Short-Term Stimulation of Lipogenesis by 3,5-L-Diiodothyronine in Cultured Rat Hepatocytes
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《内分泌学杂志》
Laboratory of Biochemistry (A.M.G., M.L., G.V.G.), Department of Biological and Environmental Sciences and Technologies, University of Lecce, 73100 Lecce, Italy
Department of Nutrition (M.J.H.G.), Graduate School of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands
Abstract
Short-term effects of 3,5-L-diiodothyronine (T2) on lipid biosynthesis were studied in cultured hepatocytes from hypothyroid rats. A comparison with the effects of T3 was routinely carried out. After T2 addition to cell cultures, a distinct stimulation of fatty acid and cholesterol syntheses, measured as incorporation of [1-14C]acetate into these lipid fractions, was observed. The T2 dose-dependent effect on both metabolic pathways, already detectable at 10–8-10–9 M, reached a 2-fold stimulation at 10–5 M T2. At this concentration, the stimulatory effect was evident within 1 h of T2 addition to the hepatocytes and increased with time up to the length of the experimental period of 4 h. T2 stimulation of lipogenesis was also confirmed by incubating hepatocytes with [3H]H2O, used as an independent index of lipogenic activity. The effects of T2 are rather specific as 3,3',5,5'-tetraiodo-D-thyronine and 3,5-diiodo-L-tyrosine were practically ineffective on both fatty acid and cholesterol synthesis. Analysis of various lipid fractions showed that T2 addition to the cells produced a significant stimulation of the incorporation of newly synthesized fatty acids into both neutral and polar lipids. By comparing the effects induced by T2 with those seen in the presence of T3, it appeared that T2 was able to mimic T3 effects. Experiments conducted in the presence of cycloheximide, a protein synthesis inhibitor, indicated that the T2 stimulatory effect on fatty acid and cholesterol synthesis was essentially independent of protein synthesis.
Introduction
THE THYROID HORMONES T3 and T4 play important roles in growth, development, cell differentiation, and metabolism through their interaction with nuclear receptors (1, 2). However, an increasing number of nonnuclear-mediated effects of thyroid hormones have been reported to occur at the plasma membrane, in mitochondria, at the cytoskeleton, and in the cytoplasm (for reviews, see Refs. 3 and 4).
In addition to T3 and T4, other iodothyronines, such as 3,5-L-diiodothyronine (T2) and 3,3'-L-diiodothyronine, produced by further peripheral deiodination of T3 and rT3, respectively, are present in biological fluids. T2 serum levels were significantly reduced in patients with hypothyroidism, and enhanced in patients with hyperthyroidism, sepsis, liver diseases, head injury, and brain tumors, suggesting a relationship between the diseases and the altered level of T3 and T2 (5). Recently, evidence has been presented indicating that T2 is able to mimic some effects of T3 on energy metabolism by acting as a biologically active analog of T3 (6, 7). It has been shown that T2 stimulates oxygen consumption in isolated perfused liver from hypothyroid rats and in mononuclear red blood cells (8, 9). T2 also increases the mitochondrial respiratory rate and cytochrome oxidase activity more rapidly than T3 (4, 10, 11), and the metabolic effects of T2 at the mitochondrial level have been observed in rats (10) and fish (12). These effects are not dependent on protein synthesis and involve a rapid and direct action of T2 with mitochondria (6, 7). The action of T2 is not limited to mitochondria as a rapid cycloheximide (CEX)-independent stimulation of glucose-6-phosphate dehydrogenase activity by T2 has been observed in rat-liver cytosol (13). Very recently the presence of cytosolic-binding proteins for T2 has been demonstrated (14). It has also been reported that T2 has significant thyromimetic effects in vivo and in vitro and it is very effective in the induction of hepatic malic enzyme gene expression (15).
It is well known that liver is a pivotal target of thyroid hormone effects. Several observations indicate that thyroid hormones greatly affect the extent to which this tissue contributes to total lipogenesis in the rats. Thyroidectomy decreases the hepatic activities of several lipogenic enzymes, such as malic enzyme, glucose-6-phosphate dehydrogenase, acetyl-coenzyme A carboxylase, and fatty acid synthase (for review see Ref. 16). Hyperthyroidism, on the other hand, increases the rate of hepatic fatty acid synthesis by raising the activities of the lipogenic enzymes (17, 18, 19, 20, 21).
However, in vivo studies cannot distinguish between direct effects of thyroid hormones on the liver and secondary effects due to endocrine or metabolite changes. The use of isolated cells can overcome this problem. Therefore, we decided to investigate whether acute effects of T2, if any, on hepatic lipogenesis occur directly by employing isolated hepatocytes.
This study presents the first demonstration of an early and direct stimulatory effect of T2 on both fatty acid and cholesterol biosynthesis in cultures of isolated rat hepatocytes.
Materials and Methods
Chemicals
[1-14C]Acetate and [3H]H2O were purchased from Amersham Pharmacia Biotech (Milan, Italy); BSA (fraction V, fatty acid-free), collagenase type I, Ham’s F-12 culture medium, and 6-n-propyl-2-thiouracil were from Sigma-Aldrich Co. (Milan, Italy); T2, T3, 3,3',5,5'-tetraiodo-D-thyronine (D-T4), 3,5-diiodo-L-tyrosine (DIT), 3-iodo-L-tyrosine, L-thyronine, and L-tyrosine were from Sigma-Aldrich and were more than 99% pure. All other reagents were of analytical grade.
Animal treatment
Male Wistar rats (250–300 g) were used throughout this study. They were kept one per cage in a temperature-controlled room under an artificial lighting regime of 12 h light, 12 h darkness. A commercial mash (Morini SpA, Milan, Italy) was available for ad libitum consumption, and the animals had free access to water. Hypothyroidism in rats was chronically produced as in Refs. 18 and 19 by continuous administration of 6-n-propyl-2-thiouracil (0.1% wt/vol, in drinking tap water) for 3 wk. All experiments were conducted in accord with local and national guidelines for animal experimentation.
Preparation of rat liver hepatocytes
Rat liver cells were isolated by perfusing the liver with collagenase as previously reported (22). The cells were suspended in Hams F-12 culture medium supplemented with 10% fetal calf serum and 1% fatty acid-free BSA, 100 U/ml penicillin, and 100 mg/ml streptomycin, and buffered (pH 7.4) with 14.5 mM sodium bicarbonate and 12.5 mM each of 2-(N-morpholino)ethanesulfonic acid and N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. Cell viability in all experiments was 90% or greater as estimated by trypan blue (0.5% wt/vol in physiological saline) exclusion. Cell suspensions were diluted with Ham’s F-12 medium to give about 5 x 105 cells/ml. Four-milliliter cell suspensions (corresponding to 1.5 mg of protein) were seeded on 60-mm vented plastic Petri dishes.
