| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Dipartimento di Fisiologia Generale ed Ambientale, Università di Napoli Federico II (A.Lo., L.B., S.D., F.G.), and Istituto Internazionale di Genetica e Biofisica (M.V.U.), 80134 Naples; and Facoltà di Scienze, Università degli Studi del Sannio Benevento (M.M., V.C.), 82100 Benevento; and Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli (A.La.), 81100 Caserta, Italy; and Department of Obstetric and Gynecology, Yale University School of Medicine (S.D.), New Haven, Connecticut 06520
Address all correspondence and requests for reprints to: Dr. F. Goglia, Dipartimento di Fisiologia Generale ed Ambientale, Università di Napoli Federico II, Via Mezzocannone 8, 80134 Naples, Italy. E-mail: goglia{at}biol.dgbm.unina.it
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As the lowest dose of 1 µg T2/100 g BW affects G6PD activity 3–5 times more than the same dose of T3, these data provide the first evidence that T2 is a factor capable of regulating G6PD activity.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Some of the nonnuclear-mediated actions of thyroid hormone depend upon naturally occurring iodothyronines with only modest affinity for the traditional T3-TR. Indeed, recent studies strongly suggest that 3,5-diiodothyronine (T2; the affinity of which for nuclear T3 receptors is very low) could be biologically relevant. T2 is able to rapidly stimulate the oxidative capacity and the respiration rate of human and rat cells (5, 6, 7, 8) by directly interacting with mitochondria. Similar results have been obtained using mitochondria isolated from fish (9), indicating that such effects are not restricted to mammalian species. Specific binding sites for T2 have been detected in rat liver mitochondria (10), and we found that T2 induced a calorigenic effect when chronically injected into hypothyroid rats by increasing their resting metabolism (11). By comparing these effects with those induced by T3, we deduced that T2 and T3 exert effects on resting metabolism through different mechanisms. The effects of T2 are rapid, independent of protein synthesis, and possibly mediated by its direct interaction with mitochondria (12). The effects elicited by T3, on the other hand, are slower, more prolonged, and dependent on induced changes in protein synthesis. Moreover, we showed that T2 has a relevant role in the response to cold, improving the cold tolerance of hypothyroid rats (13).
As well as indicating that T2 might play a role in the nonnuclear-mediated effects of thyroid hormones, the above data raise the possibility that it could be involved in some of the effects classically attributed to T3. In the present study, to confirm the thyromimetic activity of T2 and to gain insight into its potential role as a peripheral mediator of the effects of T3 and/or as a selective agonist responsible for some effects attributed to T3, we examined the effect of T2 on the activities of two T3-responsive NADPH-generating lipogenic enzymes: malic enzyme (ME) and glucose-6-phosphate-dehydrogenase (G6PD). ME is involved in the synthesis of the cytosolic reducing equivalent, and its synthesis is markedly increased by T3, primarily at the transcription level. G6PD is a ubiquitous enzyme of the pentose-phosphate pathway, which, like ME, influences the NADPH level and thus plays a crucial role in NADPH-mediated reductive processes in all cells. Previous results relating to the effects of T3 on G6PD have not indicated clearly whether the induction of this enzyme’s activity is or is not dependent on the transcriptional control exerted by T3 (14).
We chose the liver as the target tissue, as it is well known that
thyroid hormone strongly affects the extent to which this tissue
contributes to total lipogenesis in the rat. The liver’s contribution,
in fact, ranges from about 5% in the hypothyroid animal to about 35%
in the hyperthyroid animal (15). To determine whether the effect of
3,5-T2 is restricted to the liver, we also tested
the effect of 3,5-T2 on brown adipose tissue
(BAT). BAT was chosen because of its very active lipogenesis. As a
model, we used rats in which hypothyroidism had been induced by
combined treatment with propylthiouracil and iopanoic acid. This
treatment induces severe hypothyroidism and at the same time inhibits
all three known types of deiodinase enzymes (12). To compare the
effects of T2 with those of
T3, measurements were made in hypothyroid rats
receiving either T3 or T2.
To check the effectiveness of our treatment we measured
-glycerophosphate-dehydrogenase (-GPD) activity, a
specific marker of thyroid hormone action.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Male Wistar rats (250–300 g) were used throughout. Using our well established method (11), hypothyroidism was induced in some rats by the ip administration of propylthiouracil (1 mg/100 g BW) for 2 weeks together with a weekly ip injection of iopanoic acid (6 mg/100 g BW). These rats are referred to as the hypothyroid group.
To enable us to study the effect of T2 on hepatic enzymes, hypothyroid rats received, by once daily ip injection over a 2-week period, one of four different doses (1.0, 2.5, 5, or 10 µg/100 g BW) of T2. This method also allowed us to plot a dose-response relationship. In other hypothyroid rats T3 was administered by a once daily ip injection for a 2-week period, only at the lower doses (1.0 and 2.5 µg/100 g BW).
