induced significant changes in tissue (hepatic) levels of nonprotein sulfhydryls
hepatic deiodination ofT4 to T3 and rT3 and sulfhydryl (NP-SH) and reduced glutathione (GSH) are dimin-
content. There was a marked dissociation between the ished in the fasted state (9), it has been proposed that
rate of each reaction and hepatic NP-SH and GSH the effect of fasting is mediated throuigh a deficiency
levels. T4 deiodination by the alternative pathways was of these cofactors. It has been demonstrated that the
significantly higher (P < 0.01) in G > P > F. In contrast effects of fasting on T4 deiodination to T3 can be
both hepatic NP-SH and GSH concentrations were reversed with the addition ofan excess ofthiol reagents
greater (P < 0.05) in P > F > G. The lack of a relation- in vitro (9, 10). However, we and others have failed
ship between these parameters was further emphasized to induce this reversal of the fasting effect (7, 8).
on analysis of tissue from rats fed GC. Despite the To test the importance of tissue (hepatic) thiol
clearcut (P < 0.01) increase in hepatic NP-SH and GSH content in the modulation of T4 deiodination con-
conseqtuent to GC feeding, there was no alteration in sequent to dietary modification, we examined the de-
iodothyronine deiodination compared to the group fed iodinatino ofT4 to T3 and rT3 in liver homogenate from
glucose alone. rats fed a variety of diets or fasted for the same period.
These data indicate that the effects of diet on T4 The specific activity of each reaction was compared to
monodeiodination in liver are not mediated by changes
in the tissue level of sulfhydryl compounds but rather
involve alterations in the concentrations of the de- 'Abbreviations used in this paper: F, fasted animals; G,
iodinases. animals fed 20% glucose in H20; GC, animals fed gluicose plus
levels of cysteine increasing from 0.25%, G,, to 0.5%, GC2,
Receivedfor puiblication 11 December 1979 and in revised and finally to 0.75%, Gc3; GSH, reduced glutathione; NP-SH,
form 21 January 1980. nonprotein sulfhydryls; P, Purina-fed controls; T4, thyroxine;
T3, 3,5,3'-triiodothyronine; rT3, 3,3',5'-triodothyronine.
J. Clin. Invest. ( The American Society for Clinical Investigation, Inc. 0021-9738/80/04/0943/04 $1.00 943
Volume 65 April 1980 943-946
the tissue content ofNP-SH and GSH. The data suggest the amount of product was corrected by the appropriate re-
that the effect of fasting is mediated through a change covery and the amount of iodothyronine present in unin-
in the concentration of deiodinase rather than in the cubated control tubes. The concen-
availability of cofactor. Analysis ofhepatic GSH and NP-SH groups.
tration of both GSH and NP-SH was measured in all homog-
enates using a modification of the methods described by
METHODS Hissin and Hilf (13) for GSH and Sedlak and Lindsay (14)
T4 and T3 were obtained from Sigma Chemical Co., St. Louis, for NP-SH. A 2.5% homogenate was prepared in a 0.02 M
Mo. rT3 was generously provided by Dr. Eugene C. Jorgensen, EDTAsolution, (200mgliverin 8 ml 0.02 M EDTA). Aliquots
University of California, San Francisco. 1251-T3 and I251-rT3, were taken for protein estimation by the method of Lowry
ring position at specific radio- et al. (15). 4.5 ml of homogenate was mixed with 1.5 ml 25%
each labeled in the phenolic were purchased from New H3 P03 in cellulose nitrate tubes ('/2 x 21/2 in.) to precipitate
activities of 500-900 ,uCi/,Lg, proteins. This preparation was centrifuged at 4°C at 100,000 g
England Nuclear, Boston, Mass. Goat anti-rabbit gamma- for 30 min.
