Clofibrate enhancement of mitochondrial carnitine transport system of rat liver and augmentation of liver carnitine and γ-butyrobetaine hydroxylase activity by thyroxine

363

Biochimica et Biophysics Acta, 617 (1980) 0 Elsevier/North-Holland Biomedical Press

363-370

BBA 51520

CLOFIBRATE ENHANCEMENT OF MITOCHONDRIAL CARNITINE TRANSPORT SYSTEM OF RAT LIVER AND AUGMENTATION OF LIVER CARNITINE AND ?BUTYROBETAINE HYDROXYLASE ACTIVITY BY THYROXINE *

SHRI V. PANDE

and R. PARVIN

Laboratory of Intermediary H2W 1R7 (Canada) (Received

Metabotism, Clinical Research Institute of Montreal, Montreal,

July 4th, 1979)

Key words: Clofibrute enhancement, Thyroxine; (Rat liver)

Carnitine transport; y-Butyrobetaine

hydroxylase;

Summary The possibilities that the hypotriglyceridemic effect of clofibrate involves activation of carnitinedependent oxidation of fatty acids in liver and that this may be partially mediated through thyroxine have been examined. 0.25% clofibrate in diet for 10-15 days, was found to increase camitine 3-fold in livers of male as well as female rats. Liver carnitine was nearly doubled by L-thyroxine, 6 mg/kg of diet fed for 10 days, and so was the activity of y-butyrobetaine hydroxylase. Clofibrate decreased carnitine in heart and urine; thyroxine did not affect these parameters but increased serum carnitine by 26%. Clofibrate feeding doubled the concentration of hepatic long-chain acyl(-)carnitine, mitochondrial carnitine, and the rate of mitochondrial carnitine~cylc~itine translocase reaction, and enhanced acetoacetate production in liver homogenates as well as mitochondrial oxidation of palmitoylcarnitine in the presence of malonate. The ratio of esterified to free camitine in urine and serum was also increased by clofibrate. These results suggest that clofibrate and thyroxine may exert their hypotriglyceridemic effect, in part, through the activation of carnitine-mediated transport of fatty acids in liver mitochondria.

* A portion

of this work

has appeared

as an abstract

((1978)

J. Mol.

Ceil.

Cardiof.

10

(Suppl. 1). 75).

364

Introduction Clofibrate is a hypotriglyceridemic agent useful in the treatment of type III and type IV hyperlipoproteinemia. Although the serum triacylglycerol-lowering effect of clofibrate is well established, how this is accomplished is not fully understood [l-3]. Results described below show that clofibrate markedly enhances the carnitinedependent transport of fatty acids into liver mitochondria for oxidation and suggest that this effect contributes to the hypotriglyceridemic action of clofibrate. The possibility that a similar mechanism facilitates the hypotriglyceridemic effect of thyroxine [4,5] is also indicated by our data. Materials and Methods Animals and diets. 6-7-week-old male Sprague-Dawley rats were purchased from Canadian Breeding Laboratory and acclimated for 7-12 days to ground Purina rat chow diet. Then on five consecutive mornings, two rats were transferred to individual metabolic cages; one of these rats continued to receive the ground Purina rat chow diet to serve as control while the other rat received the same diet supplemented with 0.25% (w/w) clofibrate. On the fifteenth-day of this feeding regimen, a clofibrate-fed rat and its corresponding control rat were killed for various analyses. The initial body weights of control and clofibrate-fed rats on day ‘zero’ were 218 + 4 and 213 2 6 g (means ? S.E.), respectively. Urine of each rat was collected individually in flasks containing 0.5 ml 6 m HCl for the 24 h period preceding death. For following thyroxine effect, five male rats were fed ad libitum ground Purina rat chow diet supplemented with 6 mg of L-thyroxine (sodium salt, obtained from Sigma)/kg of diet while five male control rats received the same diet but without thyroxine. On completing 10 days on these diets, the rats were killed for various analyses. Carnitine analyses. Tissue extracts were prepared and total and free carnitine determined as described [6] except that 0.6 ml of water and about 300 mg of a resin (AG2-X8, 200-400 mesh, Cl- form) were used in place of charcoal suspension for the separation of acetylcarnitine from acetyl-CoA. Esterified carnitine content was obtained by subtracting value of free carnitine from that of total carnitine. For the estimation of long-chain acylcarnitine content, portions of ethanolic extract of liver (see Ref. 6), corresponding to 100 mg of tissue, were freed of solvent by evaporation under reduced pressure. To the dried residue was added 3 ml of 0.7 M HCl saturated with butanol and 1.5 ml of butanol saturated with water. The samples were vigorously mixed on a Vortex for 60 s and then the tubes were centrifuged. The butanol phase was washed with 1.5 ml of water saturated with butanol. Portions of the butanol phase were subjected to alkaline hydrolysis and the carnitine set free was estimated as described in Ref. 6. Assay of y-butyrobetaine hydroxylase and deoxycarnitine. For the assay of trimethylaminobutyrate (butyrobetaine) hydroxylase (EC 1.14.11.1), optimal assay conditions were determined based on the cofactor requirements of hydroxylase [ 71. Accordingly, liver homogenates were prepared in 110 mM