Incubation procedure
The dishes were kept in a humidified incubator in equilibrium with a 95% air-5% carbon dioxide mixture in a Heraeus incubator at 37 C. In 2 h, only viable cells became firmly attached to the bottom of the dishes so that medium could be aspirated without loss of cells. After this plating period, cell monolayer was washed and 4 ml of fresh medium without fetal calf serum with or without albumin was added together with the hormone where indicated. Thyroid hormones and their analogs were first dissolved in a small volume of 0.1 M NaOH, diluted in Ham’s F-12 medium, neutralized to pH 7.4 by HCl, and added to cultures to achieve final concentrations ranging from 10–11–10–5 M. There was no detachment of cells in a subsequent incubation period of up to 4 h.
Analytical procedure and radioactivity measurements
Fresh medium, with or without the hormone, was added together with labeled substrate 1 h before ending the incubations. Lipogenic activity was determined by monitoring the incorporation of [1-14C]acetate (0.96 mCi/mmol) into fatty acids and cholesterol. Because glycolysis and proteolysis may dilute the intracellular [14C]acetyl-CoA pool, fatty acid and cholesterol synthesis were also measured by monitoring incorporation of tritium from [3H]H2O (1 mCi/ml) into these lipid fractions as independent index of lipogenic activity (23). To terminate the lipogenic assay, the medium was aspirated and the adherent cells were washed three times with ice-cold 0.14 M KCl to remove unreacted labeled substrate, and the reaction was stopped with 1.5 ml of 0.5 N NaOH. The cells were scraped off with a rubber policeman and transferred to a test tube. Digitonin-precipitable sterols and fatty acids were extracted and counted for radioactivity as reported before (22).
Neutral lipids and phospholipids were also analyzed. After blocking the reaction with 2 ml KCl:methanol (1:2, vol/vol), total lipids were extracted according to Bligh and Dyer (24). Neutral lipids were resolved by thin layer chromatography on silica gel plates by using hexane:ethyl ether:acetic acid (70:30:10, vol/vol/vol) as a developing system. Lipid bands were visualized by iodine vapor and scraped into counting vials for measuring radioactivity. Phospholipids were separated by HPLC as previously described (25), by using a Beckman System Gold chromatograph equipped with an ultrasil-Si column (4.6 x 250 mm) (Chemtek Analytica, Bologna, Italy). The chromatographic system was programmed for gradient elution by using two mobile phases: solvent A, hexane:2-propanol (6:8, vol/vol) and solvent B, hexane:2-propanol:water (6:8:1.4, vol/vol/vol). The percentage of solvent B in solvent A was increased in 15 min from 0–100%. Flow rate was 2 ml/min and detection was at 206 nm. Eluted fractions, corresponding to the different phospholipids, were collected for radioactivity measurement.
Other methods
Protein concentration was determined by using the method of Lowry et al. (26) with BSA as a standard.
Statistical analysis
The results were computed with Excel (Microsoft 7). Comparison was made using one-way ANOVA (27) followed by a post hoc Tukey’s B test. All statistical analyses were performed using an SPSS/PC computer program (SPSS, Chicago, IL). Differences were considered statistically significant at P < 0.05.
Results
Lipogenic response to iodothyronines
Acetyl-CoA is precursor for both fatty acid and cholesterol synthesis. The capacity of hepatocytes to incorporate acetate into these lipid fractions was measured in the sixth hour of the experiment. Thyroid hormones were added after the initial 2-h plating period and the cultures were then incubated for an additional 4 h. Radiolabeled acetate was added at the third hour, and its incorporation into fatty acids and cholesterol was followed in the last hour. To maximize the response to added T2, hepatocytes isolated from hypothyroid donor rats were used throughout this study (22). A comparison with T3 effects was routinely carried out.
Fig. 1 shows that 10–5 M T2, when incubated for 4 h with hepatocytes cultured in a serum-free medium containing 1% BSA, induced a marked stimulation of both cholesterol (70%) and fatty acid (60%) synthesis. The stimulation of both these pathways induced by 10–5 M T3 was in good agreement with results previously obtained with similar methods and a similar experimental protocol as used for the present study (22).
Effect of BSA on thyroid hormone-stimulated lipogenesis
It has been demonstrated that BSA is able to bind thyroid hormone thus decreasing the concentration of the free metabolically active compound (28). Cell media supplemented with 1% BSA may influence hormone responses by altering the uptake of thyroid hormones by hepatocytes (28). Indeed, BSA (20 mg/ml) inhibits stimulation of fatty acid synthesis induced by T3 (29). Moreover, with serum (containing hormone-binding proteins) in the perfusate, the uptake of hormones in isolated rat heart was reduced (30). As expected, the presence of BSA reduced significantly the stimulation of cholesterol and fatty acid synthesis (Table 1) induced by both T2 and T3. It must be underlined that cell viability was similar in the presence and in the absence of BSA. The latter is also evidenced by the fact that control values for lipogenesis were quite similar with and without BSA in the cultures. In all further experiments, BSA was omitted from culture media after the initial 2-h plating period.
Time course of the hormone effects
In Fig. 2, a plot is presented of the rate of cholesterol (panel A) and fatty acid (panel B) synthesis as a function of time in the absence and presence of 10–5 M T2 or 10–5 M T3. Interestingly, this figure shows that within 1 h after T2 addition to hepatocytes, a significant stimulation, as compared with control incubations, of both cholesterol and fatty acid synthesis was observed. In fact, the former increased by about 30% and the latter by about 45%. The stimulation occurred already 0.5 h after T2 addition to the cells but was not yet statistically significant at that time. The stimulatory effect induced by T2 on cholesterol synthesis was statistically significant over the entire 4-h period of hormone incubation. The T2 stimulatory effect on fatty acid synthesis, significant during the whole incubation period, reached a peak at the fourth hour where a stimulation of 140% was observed. The stimulatory effect of T3 on both metabolic pathways increased steadily over the 4-h experimental period.