globulin serum was obtained from Antibodies Inc., Davis, GSH assay. To 10 ,ul of the 100,000 g supernate, 2 ml of
Calif. o-phthaladehyde was purchased from Sigma Chemical 0.1 M P04 (13.8 g Na2NPO4 + 0.73 g NaH2PO4) containing
Co., 5,5'-dithiobis-2-nitrobenzoic acid from Aldrich Chemical 0.2 M EDTA (pH 8.0) and 100 al o-phthaladehyde were
Co., Inc., Milwaukee, Wis., and EDTA was supplied by added. Afterthorough mixingand incubation atroom tempera-
Eastman Organic Chemicals Div., Eastman Kodak, Rochester, ture for 15 min, the solutions were transferred to quartz
N. Y. Other chemicals used were reagent grade and were curvettes. Fluorescence at 420 nm was determined with
purchased from commercial suppliers. the activation at 350 nm, on a Perkin-Elmer fluorescence
Animals and diets. Incuibations were performed in hepatic spectrophotometer (Perkin-Elmer Corp., Instrument Div.,
preparations obtained from male Sprague-Dawley Rats. Norwalk, Conn.). The GSH content was read off a standard
Within each experiment the rats (groups, n = 4) were closely curve (GSH: 5-100 ,M) and results expressed per milligram
matchedforweightandage. For 1 wkbefore each study period protein. 2 ml of 100,000 supernate, 4 ml of0.4 M
the animals were maintained on an ad lib intake of H2O and NP-SH assay. To 0.1 ml
Purina rodent laboratory chow; 5001 (25% protein content) Tris-HCL (pH 8.9) and of5,5'-dithiobis-2-nitrobenzoic
from Ralston Purina Co., St. Louis, Mo. Fasted animals (F) acid were added. After mixing and incubating at room air for
were totally deprived of calories (H20 ad lib only) for 72 h 5 min, the NP-SH content was determined colorimetrically at
before sacrifice, whereas fed controls were allowed access to 412 nm on a Hitachi spectrophotometer (Hitachi America,
food. In the initial experiments the controls ate Purina (P) or Ltd., San Francisco, Calif). Results were compared with those
drank 20% glucose in H2O (G). In later experiments a number obtained from prepared standards. The NP-SH concentration
of groups were fed glucose plus cysteine (Gr) and compared was expressed per milligram protein. from experi-
to the glucose fed group. Diets were enriched with cysteine Statistical methods. Meanvaltues (mean+±SE)
to increase the hepatic content of sulfhydryls. Cysteine was mental groups were compared to controls using Student's
added to glucose at the following concentrations: 0.25% (Gc,); t test for unpaired data.
0.5% (GC2), and 0.75% (Gr3). Liver was homog-
Liver homogenization and incubation. RESULTS
enized (800 g pellet discarded) and T4 incubations performed
as previously described (8). T4 (1 uM) deiodination to T3 was Effects ofdietary manipulation on body weight and
analyzed in 25% homogenate (pH 7.2), whereas T4 (1 AM) serum glucose concentration. Table I demonstrates
deiodination to rT3 was studied in 2% homogenate (pH 8.5) that body weight changes were significantly different
to facilitate optimum conditions. The buffer used for both for each dietary group. The P group gained weight,
incubations was 0.5 M Tris-HCL that contained 0.25 M whereas both the G and F groups lost weight. Despite
sucrose and 10 mM EDTA. The initial rate of each reaction
was studied; samples (100 ,ul) for analyses were removed this difference, both P and G maintained normal blood
from incubations (37°C) at 5 min (T4-rT3) and 15 min (T4-T3) glucose values. The mean serum glucose of fasted
and added to 0.9 ml of ice-cold, iodothyronine free, normal animals was significantly lower(P < 0.01) than in either
humani serum (serum extracts). The respective triiodothyro- of the fed groups.
nines in the seruim extracts were measured by the previously on serum and
described specific radioimmllnunoassays (12). In each experiment Effects ofdietary manipulation T4 T3
TABLE I and
Effects ofDietary Modification on Body Weight, Serum Glucose, T4 T3 (mean+SEM)
Percent body
Dietary Number weight Serum
group of rats change glucose T4 T3
mg/dl pgldl ng/inl
Purina (P) (12) (+) 15+2 131+6 2.7+.26 0.43+.03
Glucose (G) (12) (-) 10+1 112+7 2.5+.20 0.53+.04t
Fast (72 h) (F) (12) (-)2203 89+3* 1.2+.07* 0.24+.01*
* P < 0.01, F vs. fed.
P < 0.05, G vs. P.
944 L. A. Gavin, F. A. McMahon, and M. Moeller
The F group mean serum T4 and 40- -8
concentration. < T,
valuies were significantly lower (P 0.01) than the NP-SH z
respective valuies in the fed grouips (Table I). Althouigh z 30- OT,to T3 I -6 0
there was no difference between the meain seruim T, m
values for P and G, the T3 mean in P was significantly 0 - 0
analysis of all J -4 E
less (P < 0.05) than in G. Regression I. 20- 0
data revealed a lack of correlation between seru-m T3 c
and glucose valuies, (r = -0.3, P > 0.2). - z
Chlanges in hepatic 5' and 5 deiodination. It is 10- -2 _
clear fromi Fig. 1 (left) that T4 deiodination to T3 and rT3 0
was significantly hiigher (P < 0.01) in G coinpared to P. LO
0- t-
The rates of b)oth reactions were lowest (P < 0.001) in G GC1 GC2 GC3
the F grouip. Thuts, the total deiodination ofT4 by these FIGURE 2 The effects of feeding groups of rats (in = 4) a 20%
alternative pathways was signiificantly different for glutcose diet alone (GJ grouip) or gltucose enrichedl with cessteine
eaclh dietary groul). Fig. 1 (right) illustrates that the (G, grouip) on hepatic NP-SfI and T4 deiodinationi to T3.