365

KCl, 10 mM potassium phosphate buffer, pH 7.2, 1 mM dithiothreitol, 50 PM EDTA and the supernatant fraction was dialyzed against the same medium but having only l/lOth as much dithiothreitol. The enzyme incubation system, in a final volume of 50 ~1, contained 20 mM potassium phosphate of pH 6.7, 20 mM KCI, 0.1 mM dithiothreitol, 5 mM ascorbate, 0.5 mM ferrous ammonium sulfate, 3 mM a-ketoglutarate, 0.1 or 1 mM y-butyrobetaine, 20 pg of catalase, and between 10 and 30 pg protein from dialyzed rat liver supernatant. Incubations were for 30 min at 30°C. For deoxycarnitine analysis, samples were incubated with an excess of a partially purified y-butyrobetaine hydroxylase under conditions that permitted a quantitative conversion of deoxycamitine to carnitine (unpublished method) and the resultant carnitine estimated by coupling to carnitine acetyltransferase [6]. Assay of carnitineacylcarnitine translocase by carnitine influx. Reaction system (100 ~1) contained: 80 mM KCl, 20 mM sucrose, 20 mM Tris-HCl camitine (0.5 PCi per tube) at the concentration shown, and (PH 7.4), [‘“Cl freshly isolated mitochondria (3 mg of protein). Incubations were at 5°C for 20 s. Transport of carnitine was stopped by the addition of 150 ~1 of a mixture containing 80 mM KCl, 20 mM sucrose, 20 mM Tris-HCl (pH 7.4) and 1.67 mM mersalyl. Control incubations had 1 mM mersalyl added prior to the addition of mitochondria. Mitochondria were separated by centrifugation through a wash layer containing dextran and silicone oil and radioactivity determined as described in Ref. 8. Mitochondrial respiratory experiments. The ability of mitochondria to respire with different substrates was determined by following oxygen consumption polarographically using a Clark oxygen electrode (Oxygraph, Gilson Medical electronics, Madison, WI). The incubation systems, in a final volume of 1.95-2 ml, contained 230 mM mannitol, 70 mM sucrose, 20 mM Tris-HCl, 20 I_LMEDTA, 5 mM ADP, freshly isolated liver mitochondria (1.5-2.5 mg protein) and substrates as follows: either 1 mM pyruvate with 1 mM malate, 5 mM glutamate with 5 mM malate, 6 FM palmitoylcamitine with 1 mM malate, or 12 PM palmitoylcarnitine with 3 mM malonate. Measurement of ace toaceta te production by liver homogenates. Reaction system, in a final volume of 1 ml, contained 230 mM mannitol, 70 mM sucrose, 20 mM Tris-HCI, 20 PM EDTA, 5 mM potassium phosphate, 5 mM ADP, 3 mM potassium malonate, 25 I.IM palmitoyl(-)camitine, and liver homogenate corresponding to 5.4 mg wet weight of liver. Final pH was 7.4 and temperature was 37°C. After 5 min incubation, reactions were stopped with perchloric acid and acetoacetate was estimated in the supematants by the 3-hydroxybutyrate dehydrogenase method [ 91. To account for endogenous acetoacetate present in liver homogenates, zero min incubations were carried out and a correction was applied for these values. Preparation of liver mitochondria. Mitochondria from liver were isolated as described earlier [lo]. Statistical analyses. Statistical analyses were carried out with the two-tailed Student’s t-test for unpaired data and P values of GO.05 are considered significant. Values given throughout are mean ? S.E. of data obtained from five rats in each group.