Dose-dependent effect of thyroid hormones on lipogenesis
The dose-response curve in Fig. 3 showed that both T2 and T3 stimulated cholesterol synthesis (panel A). The magnitude of the increase was greater at supraphysiological T3 levels, reaching 140% stimulation at 10–5 M, but was already statistically significant (40%; P < 0.05) at 10–8 M. The T2 effect on cholesterolgenesis, although less pronounced, was similar to that of T3. The effect of the different iodothyronine concentrations on fatty acid synthesis is reported in panel B of Fig. 3. The rate of fatty acid synthesis increased with increasing hormone concentrations. A statistically significant effect was observed at 10–6 and 10–5 M of T2 or T3. At the latter concentration, the stimulatory effect of T3 was higher than that of T2. Although an increase in fatty acid synthesis by the two iodothyronines was also observed in the range of 10–9–10–7 M, this was not statistically relevant. At this point, it is worth underling that both cholesterol and fatty acid syntheses were not significantly influenced by T2 and T3 concentrations of 10–10 and 10–11 M (data not shown).
Effect of thyroid hormone analogs on fatty acid and cholesterol synthesis
The specificity of the effect of T2 on lipogenesis was shown by experiments carried out with some thyroid hormone analogs such as D-T4 and DIT. As shown in Fig. 4, after 4 h of incubation, neither D-T4 nor DIT induced a significant effect on cholesterol (panel A) and fatty acid (panel B) synthesis in the concentration range tested, i.e. 10–9–10–5 M. Similar negative results were obtained with 3-iodo-L-tyrosine, L- thyronine, and L-tyrosine (data not shown).
Fatty acid and cholesterol synthesis from tritiated water
Besides acetyl-CoA, fatty acid and cholesterol synthesis require NADPH+H+ as hydrogen donor in the reducing steps of their respective metabolic pathways. Incorporation of tritium from tritiated water into fatty acids and cholesterol can be used as an index of lipogenesis independent of the dilution to which intracellular acetyl-CoA pool can be subjected (23). Therefore, estimation of the rate of lipogenesis by [H3]H2O incorporation should be higher than that from labeled acetate (19, 22, 23, 31), as is the case of the results reported in Table 2 compared with those of Fig. 3. However, the important point here is that, from this Table, it can be deduced that T2 addition to liver cells increased fatty acid and cholesterol synthesis in a similar fashion whether measured by incorporation of tritium or [1-14C]acetate (see Fig. 3).
Distribution of radioactivity among various lipid classes
As shown in the experiment described in Table 3, synthesized radioactive fatty acids, in the control, were rapidly incorporated into triacylglycerols, whereas the incorporation into cholesterol ester was modest. Therefore, cholesterol remained largely in free form. After T2 addition to cells, a significant increase in fatty acid incorporation into triacylglycerols and cholesterol esters was observed. Although the effect generated by T2 was similar to the one induced by T3, it was less pronounced. It should be pointed out that the ratio of esterified fatty acids to free fatty acids was higher in T3-treated hepatocytes than in those treated with T2. Table 3 also shows the effects of T2 on the incorporation of synthesized, radioactive fatty acids into total phospholipids as well as into the main phospholipid classes. Phospholipid synthesis was greatly enhanced by T2 addition to the cells. In particular, the synthesis of phosphatidylcholine and phosphatidylethanolamine was stimulated by about 110 and 50%, respectively. Also in this case, the stimulatory effects induced by T2 were less marked as compared with the effects generated by T3. Cholesterol to phospholipid ratio is an important determinant of membrane fluidity (25, 32). Results of Table 3 show that addition of iodothyronines to hepatocytes, because of the similar increase in the levels of total cholesterol and phospholipids, did not induce a statistically significant change in the cholesterol to phospholipid ratio compared with the control.
Effect of CEX on thyroid hormone-induced lipogenesis
To investigate the nature of the effect of T2 on lipogenesis in vitro, we followed the hormone-determined rate of synthesis of cholesterol and fatty acids from labeled acetate in the presence of CEX, a well-known inhibitor of protein synthesis. The inhibitor was added to the cultures at the time of hormone addition. CEX, at a concentration of 5 x 10–5 M, had no effect on the attachment of hepatocytes in the course of the experiment. In agreement with Ref. 33 , CEX greatly inhibited lipogenesis in isolated hepatocytes (Table 4). T2 addition to the cells almost doubled the incorporation of [1-14C]acetate into cholesterol and fatty acids (see line 1 and line 3 of Table 4). Interestingly, when T2 was added to the cells together with CEX, an almost 2-fold stimulation, compared with CEX alone, of labeled acetate incorporation (expressed as nanomoles of [1-14C]acetate incorporated per hour times milligrams of protein) into both cholesterol (0.49 ± 0.01 vs. 0.23 ± 0.01) and fatty acids (1.09 ± 0.07 vs. 0.59 ± 0.02) could still be observed.
Discussion
The present study was conducted in primary cultures of rat hepatocytes to test the possibility of T2 modulation of fatty acid and cholesterol biosynthesis. The results obtained show that T2 had a significant thyromimetic effect by stimulating lipid biosynthesis in a dose and time-dependent manner. Previously we demonstrated short-term effect on fatty acid and cholesterol synthesis by T3 in hepatocytes from hypothyroid rats (22). Thus both iodothyronines may contribute to regulation of hepatic lipogenesis.
In our experiments, we did not measure the specific activity of single enzymes participating in fatty acid and cholesterol synthesis but the kinetics of appearance of radiolabeled products. In measuring fatty acid and cholesterol synthesis in our system, we need sufficient accumulation of labeled product to judge the effect of agonists. Therefore, it takes at least 30–60 min before one can terminate such an experiment. In the present study, the stimulation of lipogenesis in hepatocytes, starting from [1-14C]acetate, was already evident within 1 h after addition of T2 and reaches a peak at 4 h of hormone incubation with hepatocytes. To our knowledge, this is the first demonstration of a rapid and direct effect of T2 on hepatic lipogenesis. The observation that liver cells, in particular parenchymal cells, are responsive to thyroid hormones is quite appealing and suggests that T2 short-term effects on the liver observed in vivo (6, 7, 8, 13, 15) are direct rather than secondary.
Stimulation of fatty acid synthesis by T3 is less in the presence than in the absence of BSA (28). Our data demonstrated that BSA also reduced the stimulatory effect of T2 of fatty acid and cholesterol synthesis. At the highest hormone concentration tested (10–5 M) stimulation of fatty acid and cholesterol synthesis was reduced in the presence of BSA by about 20%. These data represent an additional demonstration of the thyromimetic action of T2.