hepatic contenit of NP-SH and GSH wvas significantly
differenit (P < 0.01) between each of the three grouips. to cysteine feeding. There were no differences in body
The su-rprisinig finding, however, was that the levels of weight changes, serum glucose, T4 or T3 valtues in the
1)oth ofthese comiipoundis were lowest in G. The hepatic G or Gc groups.
stnlfhydryl contenit was highest in P. A comparison ibe- Regression analysis ofthe data from the fouir dietary
tween the hepatic content of sulfhydryls and the grouips, P, G, G, and F failed to reveal any correlation
enzyvne activities of T4 deiodination to T3 and rT3 letween the hepatic content of sulfhydryls and the
(Fig. 1) obviously demonstrates different patterns. specific enzyme activities.
This dissociation between hepatic sulfhydryls anid T4
deiodination suggested that hepatic thiols were not DISCUSSION
regulatory uinder these conditions. a lack of correlation between
Changes in hepatic NP-SH and GSH in thte G,group). This study demonstrates and the rate of iodothyro-
Fig. 2 demonstrates the changes in hepatic NP-SH con- hepatic sulflhydryl content showved (if-
se(luent to feeding the rats 20% gltucose diets en- nine deiodincation. Each dietary grouip and
riched with increasing amotunts of cysteine. There ferent concenitrations of hepatic NP-SH, GSHi,
was an increase (P < 0.001) in hepatic suilfliydryl at specific deiodinase(s) activity, btit there was (lis-
the highest dietary cysteine intake (GC3). A similar pat- cordance between these parameters. a correlation l)e-
tern was noted for the hepatic content of GSH. How- A previouis report had stuggested T4 deiodination
ever, in spite of the increase in the tissue content of tween hepatic sulfhydryl levels aind mixed
stulfhydryls, there was no change in hepatic 5'-de- to T3. However, that sttudy compared feedinig a
iodination rate (T4 to T3), Fig. 2. Similarly, the specific diet with fasting for 48 h (9). The present report clearly
activities of T4 deiodination to rT3 were not affected dei-nonstrates a dissociation between these paramneters
by the changes in the hepatic stilfhydryls secondary when feeding of specific diets (gltucose or protein) is
compared with fasting. This is suipported by the data
AOT4 ttT3 BO NP-SH from the GC group. Thus, it is apparenit that hepatic
[flT, to RT3 SGSH NP-SHI and GSH are not the modtulators of de-
150
0 iodinase(s) activity consequient to qualitative changes
X, 125- in dietary intake. Furthermore, the data indicate that
a- these effects are mediated via alterations in deiodlinase
z 100- concentration rather than cofactor availability. Whether
0
0- or not these alterations in 5'- and 5-deiodinase activity
< 75-
Z are the primary mediators of the dietary induced
0 50-
UA changes has not been elucidated. A recent puiblication
1-1 25- demonstrated that the hepatic uptake of T4 may be
01 the critical regulatory factor (16). Fturther stuidies are
F P G F therefore warranted to determine which of these
FIGURE 1 Acomparison between hepatic T4 deiodination to changes is dominant.
T3 a-rT3 (A) and liver content of NP-SH and reduced GSH, The present data is consistent with our previous
(B). The liver homogenate preparations were obtained fromn report, which demonstrated that the addition of excess
P, G, or F groups after 72 hj. §P < 0.01, **P < 0.01, G and F sulfhydryls failed to obliterate the differences in
vs. P (A) and F and G vs. P (B), respectively. hepatic deiodinase(s) activity noted between a G and a
94
Dietart Modification of Thyroxine Deiodination 945
72 h F group (8) and a similar study of Kaplan et al. (7), Ingbar. 1978. Effect of starvation on the production and
who compared a P to a 72-h F group (7). Balsam et al. peripheral metabolism of 3,3'5'-triiodothyronine in
(10) did note that it was possible to reverse the dif- euthyroid obese subjects.j. Clin. Endocrinol. Metab. 47:
ference in T4 deiodination to T3 between a 48-h F 889-893.
group and a group fed laboratory chow, by adding GSH 4. Suda, A. K., C. S. Pittman, T. Shimizu, and J. B. Chambers.