366

Results Experiments utilizing intact liver, its slices, and isolated hepatocytes have indicated that clofibrate enhances fatty acid oxidation and ketogenesis [ll--151 but results with liver mitochondria have been at variance as Cederbaum et al. [16] have reported a decrease and Mackerer [17] an increase in fatty acid oxidation due to clofibrate. We found that clofibrate feeding did not affect the ability of liver mitochondria to respire with pyruvate, glutamate, or palmitoylcarnitine followed in the presence of malate; the respiratory control indices and ADP/O ratios, measured with pyruvate and glutamate, were also not affected. However, when palmitoylcarnitine-dependent respiration was followed in the presence of malonate, mitochondria from clofibrate group showed an enhanced respiratory ability (P < 0.05) compared to those from the controls; the rates of respiration as natoms 0 consumed/min per mg were 56 f: 1.7 for controls and 69 2 3.9 for the clofibrate group. This suggests that clofibrate increased the capacity of P-oxidation and/or the ketogenic enzymes. In support of this, we found that the formation of acetoacetate assayed using liver homogenates, in the presence of palmitoylcarnitine, was also increased (P < 0.025) by clofibrate; the rates of acetoacetate production as pmol/h per g wet liver were 24.9 + 0.97 and 34.7 +_3.23 for the controls and clofibrate groups, respectively. This difference in acetoacetate production was more marked in the absence of added palmitoylcarnitine; under these conditions, the corresponding values for controls and clofibrate group were 0.9 * 0.09 and 7.2 + 0.42, respectively. This increased production of acetoacetate from endogenous substrates could have resulted from increases in the activities of fatty acylCoA synthetase [ 181 and carnitine palmitoyltransferase [ 18,191, as well as from the increase in the amount of CoA [20] and camitine (see below) which follow clofibrate treatment. Because clofibrate has been reported not to affect the activity of hepatic triacylglycerol lipase [ 21,221, it is unlikely that endogenous lipolysis provided more fatty acids in the homogenates of clofibrate group than in those of controls. The possibility that clofibrate feeding was enhancing also the ability of mitochondria to transport fatty acids as acylcarnitine was investigated because of recent findings [lo] that conditions of enhanced fatty acid oxidation and ketogenesis show increased rates of carnitine-acylcarnitine translocase reaction brought about by increase of camitine concentration in mitochondria as well as in whole liver. We found that clofibrate feeding increased (P < 0.005) the rate of carnitine-acylcarnitine translocase reaction of isolated liver mitochondria; the rates of camitine transport as nmol/min per mg protein were, respectively, 0.4 + 0.07 and 0.9 +_0.08 for controls and clofibrate group at 0.3 mM medium camitine, and 0.8 + 0.06 and 1.7 + 0.2 at 1 .O mM medium carnitine. Clofibrate feeding increased also the carnitine content of liver (Table I) as well as that of liver mitochondria; the values of the latter expressed as nmol/mg protein for control and clofibrate group were, respectively, 0.25 + 0.04 and 0.6 2 0.13 for conventionally prepared mitochondria and 0.6 + 0.07 and 1.47 f 0.16 for mitochondria isolated by a silicone oil method [lo]. Clofibrate increased (P < 0.001) also the concentration of long-chain acylcarnitine in liver from a control value of 41 + 6 to 98 + 7 nmol/lOO g body weight.