To enable us to study the time course of the effects of the iodothyronines on hepatic enzymes, hypothyroid animals were acutely ip injected with a single dose of 25 µg/100 g BW of either T3 or T2. As a control for the possible effects of the injection itself, saline was injected ip into hypothyroid control rats.
To enable evaluation of the possible involvement of changes in protein synthesis in the actions of the diiodothyronine tested, some hypothyroid animals were injected ip with cycloheximide (0.5 mg/100 g BW) in combination with 25 µg/100 g BW T2, then killed after 3 h.
Enzyme assays
Cytosolic malic enzyme activity was assayed by measuring NADPH
formation from K-malate and NADP, using a slightly modified version of
the method of Hsu and Lardy (16). Cytosolic G6PD activity was assayed
by measuring NADPH formation from glucose-6-phosphate and NADP, as
described by Lee (17).
We did not correct G6PD activity for the contribution to NADPH accumulation made by phosphogluconate dehydrogenase (the second enzyme in the pentose phosphate shunt that catalyses the conversion of 6-phosphogluconic acid to ribulose-5-phosphate, generating an additional molecule of NADPH) for the following reasons. 1) In all of our assays the coefficient of correlation for accumulated NADPH was more than 0.998, indicating a negligible deviation from linearity under our assay conditions. A significant contribution of phosphogluconate to NADPH formation will, in fact, lead to a nonlinearity of NADPH accumulation as a function of the time. 2) It has already been shown that under our conditions, the NADPH accumulated represents largely G6PD activity (90%) (18), and calculating G6PD activity while taking account of the activity of phosphogluconate dehydrogenase could add more potential sources of error. In both assays, the in vitro formation of NADPH was measured spectrophotometrically at 25 ± 2 C at 340 nm for 5 or 3 min at 30-sec intervals. Blanks without added substrate were routinely run to correct for the formation of NADPH from endogenous substrates.
Mitochondrial -GPD activity was assayed using rat liver mitochondria
obtained at 3000 x g, as reported in Lanni et
al. (7). This method uses phenazine methosulfate and
2-p-iodophenyl-3-p-nitrophenyl-5-phenyl-monotetrazolium
chloride as electron acceptors, as reported by Lee and Lardy (19).
Protein content was determined by the method of Hartree (20), using BSA as the protein standard.
G6PD expression
Total liver RNA was isolated by the method of Chomczynski and
Sacchi (21). For Northern blot analysis, a standard method was used
(22). Briefly, 20 µg total RNA were transferred onto nylon membranes
(Hybond-N+, Amersham Pharmacia Biotech, Arlington Heights, IL) with 20 x SSC (1 x
SSC = 150 mM NaCl and 15 mM sodium
citrate, pH 7.0).
To detect specific messenger RNA (mRNA), we used a 150-bp probe derived from human G6PD complementary DNA [sequence previously reported (23)] labeled with 32P using a random priming system. An 18S-derived oligonucleotide was used to normalize the amount of RNA for each line. Quantitative data were obtained by analysis of Northern blot experiments using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Statistical analysis
Values are presented as the mean ± SE. A
Student-Newman-Keuls test was used for multiple comparisons. Results
were considered significant when P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-GPD, -48% for ME, and -28% for G6PD).
Administration of T2 or T3
to hypothyroid rats enhanced the activities of all of the above
enzymes, but there were important differences among the effects.
-GPD
Figure 1
shows the effects of
T3 and T2 on -GPD
activity when chronically (upper panel) or acutely
(lower panel) administered to hypothyroid rats. The chronic
administration of 3,5-T2 to hypothyroid rats
induced a dose-dependent increase in -GPD activity that reached
significance (+120%) at a dose of 5 µg/100 g BW. As expected, the
chronic administration of T3 to hypothyroid rats
induced a significant stimulation of -GPD activity even at a dose of
1 µg/100 g BW. At this dose, T3 increased
-GPD activity to a value not significantly different from the
euthyroid level. At a dose of 2.5 µg/100 g BW,
T3 increased -GPD activity by 144%.
|
-GPD activity, with T3
showing the greater effect. Both effects were slow to develop. A
significant effect of T3 on -GPD was first
evident at 24 h, whereas the effect of
3,5-T2 was slower; a significant increase was
first detected 48 h after its injection into hypothyroid rats.