It could be argued that T2 might affect the specific radioactivity of the intracellular [14C]acetyl-CoA pool and thereby seemingly stimulate lipogenesis. Therefore, studies were initiated using incorporation of [3H]H2O into lipids as an independent index of lipogenesis (23). The outcome of the experiment presented in Table 2 permitted us to exclude this possibility as a similar pattern of fatty acid and cholesterol synthesis was observed in both cases. This result is analogous to the one for T3 observed earlier (22).
One problem that plagues consideration of the literature on thyroid hormone action is that of "dose". It must be underlined that in our experiments, after a 2 h plating period, serum-free medium with the appropriate hormone concentration was added to the cells. Unlike the in vivo situation, where thyroid hormones exert their effect in a complex network of serum hormones, the stimulation of lipogenesis observed in our isolated cells may only be attributed to the thyroid hormone added to the cells. The effect on different enzymatic activities at supra-physiological thyroid hormone concentrations, similar to those used in this study, is not uncommon at least as far as various cultured cell types are concerned (34, 35, 36). The concentration of T3 that is effective in stimulating lipogenesis in this study is within the range that stimulates glycogen synthesis and glycogen deposition in rat hepatocyte cultures (37). Mariash and Oppenheimer (38) noted that, in cultured rat hepatocytes, a severalfold higher initial free T3 concentration was required to achieve a given level of malic enzyme induction when serum was absent from the medium. This phenomenon was ascribed to an accelerated rate of T3 metabolism in the absence of serum (38). Therefore, taking into account that 96% of the added T3 is bound to albumin present in the medium (39) and that T3 is rapidly metabolized by the hepatocyte monolayer (38, 40), the concentration of the active hormone could be considered two to three orders of magnitude lower than the added ones. Anyway, even if the maximum effect shown by the iodothyronines on fatty acid and cholesterol synthesis is observed in this study at supraphysiological or pharmacological hormone concentrations, this effect can be considered specific as the same concentrations of thyroid hormone analogs, such as D-T4 and DIT (Fig. 4), had no significant effect on both metabolic pathways.
Moreover, in the present study, hepatocytes treated with T2 have almost 70% of fatty acid-associated radioactivity in the triacylglycerol and phospholipid fractions, whereas most of synthesized cholesterol remained unesterified. These results show that T2, similarly to T3, affects channeling of fatty acids. The T2-induced stimulation of fatty acid synthesis that we observed could create a greater substrate availability for neutral and phospholipid synthesis. Therefore, it is reasonable to suppose that also some enzymatic activities of complex lipid biosynthesis could be influenced by iodothyronines. Furthermore, thyroid hormone has been reported to stimulate synthesis and oxidation of fatty acids simultaneously (17, 41), thus creating an energy-using substrate cycle. The reduced stimulation of esterification compared with the stimulation of de novo synthesis as induced by T2 (ratio of esterified fatty acids to free fatty acids in Table 3) suggests that more fatty acids synthesized in the presence of T2 remain available for the oxidation pathway. This notion is in line with reports in which it was demonstrated that T2 induced a short-term calorigenic effect in hypothyroid rats by increasing their resting metabolism (6). Cholesterol is not normally a large component of intracellular membranes but is an important constituent of plasma membranes. Although thyroid status has dramatic effects on plasma cholesterol levels (42), it appears not to have the same effects on membrane cholesterol levels. In rats, hyperthyroidism resulted in an increase in both cholesterol and phospholipid content of erythrocyte membranes but with no change in the cholesterol to phospholipid ratio (43), whereas, in hypothyroidism, the decrease in phospholipid content is greater than the decrease in cholesterol content, with a consequent small increase in the cholesterol to phospholipid ratio (44). In our experiments, the contemporaneous increase in the radiolabeled cholesterol and phospholipid amount induced by hormone addition to the cells did not modify the cholesterol to phospholipid ratio significantly.
In agreement with previous observations (33), CEX inhibited lipogenesis in isolated rat hepatocytes. Therefore, the lower rate of lipogenesis in the presence of CEX shown in Table 4 can be due to blocking of the turnover of key enzymes in this pathway. However, compared with the data with CEX alone, an almost doubled stimulation of both cholesterol and fatty acid synthesis was still observed when T2 was added together with the inhibitor, thus suggesting that the stimulatory effect of T2 was independent of protein synthesis. This consideration is in line with a rapid nongenomic effect of T2 occurring on enzymatic activities in rat-liver mitochondria after in vivo T2 administration to hypothyroid rats (4, 8).
More than a decade ago, a direct action of thyroid hormone on cell membranes was observed (45, 46). This rapid mechanism, recently recognized (47), occurred by direct hormonal stimulation that is independent of nuclear events or protein synthesis. A short-term nongenomic action of T2, which activate signal transducing kinases has been recently demonstrated in chick embryo hepatocytes (34). The important point is that the response of lipogenesis to the thyroid hormone that we observed in this study is rather rapid, indicating that early modulating mechanisms may make a contribution to the overall enhancement of the process, e.g. by interconversion of preexisting lipogenic enzyme(s) to a more active form. In keeping with the notion that mechanisms different from protein synthesis are involved in the early T2 actions on lipogenesis is our preliminary result suggesting that the mass of fatty acid synthase was not affected by treatment of hepatocytes with the iodothyronines (data not shown).
Our data on the early T2 stimulation of fatty acid and cholesterol synthesis add further support to the recent findings that in vivo T2 administration determines, in rat liver, a short-term increase in the activities of malic enzyme (39), generally considered a model system to study the effect of thyroid hormones, and glucose-6-phosphate dehydrogenase (13). Both these enzymes represent the most important sources of NADPH for reductive steps of hepatic lipogenic activities. Although at the moment we do not have clear evidence concerning the actual T2 mechanism of action, we believe that results of this study provide new data that improve our understanding of how the single iodothyronines might regulate the metabolic rate. Lastly, our results and those in Refs. 5, 6, 7, 8, 9, 10, 11, 12 raise important questions regarding the uniqueness of T3 as the only active thyroid hormone.
Acknowledgments
The technical support of Aventis Bulk is gratefully acknowledged.
Footnotes
This study was supported in part by the Ministero Istruzione Università Ricerca and the Stichting Toxicologisch Onderzoek Utrecht.