361

TABLE EFFECT

I OF CLOFIBRATE

ON TOTAL

OF CERTAIN

TISSUES

nmol/lOO Male Female

Liver **

Controls Clofibrate Controls Clofibrate

Liver

1732 5527 * 1085 3157 *

Heart

Male

Skeletal muscle

g body

weight

f 172 ? 548 t 57 * 126

nmol/g Male

OF RATS

Camitine

Group

Tissue

Sex of rats

CARNITINE

wet tissue

1712+ 24 1434 * t 60 1199 * 55 1098 f 75

Controls Clofibrate Controls Clofibrate

* P < or 4 0.005 compared to the corresponding controls. ** Clofibrate diet was fed for only 10 days unlike 15 days to male rats.

Recently we [lo] and others [23,24] have observed that the ratio of acylcamitine to free camitine in serum and urine rises on starvation suggesting that an increase of fatty acid oxidation in liver not only accompanies enhanced export of acetyl groups as ketone bodies but also as acetylcamitine. The present finding that clofibrate increased this ratio in serum and urine (Table II), therefore, indicates that clofibrate enhanced fatty acid oxidation in vivo as well. Table III shows that dietary thyroxine increased carnitine by 86% in liver and 26% in serum; camitine levels in heart, skeletal muscle, and the urinary excretion of camitine were not affected. Thyroxine treatment ha8 been reported to enhance carnitine in hearts of guinea-pigs [25] but to decrease it in heart and skeletal muscle of mice [26]. Because of differences in animals, and dose and duration of thyroxine treatment, a direct comparison of these TABLE EFFECT URINE

II OF CLOFIBRATE

FEEDING

Total deoxycarnitine

Serum concentration Controls Clofibrate

* P < or < 0.05.

Total

nmol/lOO 86 * 16 20*10’

AND

DEOXYCARNITINE

OF SERUM

Camitine

57-t 47*

Urinary concentration Controls Clofibrate

ON CARNITINE

Free

2 4* g body

806 * 124 419 * 22*

42i 26~

Esterified

2 2*

15-t: 21*

2 3

AND

Esterlfied to free carnitine ratio

0.36 0.79

f 0.08 * 0.13

*

0.45 + 0.08 2.30 f 0.49

*

wt. in the last 24 h 582 f 124 138* 19 *

224 f 12 282 f 18 *

368

TABLE EFFECT

III OF

L-THYROXINE

ON

CARNITINE

OF

CERTAIN

TISSUES,

SERUM,

AND

URINE

OF

RATS n.s..

not

significant. Total

carnitine

Skeletal

Thyroxine

(n = 5)

(n = 5)

329 muscle

Heart

P

Control

nmol/g

Liver

content

wet

+

1132+ 1484

t

tissue

+

59

46

1010

+

78

ll.S.

54

1408

f

45

Il.S.

24

612

co.005

IrM

Serum

68

+

nmol/lOO Urine

1220

f 210

2.0 g body

86 wt.

f

in the last 1471

3.4

co.005

24 h

f 183

Il.S.

