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General and Comparative Endocrinology 104, 61–66 (1996) Article No. 0141

Direct Effects of 3,5,38-Triiodothyronine and 3,5-Diiodothyronine on Mitochondrial Metabolism in the Goldfish Carassius auratus S. C. Leary, K. N. Barton, and J. S. Ballantyne Department of Zoology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Accepted May 13, 1996

Direct effects of 3,5,38-triiodothyronine (T3) and 3,5diiodothyronine (T2) on the metabolism of the goldfish (Carassius auratus) were assessed using mitochondria isolated from liver and red muscle. Following a 5-min incubation with either T3 or T2, the oxidation rates of substrates involved in amino acid and carbohydrate metabolism and lipid catabolism were measured. State 3 oxidation of pyruvate was significantly higher for liver mitochondria treated with T2 and for red muscle mitochondria incubated with T3 when compared to control mitochondria. Rapid elevation of state 3 rates of substrate oxidation by thyroid hormones may be important in mediating diurnal changes in mitochondrial metabolism. Significant increases in liver and red muscle mitochondrial state 4 rates were also observed for pyruvate in T2- and T3-treated mitochondria and for glutamate in T3-treated mitochondria. r 1996 Academic Press, Inc. Thyroid hormones (THs) mediate both long- and short-term changes in metabolism (Soboll, 1993b). These effects involve the binding of 3,5,38-triiodothyronine (T3) to nuclear receptors and include increased rates of protein synthesis and lipolysis (Soboll, 1993a). DeNayer (1987) and Soboll (1993a) have suggested that in mammalian systems, T3 may also directly affect the activity of cytosolic enzymes and mitochondria through extranuclear pathways. These pathways may be important in the short-term responses to THs. In

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addition, it has been suggested that 3,5-diiodothyronine (T2), produced in vivo by the action of iodothyronine deiodinases on T3, also has a short-term effect on oxidative metabolism which may exceed that of T3 (Horst et al., 1989; Kvetny, 1992; Lanni et al., 1992; O’Reilly and Murphy, 1992). Little is known of the short-term effects of THs in fish. Past studies have dealt almost exclusively with the long-term effects of T3 (Eales, 1988; Eales and Brown, 1993). A few recent studies in fish have demonstrated short-term changes in the activities of cytosolic and membrane-bound enzymes (Ballantyne et al., 1992; Battersby et al., 1996) and altered mitochondrial respiration rates in T3-treated fish (Ballantyne et al., 1992). It is not known if a direct mitochondrial effect is responsible for these changes. Isolated mitochondria retain much of their in vivo metabolic organization and have been used extensively in mammalian systems to study direct effects of THs (O’Reilly and Murphy, 1992; Sterling and Brenner, 1995). To determine if a direct mitochondrial effect is responsible for short-term effects of THs, we examined the in vitro effects of T2 and T3 on the metabolism of mitochondria isolated from the red muscle and liver of the goldfish (Carassius auratus). Since the effect may vary depending on the metabolic pathway involved, we examined the effects on the oxidation of substrates from pathways involved in amino acid, lipid, and carbohydrate oxidation.

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MATERIALS AND METHODS Animals Goldfish (40–80 g) were purchased from Grassy Forks, Inc. (Grassy Forks, IN). All fish were acclimated in constantly flowing water at 20 6 2° with a 12-hr light:12-hr dark photoperiod for 3 weeks prior to experimentation. Fish were fed a commercial diet (quality feed, Zeigler Aquaculture) once daily ad libitum. All experiments were conducted 4 hr after the onset of the dark phase since there are significant diurnal variations in the levels of circulating plasma thyroid hormones in goldfish (Speiler and Noeske, 1979).

Mitochondrial Isolation Fish were killed by decapitation and the liver and red muscle were rapidly excised, minced, and placed in ice-cold isolation medium (pH 7.2 at 20°, consisting of 250 mM sucrose, 30 mM Hepes, 1 mM EGTA (ethylene glycol-bis(b-aminoethyl ether)-N,N,N8,N8tetraacetic acid), and 1% essentially fatty acid-free bovine serum albumin (BSA)). All tissues were homogenized with three passes of a loosely fitting Teflon pestle of a Potter–Elvehjem homogenizer. The homogenate was then centrifuged at 475g for 10 min. The resulting pellet was removed and centrifuged at 5850g for 10 min. The mitochondrial pellet was removed and resuspended in the same medium and again centrifuged at 5850g for 10 min. The resulting mitochondrial pellet was then resuspended in the isolation medium and used in subsequent assays.