ME
Figure 2 shows the effects of
T3 and T2 on ME activity
when chronically (upper panel) or acutely (lower
panel) administered to hypothyroid rats. The chronic
administration of T2 to hypothyroid rats restored
ME activity to euthyroid values at a dose of 1 µg/100 g BW, with
higher doses being no more effective. In contrast, chronic
administration of T3 had a dose-related effect on
ME activity, increasing it by 123% and 288% at doses of 1 and 2.5
µg/100 g BW, respectively.
|
-GPD, both effects
were slow to develop. A significant effect of T3
on ME was first detected at 24 h, whereas the effect of
T2 was slower; a significant increase was first
detected 48 h after its injection into hypothyroid rats.
G6PD
Figure 3 shows the effects of
T3 and T2 on liver G6PD
activity when chronically (upper panel) or acutely
(lower panel) administered to hypothyroid rats. The chronic
administration of T2 to hypothyroid rats
increased liver G6PD activity by 176% at the lowest dose used (1
µg/100 g BW). The same dose of T3 increased
liver G6PD activity by only 43%. The acute administration of
T3 or T2 evoked temporally
different patterns of response. The injection of a single dose of
T2 (25 µg/100 g BW) caused a significant
increase (of about 70%) as early as 1 h after the injection,
rising to a 300% increase at 48 h. However, for
T3, the increase was significant only at 24 and
48 h.
|
OD/min mg protein vs. 1.3
± 0.14 OD/min mg protein; n = 6). Chronic administration of
3,5-T2 to hypothyroid rats increased BAT G6PD
activity significantly, and it reached values of 1.48 ± 0.2,
1.48 ± 0.23, 1.51 ± 0.166, and 2.07 ± 0.2 at doses of
1, 2.5, 5, and 10 µg/100 g BW, respectively. Chronic administration
of T3 had a lesser effect than
3,5-T2; in fact, the highest dose of
T3 used in this study (2.5 µg/100 g BW) was
sufficient to restore the level to euthyroid values, the BAT G6PD
activity at that dose was 1.4 ± 0.096 A/min mg protein.
As the effect of T2 on liver G6PD occurred
earlier than that of T3, we investigated both
effects to determine whether they were exerted at the pretranslational
or translational level. The liver G6PD mRNA level was much lower in
hypothyroid than in euthyroid rats (Fig. 4). Although T2 was
ineffective in increasing the liver G6PD mRNA level in hypothyroid
rats, T3 enhanced the level to values higher than
the euthyroid value. As the enhancement of liver G6PD activity by
T2 is thus independent of the transcription
mechanism, regulation by T2 could occur at the
translational level. To test this hypothesis, cycloheximide was
simultaneously injected with T2. This treatment
did not cause any inhibition of the early effect of
T2; the liver G6PD activity was increased by
about 160% 3 h after its injection into hypothyroid rats. We also
tried to perform a Western blot analysis of G6PD protein in rat liver
cytosol in control and T2- or
T3-treated animals, but, unfortunately, we have
not been able to detect a rat G6PD signal in Western blots with our
rabbit antihuman G6PD antibody. Although this antibody has been
successfully used against both human and mouse G6PD (24), we conclude
that it does not work in the case of rat G6PD. Because of this, we
cannot entirely exclude the possibility that the later effect of
T2 on G6PD could be affected by changes in G6PD
protein.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The mechanisms involved in the regulation of hepatic G6PD by thyroid hormone have not yet been conclusively explained. Mariash et al. (27) demonstrated that G6PD activity increased after T3 administration and that the increase was correlated with an increase in protein synthesis. Miksicek and Towle (28), using an in vitro translation assay (to indirectly quantify mRNA levels), reported that the increase in G6PD protein synthesis observed after T3 administration was secondary to an increase in G6PD mRNA. Others (29) reported that T3 had very little effect on the expression of the gene for G6PD; the enzyme’s activity was induced without a concomitant increase in its mRNA. These discrepancies may to some extent reflect differences in the thyroid hormone treatment given or in the thyroid status of the animal (euthyroid, hypothyroid, or hyperthyroid), the consequence being that deiodinase activities may be affected differently in different studies (11).
The levels of -GPD activity in our hypothyroid rats fit well with
those in previous reports in which hypothyroidism was induced in
different ways; this suggests that, in this sense, our model of
hypothyroidism is comparable with the previous ones. In our hypothyroid
rats, on the other hand, all three deiodinase enzymes were inhibited,
enabling us to attribute the observed effects to the injected
iodothyronines themselves rather than to their metabolic products
(12).
In the present report we show that 3,5-T2 administration to hypothyroid rats exerts a thyromimetic effect by stimulating ME and G6PD. Concerning the effect on ME, it has already been shown that 3,5-T2, like T3, is able to increase ME mRNA expression when injected in vivo into hypothyroid rats (30). The doses of T3 and T2 used in that study, however, were not comparable, the doses of T2 were 100 times higher than those of T3. When we used similar doses, T3 had a more pronounced effect than T2. Moreover, the effect elicited by T2 reached significance at 48 h after its injection, and it was actinomycin D sensitive (data not shown), confirming that the effect of T2 is exerted at the transcriptional level.