Abbreviations: CEX, Cycloheximide; DIT, 3,5-diiodo-L-tyrosine; D-T4, 3,3',5,5'-tetraiodo-D-thyronine; T2, 3,5-L-diiodothyronine.
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Department of Nutrition (M.J.H.G.), Graduate School of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands
Abstract
Short-term effects of 3,5-L-diiodothyronine (T2) on lipid biosynthesis were studied in cultured hepatocytes from hypothyroid rats. A comparison with the effects of T3 was routinely carried out. After T2 addition to cell cultures, a distinct stimulation of fatty acid and cholesterol syntheses, measured as incorporation of [1-14C]acetate into these lipid fractions, was observed. The T2 dose-dependent effect on both metabolic pathways, already detectable at 10–8-10–9 M, reached a 2-fold stimulation at 10–5 M T2. At this concentration, the stimulatory effect was evident within 1 h of T2 addition to the hepatocytes and increased with time up to the length of the experimental period of 4 h. T2 stimulation of lipogenesis was also confirmed by incubating hepatocytes with [3H]H2O, used as an independent index of lipogenic activity. The effects of T2 are rather specific as 3,3',5,5'-tetraiodo-D-thyronine and 3,5-diiodo-L-tyrosine were practically ineffective on both fatty acid and cholesterol synthesis. Analysis of various lipid fractions showed that T2 addition to the cells produced a significant stimulation of the incorporation of newly synthesized fatty acids into both neutral and polar lipids. By comparing the effects induced by T2 with those seen in the presence of T3, it appeared that T2 was able to mimic T3 effects. Experiments conducted in the presence of cycloheximide, a protein synthesis inhibitor, indicated that the T2 stimulatory effect on fatty acid and cholesterol synthesis was essentially independent of protein synthesis.
Introduction
THE THYROID HORMONES T3 and T4 play important roles in growth, development, cell differentiation, and metabolism through their interaction with nuclear receptors (1, 2). However, an increasing number of nonnuclear-mediated effects of thyroid hormones have been reported to occur at the plasma membrane, in mitochondria, at the cytoskeleton, and in the cytoplasm (for reviews, see Refs. 3 and 4).
In addition to T3 and T4, other iodothyronines, such as 3,5-L-diiodothyronine (T2) and 3,3'-L-diiodothyronine, produced by further peripheral deiodination of T3 and rT3, respectively, are present in biological fluids. T2 serum levels were significantly reduced in patients with hypothyroidism, and enhanced in patients with hyperthyroidism, sepsis, liver diseases, head injury, and brain tumors, suggesting a relationship between the diseases and the altered level of T3 and T2 (5). Recently, evidence has been presented indicating that T2 is able to mimic some effects of T3 on energy metabolism by acting as a biologically active analog of T3 (6, 7). It has been shown that T2 stimulates oxygen consumption in isolated perfused liver from hypothyroid rats and in mononuclear red blood cells (8, 9). T2 also increases the mitochondrial respiratory rate and cytochrome oxidase activity more rapidly than T3 (4, 10, 11), and the metabolic effects of T2 at the mitochondrial level have been observed in rats (10) and fish (12). These effects are not dependent on protein synthesis and involve a rapid and direct action of T2 with mitochondria (6, 7). The action of T2 is not limited to mitochondria as a rapid cycloheximide (CEX)-independent stimulation of glucose-6-phosphate dehydrogenase activity by T2 has been observed in rat-liver cytosol (13). Very recently the presence of cytosolic-binding proteins for T2 has been demonstrated (14). It has also been reported that T2 has significant thyromimetic effects in vivo and in vitro and it is very effective in the induction of hepatic malic enzyme gene expression (15).
It is well known that liver is a pivotal target of thyroid hormone effects. Several observations indicate that thyroid hormones greatly affect the extent to which this tissue contributes to total lipogenesis in the rats. Thyroidectomy decreases the hepatic activities of several lipogenic enzymes, such as malic enzyme, glucose-6-phosphate dehydrogenase, acetyl-coenzyme A carboxylase, and fatty acid synthase (for review see Ref. 16). Hyperthyroidism, on the other hand, increases the rate of hepatic fatty acid synthesis by raising the activities of the lipogenic enzymes (17, 18, 19, 20, 21).
However, in vivo studies cannot distinguish between direct effects of thyroid hormones on the liver and secondary effects due to endocrine or metabolite changes. The use of isolated cells can overcome this problem. Therefore, we decided to investigate whether acute effects of T2, if any, on hepatic lipogenesis occur directly by employing isolated hepatocytes.
This study presents the first demonstration of an early and direct stimulatory effect of T2 on both fatty acid and cholesterol biosynthesis in cultures of isolated rat hepatocytes.
Materials and Methods
Chemicals
[1-14C]Acetate and [3H]H2O were purchased from Amersham Pharmacia Biotech (Milan, Italy); BSA (fraction V, fatty acid-free), collagenase type I, Ham’s F-12 culture medium, and 6-n-propyl-2-thiouracil were from Sigma-Aldrich Co. (Milan, Italy); T2, T3, 3,3',5,5'-tetraiodo-D-thyronine (D-T4), 3,5-diiodo-L-tyrosine (DIT), 3-iodo-L-tyrosine, L-thyronine, and L-tyrosine were from Sigma-Aldrich and were more than 99% pure. All other reagents were of analytical grade.
Animal treatment
Male Wistar rats (250–300 g) were used throughout this study. They were kept one per cage in a temperature-controlled room under an artificial lighting regime of 12 h light, 12 h darkness. A commercial mash (Morini SpA, Milan, Italy) was available for ad libitum consumption, and the animals had free access to water. Hypothyroidism in rats was chronically produced as in Refs. 18 and 19 by continuous administration of 6-n-propyl-2-thiouracil (0.1% wt/vol, in drinking tap water) for 3 wk. All experiments were conducted in accord with local and national guidelines for animal experimentation.
Preparation of rat liver hepatocytes
Rat liver cells were isolated by perfusing the liver with collagenase as previously reported (22). The cells were suspended in Hams F-12 culture medium supplemented with 10% fetal calf serum and 1% fatty acid-free BSA, 100 U/ml penicillin, and 100 mg/ml streptomycin, and buffered (pH 7.4) with 14.5 mM sodium bicarbonate and 12.5 mM each of 2-(N-morpholino)ethanesulfonic acid and N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. Cell viability in all experiments was 90% or greater as estimated by trypan blue (0.5% wt/vol in physiological saline) exclusion. Cell suspensions were diluted with Ham’s F-12 medium to give about 5 x 105 cells/ml. Four-milliliter cell suspensions (corresponding to 1.5 mg of protein) were seeded on 60-mm vented plastic Petri dishes.