1978. The production and metabolism of 3,5,3'-triiodo-
in vitro; however, these stuidies were performed in thyronine and 3,3',5'-triiodothyronine in normal and
tissue preparations from T4-treated animals (10). fasting subjects. J. Clin. Endocrinol. Metab. 47: 1131-
Therefore, in the rat, the combination of "chemical 1319. M. and R. D. 1978.
hypothyroidism" (low serum T4) and the caloric dep- 5. Kaplan, M., Utiger. lodothyronine
rivation induced by fasting apparently both metabolism in rat liver homogenates. J. Clin Invest. 61:
affects 459-471.
deiodinase and cofactor (sulfhydryl) concentration. 6. Harris, A. R., S. W. Fang, A. G. Vagenakis, and L. E.
However, the difference between G and P cannot be Braverman. 1978. Effect of starvation, nutrient replace-
attributed to a hypothyroid state as the mean serum ment and hypothyroidism on in vitro hepatic T4 to T,
T4 was the same in both groups. Furthermore, the conversion in the rat. Aletab. Clin. Exp. 27: 1680-1690.
7. Kaplan, M. M. 1979. Subcellular alterations causing
hepatic sulfhydryl concentration was less (P < 0.001) reduced hepatic thyroxine 5'-monodeiodination activity
in G compared to P and in addition the diet-induced 8. in fasted rats. Endocrinology. 104: 58-64.
increase (glucose plus cysteine) in hepatic NP-SH and Gavin, L. A., F. Bui, F. McMahon, and R. R. Cavalieri,
GSH did not alter the iodothyronine deiodination. 1980. Sequential deiodination of thyroxine to 3,3'-
diiodothyronine via 3,5,3'-triiodothyronine and 3,3',5'-
The greater activity of T4 deiodination (to T3 and to triiodothyronine in rat liver homogenate: the effects of
rT3) in G cannot be attributed to differences in triiodo- fasting vs glucose feeding. J. Biol. Chem. 255: 49-54.
thyronine degradation. The metabolism ofboth T3 and 9. Harris, A. R., S. L. Fang, L. Hinerfeld, L. Braverman,
rT3 to 3,3'-T2 were similar in G and P (unpublished and A. G. Vagenakis. 1979. The role ofsulfhydryl groups
observation). on the impaired hepatic 3,5,3'-triiodothyronine genera-
tion from thyroxine in the hypothyroid, starved, fetal
The observed alterations in hepatic deiodinase and neonatal rodent.J. Clin. Invest. 63: 516-524.
activities (G > P) can account for the higher serum 10. Balsam, A. and S. H. Ingbar. 1979. Observations on the
T3 values in G compared to P. The previously noted factors that control the generation of T3 from T4 in rat
higher serum T3 values in man (17) and rat (18) fed liver and the natuire of the defect induced by fasting. J.
Clin. Invest. 63: 1145-1156.
carbohydrates compared to protein are probably due to 11. Visser, T. J., I. V. Does-Tobe, R. Doctor, and G. Henne-
a similar mechanism. It has also been demonstrated mann. 1976. Subcellular localization of a rat liver
that refeeding with carbohydrate rather than protein enzyme converting thyroxine to triiodothyronine and
in fasted man and rat reverses the effects of fasting possible involvement ofessential thiol groups. Biochem.
on serum T3 and T3 generation from T4 (1, 6). J. 157: 479-482.
12. Gavin, L. A., J. N. Castle, F. A. McMahon, P. Martin,
In conclusion, this report shows that there is a lack of M. E. Hammond, and R. R. Cavalieri. 1977. Extrathyroidal
correlation between hepatic sulfhydryls and iodothyro- conversion of thyroxine to 3,3',5'-triiodothyronine (re-
nine deiodinase activity in groups of rats fed a variety verse-T3) and to 3,5,3'-triiodothyronine (T3) in humans.
of diets or fasted for an equivalent period and that the J. Clin. Endocrinol. Metab. 44: 733-742.
13. Hissin, P. J., and R. Hilf. 1976. A fluorometric method
dietary effects are not mediated via alterations in for determination of oxidized and reduced glutathione
hepatic thiols but are probably modulated through in tissues. Anal. Biochem. 74: 214-226.
changes in the concentration of deiodinase enzymes. 14. Sedlak, J., and R. H. Lindsay. 1968. Estimation of total,
protein-bound, and non-protein sulfhydryl groups in
ACKNOWLEDGMENTS tissue with Ellman's Reagent. Anal. Biochem. 25:
192-205.
This study was supported by the research division of the 15. Lowry, 0. H., N. J. Rosebough, A. L. Farr, and J. Randall.
Veterans Administration and grant AM 24013-01 from the 1951. Protein measurement with Folin phenol reagent.
National Instituites of Health. 16. J. Biol. Chem. 193: 265-275.
Jennings, A. S., D. C. Ferguson, and R. D. Utiger.
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946 L. A. Gavin, F. A. McMahon, and M. Moeller
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