Mitochondrial Respiration The concentration of T3 in a number of tissues has been shown to be similar in both the rainbow trout and the rat (Fok et al., 1990). The range of doses used in the present study (0.3, 3.0, and 30 nM T3) was based on those reported to have elicited a physiological response in several isolated rat liver mitochondria studies (Sterling et al., 1977, 1980; Sterling and Brenner, 1995). Doses of T2 in the same range were used to facilitate comparisons with T3. One volume of mitochondrial suspension (10–60 mg

Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

Leary, Barton, and Ballantyne

protein/ml) was added to 9 vol of reaction medium (pH 7.2 at 20°, consisting of 150 mM KCl, 30 mM Hepes, 10 mM KH2PO4, and 1% BSA). Mitochondrial preparations were incubated for 5 min in one of three doses of either T3 or T2, with each dose of the hormone run parallel in time to a control. Oxygen consumption was measured at 20°, according to the method for lake char described by Ballantyne et al. (1989). Substrates were chosen as indices of amino acid (glutamate, 5 mM ) and carbohydrate (pyruvate, 10 mM) metabolism and lipid catabolism (palmitoyl carnitine, 0.25 mM ). State 3 respiration rates (Chance and Williams, 1956) in the presence of ADP (0.50 mM) and saturating substrate concentrations were used to estimate maximal rates of substrate oxidation. Rates of oxygen uptake after added ADP was phosphorylated to ATP in the presence of substrate, or state 4 oxidation rates (Chance and Williams, 1956), were also determined. This method yields mitochondria with respiratory control ratios (state 3/state 4) of 9.10 6 1.51 (n 5 8) for liver mitochondria oxidizing glutamate and 11.56 6 1.67 (n 5 8) for red muscle mitochondria oxidizing palmitoyl carnitine. Both state 3 and state 4 respiration rates were represented as a percentage of a control run simultaneously for each trial.

Statistical Analysis Analysis of variance was performed using the GLM (general linear model) procedure (SAS, Gary, NC). To establish if the data were normally distributed the assumption of normality was tested using Proc Univariate (SAS). State 3 respiration rates were found to be normally distributed while state 4 respiration rates were log transformed in order to normalize the data (Steel and Torrie, 1980). Significant differences (P , 0.05) in mitochondrial respiration rates between treatment groups and the control for dose and hormone were detected using one-way ANOVAs and identified using Tukey’s HSD (Steel and Torrie, 1980).

RESULTS State 3 oxidation of pyruvate was significantly higher in liver mitochondria incubated with T2 than in control mitochondria (Fig. 1). By contrast, incubations with the

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T3 and T2 Effects on Mitochondrial Metabolism in Goldfish

two lower doses of T3 resulted in significant increases in state 3 oxidation of pyruvate for red muscle mitochondria when compared to control mitochondria (Fig. 2). State 3 oxidation of palmitoyl carnitine and glutamate were unaffected by T2 or T3 treatment (Figs. 1 and 2). T2 treatment resulted in significantly higher state 4 rates for pyruvate oxidation when red muscle and liver mitochondria were compared to control mitochondria (Fig. 3). A significant increase in state 4 rates for palmitoyl carnitine was observed when red muscle mitochondria were incubated in 0.3 nM T2 (Fig. 3). T3 treatment of red muscle mitochondria resulted in significant increases in state 4 rates for pyruvate at all doses, while liver mitochondrial pyruvate oxidation was unaffected (Fig. 4). State 4 rates for glutamate oxidation were significantly elevated by T3 in both red muscle and liver mitochondria (Fig. 4).

FIG. 2. State 3 respiration rates (% of control) of isolated red muscle (A) and liver (B) mitochondria for pyruvate, glutamate, and palmitoyl carnitine at three T3 doses (0.3, 3.0, and 30.0 nM). Values are presented as the means 6 SE (n 5 7). An asterisk above a bar indicates significant difference from control.

DISCUSSION

FIG. 1. State 3 respiration rates (% of control) of isolated red muscle (A) and liver (B) mitochondria for pyruvate, glutamate, and palmitoyl carnitine at three T2 doses (0.3, 3.0, and 30.0 nM). The values are presented as the means 6 SE (n 5 8). An asterisk above a bar indicates significant difference from control.

The primary finding of the present study is the demonstration of a direct effect of T2 and T3 on goldfish liver and red muscle mitochondria. State 3 oxidation of pyruvate increased significantly when mitochondria were incubated with either T2 or T3; however, T2 incubations lead to significant increases in state 3 respiration rates in liver mitochondria, whereas T3 treatment resulted in significant increases in state 3 respiration rates in red muscle mitochondria. Similarly, Ballantyne et al. (1992) found that T3 treatment stimulated significant increases in state 3 respiration rates in red muscle mitochondria but not in liver mitochondria of the lake char (Salvelinus namaycush). Since pyruvate was the only substrate in the present study whose oxidation was responsive to T2 or T3, the unique components of