Incubation procedure
The dishes were kept in a humidified incubator in equilibrium with a 95% air-5% carbon dioxide mixture in a Heraeus incubator at 37 C. In 2 h, only viable cells became firmly attached to the bottom of the dishes so that medium could be aspirated without loss of cells. After this plating period, cell monolayer was washed and 4 ml of fresh medium without fetal calf serum with or without albumin was added together with the hormone where indicated. Thyroid hormones and their analogs were first dissolved in a small volume of 0.1 M NaOH, diluted in Ham’s F-12 medium, neutralized to pH 7.4 by HCl, and added to cultures to achieve final concentrations ranging from 10–11–10–5 M. There was no detachment of cells in a subsequent incubation period of up to 4 h.
Analytical procedure and radioactivity measurements
Fresh medium, with or without the hormone, was added together with labeled substrate 1 h before ending the incubations. Lipogenic activity was determined by monitoring the incorporation of [1-14C]acetate (0.96 mCi/mmol) into fatty acids and cholesterol. Because glycolysis and proteolysis may dilute the intracellular [14C]acetyl-CoA pool, fatty acid and cholesterol synthesis were also measured by monitoring incorporation of tritium from [3H]H2O (1 mCi/ml) into these lipid fractions as independent index of lipogenic activity (23). To terminate the lipogenic assay, the medium was aspirated and the adherent cells were washed three times with ice-cold 0.14 M KCl to remove unreacted labeled substrate, and the reaction was stopped with 1.5 ml of 0.5 N NaOH. The cells were scraped off with a rubber policeman and transferred to a test tube. Digitonin-precipitable sterols and fatty acids were extracted and counted for radioactivity as reported before (22).
Neutral lipids and phospholipids were also analyzed. After blocking the reaction with 2 ml KCl:methanol (1:2, vol/vol), total lipids were extracted according to Bligh and Dyer (24). Neutral lipids were resolved by thin layer chromatography on silica gel plates by using hexane:ethyl ether:acetic acid (70:30:10, vol/vol/vol) as a developing system. Lipid bands were visualized by iodine vapor and scraped into counting vials for measuring radioactivity. Phospholipids were separated by HPLC as previously described (25), by using a Beckman System Gold chromatograph equipped with an ultrasil-Si column (4.6 x 250 mm) (Chemtek Analytica, Bologna, Italy). The chromatographic system was programmed for gradient elution by using two mobile phases: solvent A, hexane:2-propanol (6:8, vol/vol) and solvent B, hexane:2-propanol:water (6:8:1.4, vol/vol/vol). The percentage of solvent B in solvent A was increased in 15 min from 0–100%. Flow rate was 2 ml/min and detection was at 206 nm. Eluted fractions, corresponding to the different phospholipids, were collected for radioactivity measurement.
Other methods
Protein concentration was determined by using the method of Lowry et al. (26) with BSA as a standard.
Statistical analysis
The results were computed with Excel (Microsoft 7). Comparison was made using one-way ANOVA (27) followed by a post hoc Tukey’s B test. All statistical analyses were performed using an SPSS/PC computer program (SPSS, Chicago, IL). Differences were considered statistically significant at P < 0.05.
Results
Lipogenic response to iodothyronines
Acetyl-CoA is precursor for both fatty acid and cholesterol synthesis. The capacity of hepatocytes to incorporate acetate into these lipid fractions was measured in the sixth hour of the experiment. Thyroid hormones were added after the initial 2-h plating period and the cultures were then incubated for an additional 4 h. Radiolabeled acetate was added at the third hour, and its incorporation into fatty acids and cholesterol was followed in the last hour. To maximize the response to added T2, hepatocytes isolated from hypothyroid donor rats were used throughout this study (22). A comparison with T3 effects was routinely carried out.
Fig. 1 shows that 10–5 M T2, when incubated for 4 h with hepatocytes cultured in a serum-free medium containing 1% BSA, induced a marked stimulation of both cholesterol (70%) and fatty acid (60%) synthesis. The stimulation of both these pathways induced by 10–5 M T3 was in good agreement with results previously obtained with similar methods and a similar experimental protocol as used for the present study (22).
Effect of BSA on thyroid hormone-stimulated lipogenesis
It has been demonstrated that BSA is able to bind thyroid hormone thus decreasing the concentration of the free metabolically active compound (28). Cell media supplemented with 1% BSA may influence hormone responses by altering the uptake of thyroid hormones by hepatocytes (28). Indeed, BSA (20 mg/ml) inhibits stimulation of fatty acid synthesis induced by T3 (29). Moreover, with serum (containing hormone-binding proteins) in the perfusate, the uptake of hormones in isolated rat heart was reduced (30). As expected, the presence of BSA reduced significantly the stimulation of cholesterol and fatty acid synthesis (Table 1) induced by both T2 and T3. It must be underlined that cell viability was similar in the presence and in the absence of BSA. The latter is also evidenced by the fact that control values for lipogenesis were quite similar with and without BSA in the cultures. In all further experiments, BSA was omitted from culture media after the initial 2-h plating period.
Time course of the hormone effects
In Fig. 2, a plot is presented of the rate of cholesterol (panel A) and fatty acid (panel B) synthesis as a function of time in the absence and presence of 10–5 M T2 or 10–5 M T3. Interestingly, this figure shows that within 1 h after T2 addition to hepatocytes, a significant stimulation, as compared with control incubations, of both cholesterol and fatty acid synthesis was observed. In fact, the former increased by about 30% and the latter by about 45%. The stimulation occurred already 0.5 h after T2 addition to the cells but was not yet statistically significant at that time. The stimulatory effect induced by T2 on cholesterol synthesis was statistically significant over the entire 4-h period of hormone incubation. The T2 stimulatory effect on fatty acid synthesis, significant during the whole incubation period, reached a peak at the fourth hour where a stimulation of 140% was observed. The stimulatory effect of T3 on both metabolic pathways increased steadily over the 4-h experimental period.