divergent results is impossible; nonetheless, we should stress that in our study, effects of thyroxine were followed under conditions where overt signs of thyrotoxicosis were not evident inasmuch relative to that of controls, thyroxine neither affected the weight gain of rats nor their final liver weights. The activity of y-butyrobetaine hydroxylase was increased (P << 0.001) by thyroxine: the enzyme activities expressed as nmol carnitine formed/min per mg protein, for control and clofibrate groups were, respectively, 0.44 + 0.021, and 0.75 + 0.02 when the assay system contained 0.1 mM y-butyrobetaine; the corresponding values in the presence of 1 mM butyrobetaine were 0.16 +_0.009 and 0.32 ? 0.021. Discussion Recently we have found that carnitine concentration in rat liver mitochondria remains below saturating for the camitine-acylcarnitine translocase and that the increased rates of the translocase reaction, observed with mitochondria of starved or diabetic rats, can be related to the elevation of mitochondrial carnitine [lo]. The present work suggests that a similar mechanism is involved in the enhancement of carnitine-acylcarnitine translocase reaction on clofibrate feeding. It is probable that the translocase remains subsaturated also with respect to the concentration of free long-chain acylcamitine in cytosol in vivo. If so, a further enhancement of acylcamitine import would result when the concentrations of long-chain acylcarnitines rise, as they do on starvation, in diabetes 1271 and on clofibrate feeding (this paper). The observed decreases in the urinary excretion of carnitine and y-butyrobetaine suggest that clofibrate favoured the retention of carnitine in liver as well as its enhanced synthesis. Increased mobilization of carnitine from extrahepatic tissues may also have been involved insofar as clofibrate decreased camitine in heart and serum, and a similar trend (but P > 0.05) was seen in skeletal muscle

369

(Table I). However, unlike thyroxine, clofibrate feeding did not affect the activity of y-butyrobetaine hydroxylase in liver. The finding that clofibrate decreased cardiac carnitine warrants further investigation in view of the reports that carnitine shows an antiarrhythmic effect [28] and that clofibrate therapy increases the incidence of arrhythmia as a side effect [ 31. Clofibrate is known to cause proliferation of peroxisomes in liver and Lazarow has suggested that the hypotriglyceridemic effect of clofibrate may be exerted by the activation of peroxisomal fatty acid oxidation [29]. Although the proliferation of peroxisomes is more marked in livers of male than of female rats [ 301, the hypotriglyceridemic effect of clofibrate is seen equally well in both sexes [ 311. The possibility that clofibrate enhances peroxisomal oxidation of fatty acids in livers of female rats just as well as it does in males but without causing overall peroxisomal proliferation in females, is weakened by the observation [ 321 that di-( 2ethyl)phthalate, another peroxisomeproliferating hypolipidemic drug, causes lesser activation of peroxisomal palmitoyl-CoA oxidation in livers of female than of male rats. Moreover, recent work indicates that compared to that of mitochondria the contribution of peroxisomal system to fatty acid oxidation is less than 10% in liver cells obtained from clofibrate-treated rats [ 331. These considerations minimize (cf. Ref. 34) the possibility of peroxisomal fatty acid oxidation being a major factor responsible for the hypotriglyceridemic action of clofibrate. Conversely, if the hepatic carnitineenhancing effect of clofibrate were related to the hypotriglyceridemic effect, then one would expect to see it also in female rats and this was presently observed (Table I). It has been suggested that some of the clofibrate effects may be related to the ability of this drug to displace thyroxine from plasma proteins to liver [35,36]. Although this possibility cannot be entirely excluded inasmuch as thyroxine enhanced liver carnitine, it is lessened by the observations that in all other respects the effects of thyroxine differed horn those of clofibrate. Although in appropriate doses thyroxine itself acts as a hypolipidemic drug, its mechanism of hypotriglyceridemic effect has not been explored [37]. Our finding of the enhancement of liver camitine together with the reported augmentation of carnitine palmitoyltransferase [38] suggest that the stimulation of hepatic fatty acid oxidation by thyroid hormones (cf. Ref. 39) may also be aided by an acceleration of the carnitinedependent transport of fatty acids into mitochondria. Unlike clofibrate, however, thyroxine did not increase the ratio of esterified to free carnitine in serum and urine of rats, and this may relate to the fact that whereas clofibrate enhances camitine acetyltransferase activity several fold in liver, thyroxine does not [ 401. Acknowledgements We thank Ayerst Laboratories of Montreal for supply of clofibrate and Miss Denise Guertin and Mr. Antoine Brault for excellent technical help. This work was supported by grants from the Quebec Heart Foundation and the Medical Research Council of Canada (MT-4264).

370

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