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its metabolism may indicate possible sites of action. Transport of pyruvate and the pyruvate dehydrogenase (PDH) reaction are the two components of the oxidation of pyruvate not shared by the other two substrates. PDH is a complex enzyme known to be regulated by a variety of factors (Stryer, 1988) and is a likely site for interaction with THs. Enhanced oxidation rates of specific substrates such as pyruvate may thus be an important short-term function of T2 and T3 and may be of significance in mediating diurnal changes in metabolism. The in vitro stimulation of respiration observed in state 4 is congruent with in vivo studies of the shortterm effects of T3 in other fish species. Ballantyne et al. (1992) reported significantly higher state 4 respiration rates in red muscle mitochondria of S. namaycush from T3-treated versus control groups. There are several possible explanations for the in-

FIG. 4. State 4 respiration rates (% of control) of isolated red muscle (A) and liver (B) mitochondria for pyruvate, glutamate, and palmitoyl carnitine at three T3 doses (0.3, 3.0, and 30.0 nM). Values are presented as the means 6 SE (n 5 7). An asterisk above a bar indicates a significant difference from control.

FIG. 3. State 4 respiration rates (% of control) of isolated red muscle (A) and liver (B) mitochondria for pyruvate, glutamate, and palmitoyl carnitine at three T2 doses (0.3, 3.0, and 30.0 nM). Values are presented as the means 6 SE (n 5 8). An asterisk above a bar indicates a significant difference from control.


creased state 4 oxidation rates with T2 or T3 treatment. Increased rates of state 4 respiration associated with hyperthyroid states in mammals have been found to be due to an increased cation leak across the inner mitochondrial membrane (Harper et al., 1993). As cations leak into the mitochondrial matrix the membrane potential partially collapses: mitochondrial respiration increases to augment the pumping of protons out of the mitochondrial matrix to restore the membrane potential. A similar mechanism may be responsible for the observed effect in fish. In mammals, increased proton leak is linked to heat production (Brand et al., 1994) and is associated with thermogenic processes. Since thermogenesis is not significant in goldfish, the short-term changes observed in an enhanced proton leak in fish must have another physiological function. The EGTA used in the isolation medium in the present study would have chelated all the extramito-

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T3 and T2 Effects on Mitochondrial Metabolism in Goldfish

chondrial Ca21 in solution, precluding a calciummediated response and suggesting a direct effect of THs on a binding protein in the inner mitochondrial membrane. Several researchers have proposed that THs interact directly with the AdNt (see Hafner, 1987; Sterling et al., 1980; Sterling and Brenner, 1995). An effect of THs on the AdNt would affect the oxidation of all substrates to the same extent. The substrate-specific stimulation of state 4 rates observed in the present study, however, suggests that the AdNt is not involved in the observed thyroid hormone effects unless it has a preferential association with enzymes or transporters of specific pathways. An enhancement of components of the electron transport chain by THs would also be consistent with increased state 4 rates of respiration. Such effects should result in enhanced state 4 rates with all substrates unless, as indicated above, substrate-specific interactions with the electron transport chain are involved. Another possible consequence of an increased proton leak may be the associated changes in the pH of the matrix. It is well established that small shifts in pH may lead to considerable changes in metabolic flux (Hochachka and Somero, 1984). Increased proton content within the mitochondrial matrix may lead to shifts in metabolic flux rates through the differential effects of pH on the Vmax of various enzymes in different pathways. Another important finding of the present study is that, similar to the case in mammals, T2 may be an important intracellular signal in lower vertebrates. While it appears that T2 invokes a more rapid stimulation of mitochondrial metabolism than T3 in a number of mammalian tissues (Horst et al., 1989; Lanni et al., 1992; Kvetny, 1992), analogous studies involving lower vertebrates have yet to be conducted. The present study suggests that both T2 and T3 can have a direct, rapid effect on mitochondrial metabolism. Where T2 and T3 effects on state 3 and state 4 oxidation rates of a particular substrate were observed, they were observed for most doses. The one notable exception was the increase in the state 4 rate of palmitoyl carnitine oxidation in red muscle in response to T2 treatment. The basis for this dose effect remains to be explained. However, doses used in the present study were derived from those reported to

have elicited physiological responses in mammalian studies of isolated rat mitochondria (Sterling et al., 1977, 1980; Sterling and Brenner, 1995) based on the fact that T3 distribution within the rainbow trout and the rat was shown to be comparable (Fok et al., 1990). The doses used for T3 are within the range reported for liver and muscle of rainbow trout (Fok et al., 1990). Tissue values for T2 are not available at this time. Altered rates of state 3 and state 4 respiration indicate that T2 and T3 exert rapid, direct effects on mitochondrial metabolism independent of those involving the binding of THs to nuclear receptors. In goldfish, these effects appear to be tissue specific; changes in state 3 oxidation of pyruvate were observed in liver mitochondria following T2 treatment and in red muscle mitochondria following T3 incubation. Further studies are required to establish the specific site(s) and mechanism(s) of action.

ACKNOWLEDGMENTS We thank P. J. LeBlanc for his assistance. This work was supported by an NSERC operating grant to JSB.

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