Dose-dependent effect of thyroid hormones on lipogenesis
The dose-response curve in Fig. 3 showed that both T2 and T3 stimulated cholesterol synthesis (panel A). The magnitude of the increase was greater at supraphysiological T3 levels, reaching 140% stimulation at 10–5 M, but was already statistically significant (40%; P < 0.05) at 10–8 M. The T2 effect on cholesterolgenesis, although less pronounced, was similar to that of T3. The effect of the different iodothyronine concentrations on fatty acid synthesis is reported in panel B of Fig. 3. The rate of fatty acid synthesis increased with increasing hormone concentrations. A statistically significant effect was observed at 10–6 and 10–5 M of T2 or T3. At the latter concentration, the stimulatory effect of T3 was higher than that of T2. Although an increase in fatty acid synthesis by the two iodothyronines was also observed in the range of 10–9–10–7 M, this was not statistically relevant. At this point, it is worth underling that both cholesterol and fatty acid syntheses were not significantly influenced by T2 and T3 concentrations of 10–10 and 10–11 M (data not shown).
Effect of thyroid hormone analogs on fatty acid and cholesterol synthesis
The specificity of the effect of T2 on lipogenesis was shown by experiments carried out with some thyroid hormone analogs such as D-T4 and DIT. As shown in Fig. 4, after 4 h of incubation, neither D-T4 nor DIT induced a significant effect on cholesterol (panel A) and fatty acid (panel B) synthesis in the concentration range tested, i.e. 10–9–10–5 M. Similar negative results were obtained with 3-iodo-L-tyrosine, L- thyronine, and L-tyrosine (data not shown).
Fatty acid and cholesterol synthesis from tritiated water
Besides acetyl-CoA, fatty acid and cholesterol synthesis require NADPH+H+ as hydrogen donor in the reducing steps of their respective metabolic pathways. Incorporation of tritium from tritiated water into fatty acids and cholesterol can be used as an index of lipogenesis independent of the dilution to which intracellular acetyl-CoA pool can be subjected (23). Therefore, estimation of the rate of lipogenesis by [H3]H2O incorporation should be higher than that from labeled acetate (19, 22, 23, 31), as is the case of the results reported in Table 2 compared with those of Fig. 3. However, the important point here is that, from this Table, it can be deduced that T2 addition to liver cells increased fatty acid and cholesterol synthesis in a similar fashion whether measured by incorporation of tritium or [1-14C]acetate (see Fig. 3).
Distribution of radioactivity among various lipid classes
As shown in the experiment described in Table 3, synthesized radioactive fatty acids, in the control, were rapidly incorporated into triacylglycerols, whereas the incorporation into cholesterol ester was modest. Therefore, cholesterol remained largely in free form. After T2 addition to cells, a significant increase in fatty acid incorporation into triacylglycerols and cholesterol esters was observed. Although the effect generated by T2 was similar to the one induced by T3, it was less pronounced. It should be pointed out that the ratio of esterified fatty acids to free fatty acids was higher in T3-treated hepatocytes than in those treated with T2. Table 3 also shows the effects of T2 on the incorporation of synthesized, radioactive fatty acids into total phospholipids as well as into the main phospholipid classes. Phospholipid synthesis was greatly enhanced by T2 addition to the cells. In particular, the synthesis of phosphatidylcholine and phosphatidylethanolamine was stimulated by about 110 and 50%, respectively. Also in this case, the stimulatory effects induced by T2 were less marked as compared with the effects generated by T3. Cholesterol to phospholipid ratio is an important determinant of membrane fluidity (25, 32). Results of Table 3 show that addition of iodothyronines to hepatocytes, because of the similar increase in the levels of total cholesterol and phospholipids, did not induce a statistically significant change in the cholesterol to phospholipid ratio compared with the control.
Effect of CEX on thyroid hormone-induced lipogenesis
To investigate the nature of the effect of T2 on lipogenesis in vitro, we followed the hormone-determined rate of synthesis of cholesterol and fatty acids from labeled acetate in the presence of CEX, a well-known inhibitor of protein synthesis. The inhibitor was added to the cultures at the time of hormone addition. CEX, at a concentration of 5 x 10–5 M, had no effect on the attachment of hepatocytes in the course of the experiment. In agreement with Ref. 33 , CEX greatly inhibited lipogenesis in isolated hepatocytes (Table 4). T2 addition to the cells almost doubled the incorporation of [1-14C]acetate into cholesterol and fatty acids (see line 1 and line 3 of Table 4). Interestingly, when T2 was added to the cells together with CEX, an almost 2-fold stimulation, compared with CEX alone, of labeled acetate incorporation (expressed as nanomoles of [1-14C]acetate incorporated per hour times milligrams of protein) into both cholesterol (0.49 ± 0.01 vs. 0.23 ± 0.01) and fatty acids (1.09 ± 0.07 vs. 0.59 ± 0.02) could still be observed.
Discussion
The present study was conducted in primary cultures of rat hepatocytes to test the possibility of T2 modulation of fatty acid and cholesterol biosynthesis. The results obtained show that T2 had a significant thyromimetic effect by stimulating lipid biosynthesis in a dose and time-dependent manner. Previously we demonstrated short-term effect on fatty acid and cholesterol synthesis by T3 in hepatocytes from hypothyroid rats (22). Thus both iodothyronines may contribute to regulation of hepatic lipogenesis.
In our experiments, we did not measure the specific activity of single enzymes participating in fatty acid and cholesterol synthesis but the kinetics of appearance of radiolabeled products. In measuring fatty acid and cholesterol synthesis in our system, we need sufficient accumulation of labeled product to judge the effect of agonists. Therefore, it takes at least 30–60 min before one can terminate such an experiment. In the present study, the stimulation of lipogenesis in hepatocytes, starting from [1-14C]acetate, was already evident within 1 h after addition of T2 and reaches a peak at 4 h of hormone incubation with hepatocytes. To our knowledge, this is the first demonstration of a rapid and direct effect of T2 on hepatic lipogenesis. The observation that liver cells, in particular parenchymal cells, are responsive to thyroid hormones is quite appealing and suggests that T2 short-term effects on the liver observed in vivo (6, 7, 8, 13, 15) are direct rather than secondary.
Stimulation of fatty acid synthesis by T3 is less in the presence than in the absence of BSA (28). Our data demonstrated that BSA also reduced the stimulatory effect of T2 of fatty acid and cholesterol synthesis. At the highest hormone concentration tested (10–5 M) stimulation of fatty acid and cholesterol synthesis was reduced in the presence of BSA by about 20%. These data represent an additional demonstration of the thyromimetic action of T2.