The most important result in this paper is that the activity of G6PD is primarily regulated by T2. Chronic administration of T2 to hypothyroid rats did not affect the levels of hepatic G6PD mRNA. In addition, cycloheximide administration did not block the early stimulatory effect of T2 on G6PD activity.
The increase in G6PD activity, and in general in the activity of lipogenic enzymes, that is induced by T2 fits well with our recent data showing that 3,5-T2 is able to increase the metabolic rate of hypothyroid rats and improve their cold tolerance (13). In fact, the idea that an increased synthesis of fatty acids may play a role in the thermogenic action of thyroid hormone is well established as well as the correlation between the ability of thyroid hormone to stimulate oxygen consumption, and lipogenesis (31). The very rapid effect of T2 on G6PD, independent of protein synthesis, seems to confirm our hypothesis that T2 may be involved in physiological situations in which very rapid extra energy utilization is required, such as cold exposure (13). The action of T2 may be exerted through activation of G6PD enzyme molecules preexisting in an inactive form, according to cellular need. Further evidence for a rapid posttranslational mechanism that leads to a 1.4-fold increase in G6PD expression has been reported in cultured kidney epithelial cells stimulated with platelet-derived growth factor (32, 33). In addition, a posttranslational mechanism leading to activation of G6PD activity expression has been hypothesized in cells originating from various tissues exposed to oxidative stress (24). As the addition of T2 to the enzyme assay did not reproducibly increase the G6PD activity, we suggest that the effect of T2 may be mediated by a signaling pathway at the plasma membrane level.
We can conclude that T2 is a factor capable of regulating the activity of this important enzyme, but the physiological significance of this regulation is still an open question. Measurement of the level of T2 in the serum and in tissues would improve our understanding of the physiological meaning of G6PD regulation by T2. Unfortunately, some methodological problems exist that do not permit us to obtain reliable values for these levels in the rat.
In the absence of direct evidence, three hypotheses may be put forward. 1) G6PD is the first and key regulatory enzyme of the pentose-phosphate pathway. By influencing the activity of this enzyme, 3,5-T2 may act as a factor that, modulating ribose phosphates synthesis, regulates at the posttranscriptional level some processes under the influence of the thyroid gland. 2) T2 could protect against oxidative stress by enhancing G6PD activity (34). A recent report has, in fact, demonstrated that G6PD activity is necessary to ensure cell survival when damage is produced by reactive oxygen intermediates (34). 3) T2, by influencing G6PD activity, could be involved in cellular differentiation. The existing evidence that inhibition of G6PD is associated with a block of the differentiation of mouse embryo fibroblasts to adipocytes provides support for this possibility (35). Further investigations, however, will be required to fully understand the physiological role played by the regulatory action of T2 on G6PD activity.
|
Footnotes |
|---|
Received July 27, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-glycerophosphate dehydrogenase and other
dehydrogenase in various organs of the rat. J Biol Chem 240:1427–1436This article has been cited by other articles:
 |
|
 |
|
  A. M. Giudetti, M. Leo, M. J. H. Geelen, and G. V. Gnoni Short-Term Stimulation of Lipogenesis by 3,5-L-Diiodothyronine in Cultured Rat Hepatocytes Endocrinology, September 1, 2005; 146(9): 3959 - 3966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Moreno, E. Silvestri, A. Lombardi, T. J. Visser, F. Goglia, and A. Lanni Identification of 3,5-Diiodo-L-Thyronine-Binding Proteins in Rat Liver Cytosol by Photoaffinity Labeling Endocrinology, June 1, 2003; 144(6): 2297 - 2303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. M. Frederiks, K. S. Bosch, J. S.S.G. De Jong, and C. J.F. Van Noorden Post-translational Regulation of Glucose-6-phosphate Dehydrogenase Activity in (Pre)neoplastic Lesions in Rat Liver J. Histochem. Cytochem., January 1, 2003; 51(1): 105 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Pinna, O. Brodel, T. Visser, A. Jeitner, H. Grau, M. Eravci, H. Meinhold, and A. Baumgartner Concentrations of Seven Iodothyronine Metabolites in Brain Regions and the Liver of the Adult Rat Endocrinology, May 1, 2002; 143(5): 1789 - 1800. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Moreno, A. Lombardi, L. Beneduce, E. Silvestri, G. Pinna, F. Goglia, and A. Lanni Are the Effects of T3 on Resting Metabolic Rate in Euthyroid Rats Entirely Caused by T3 Itself? Endocrinology, February 1, 2002; 143(2): 504 - 510. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