It could be argued that T2 might affect the specific radioactivity of the intracellular [14C]acetyl-CoA pool and thereby seemingly stimulate lipogenesis. Therefore, studies were initiated using incorporation of [3H]H2O into lipids as an independent index of lipogenesis (23). The outcome of the experiment presented in Table 2 permitted us to exclude this possibility as a similar pattern of fatty acid and cholesterol synthesis was observed in both cases. This result is analogous to the one for T3 observed earlier (22).
One problem that plagues consideration of the literature on thyroid hormone action is that of "dose". It must be underlined that in our experiments, after a 2 h plating period, serum-free medium with the appropriate hormone concentration was added to the cells. Unlike the in vivo situation, where thyroid hormones exert their effect in a complex network of serum hormones, the stimulation of lipogenesis observed in our isolated cells may only be attributed to the thyroid hormone added to the cells. The effect on different enzymatic activities at supra-physiological thyroid hormone concentrations, similar to those used in this study, is not uncommon at least as far as various cultured cell types are concerned (34, 35, 36). The concentration of T3 that is effective in stimulating lipogenesis in this study is within the range that stimulates glycogen synthesis and glycogen deposition in rat hepatocyte cultures (37). Mariash and Oppenheimer (38) noted that, in cultured rat hepatocytes, a severalfold higher initial free T3 concentration was required to achieve a given level of malic enzyme induction when serum was absent from the medium. This phenomenon was ascribed to an accelerated rate of T3 metabolism in the absence of serum (38). Therefore, taking into account that 96% of the added T3 is bound to albumin present in the medium (39) and that T3 is rapidly metabolized by the hepatocyte monolayer (38, 40), the concentration of the active hormone could be considered two to three orders of magnitude lower than the added ones. Anyway, even if the maximum effect shown by the iodothyronines on fatty acid and cholesterol synthesis is observed in this study at supraphysiological or pharmacological hormone concentrations, this effect can be considered specific as the same concentrations of thyroid hormone analogs, such as D-T4 and DIT (Fig. 4), had no significant effect on both metabolic pathways.
Moreover, in the present study, hepatocytes treated with T2 have almost 70% of fatty acid-associated radioactivity in the triacylglycerol and phospholipid fractions, whereas most of synthesized cholesterol remained unesterified. These results show that T2, similarly to T3, affects channeling of fatty acids. The T2-induced stimulation of fatty acid synthesis that we observed could create a greater substrate availability for neutral and phospholipid synthesis. Therefore, it is reasonable to suppose that also some enzymatic activities of complex lipid biosynthesis could be influenced by iodothyronines. Furthermore, thyroid hormone has been reported to stimulate synthesis and oxidation of fatty acids simultaneously (17, 41), thus creating an energy-using substrate cycle. The reduced stimulation of esterification compared with the stimulation of de novo synthesis as induced by T2 (ratio of esterified fatty acids to free fatty acids in Table 3) suggests that more fatty acids synthesized in the presence of T2 remain available for the oxidation pathway. This notion is in line with reports in which it was demonstrated that T2 induced a short-term calorigenic effect in hypothyroid rats by increasing their resting metabolism (6). Cholesterol is not normally a large component of intracellular membranes but is an important constituent of plasma membranes. Although thyroid status has dramatic effects on plasma cholesterol levels (42), it appears not to have the same effects on membrane cholesterol levels. In rats, hyperthyroidism resulted in an increase in both cholesterol and phospholipid content of erythrocyte membranes but with no change in the cholesterol to phospholipid ratio (43), whereas, in hypothyroidism, the decrease in phospholipid content is greater than the decrease in cholesterol content, with a consequent small increase in the cholesterol to phospholipid ratio (44). In our experiments, the contemporaneous increase in the radiolabeled cholesterol and phospholipid amount induced by hormone addition to the cells did not modify the cholesterol to phospholipid ratio significantly.
In agreement with previous observations (33), CEX inhibited lipogenesis in isolated rat hepatocytes. Therefore, the lower rate of lipogenesis in the presence of CEX shown in Table 4 can be due to blocking of the turnover of key enzymes in this pathway. However, compared with the data with CEX alone, an almost doubled stimulation of both cholesterol and fatty acid synthesis was still observed when T2 was added together with the inhibitor, thus suggesting that the stimulatory effect of T2 was independent of protein synthesis. This consideration is in line with a rapid nongenomic effect of T2 occurring on enzymatic activities in rat-liver mitochondria after in vivo T2 administration to hypothyroid rats (4, 8).
More than a decade ago, a direct action of thyroid hormone on cell membranes was observed (45, 46). This rapid mechanism, recently recognized (47), occurred by direct hormonal stimulation that is independent of nuclear events or protein synthesis. A short-term nongenomic action of T2, which activate signal transducing kinases has been recently demonstrated in chick embryo hepatocytes (34). The important point is that the response of lipogenesis to the thyroid hormone that we observed in this study is rather rapid, indicating that early modulating mechanisms may make a contribution to the overall enhancement of the process, e.g. by interconversion of preexisting lipogenic enzyme(s) to a more active form. In keeping with the notion that mechanisms different from protein synthesis are involved in the early T2 actions on lipogenesis is our preliminary result suggesting that the mass of fatty acid synthase was not affected by treatment of hepatocytes with the iodothyronines (data not shown).
Our data on the early T2 stimulation of fatty acid and cholesterol synthesis add further support to the recent findings that in vivo T2 administration determines, in rat liver, a short-term increase in the activities of malic enzyme (39), generally considered a model system to study the effect of thyroid hormones, and glucose-6-phosphate dehydrogenase (13). Both these enzymes represent the most important sources of NADPH for reductive steps of hepatic lipogenic activities. Although at the moment we do not have clear evidence concerning the actual T2 mechanism of action, we believe that results of this study provide new data that improve our understanding of how the single iodothyronines might regulate the metabolic rate. Lastly, our results and those in Refs. 5, 6, 7, 8, 9, 10, 11, 12 raise important questions regarding the uniqueness of T3 as the only active thyroid hormone.
Acknowledgments
The technical support of Aventis Bulk is gratefully acknowledged.
Footnotes
This study was supported in part by the Ministero Istruzione Università Ricerca and the Stichting Toxicologisch Onderzoek Utrecht.
Abbreviations: CEX, Cycloheximide; DIT, 3,5-diiodo-L-tyrosine; D-T4, 3,3',5,5'-tetraiodo-D-thyronine; T2, 3,5-L-diiodothyronine.
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