butyrobetaine availability in liver is a regulatory factor for carnitine biosynthesis in rat : flux...

Eur. J. Biochem. 185,671 -675 (1989) 0 FEBS 1989

Butyrobetaine availability in liver is a regulatory factor for carnitine biosynthesis in rat Flux through butyrobetaine hydroxylase in fasting state

Attila SANDOR' and Charles L. FfOPPEL'

Institute of Biochemistry, University Medical School, Pecs, Hungary Medical Research, Vcterans Administration Medical Center, Cleveland

(Received January 9/July 17, 1989) - EJB 89 0029

Urinary excretion of total carnitine in 48-h fasted rats dropped to 0.30 0.01 ymol/day from 2.23 0.4 pmol/ day found in fed. control animals (mean SEM). Despite this marked retention, the total carnitine content of the whole body remained constant, about 83 pmol, predicting a slow-down in biosynthesis.

The conversion of butyrobetaine into carnitine takes place only in the liver in rats. 48 h of starvation caused a decrease in the liver butyrobctaine levcl from 11.6 f 1.19 nmol/g to 9.30 & 1.19 nmol/g, which in whole livers corresponds to a decrease from 138 nmol to 61.3 nmol. The convcrsion rate of butyrobetaine into carnitine was studied with radiolabelled butyrobetaine. 30 min aftcr injcction of ['Hlbutyrobetaine the carnitine pool in the liver of fasted rats was labelled to about the same extent as that in fed rats. but from a butyrobetaine pool with higher specific radioactivity. Therefore, the conversion rate of butyrobetaine into carnitine was reduced. The newly formed carnitine found in the whole body of fasted rats was estimated to be 59% of controls. We conclude that the biosynthesis of carnitine in fasted rats slows down, for which a decreased availability of butyrobetaine in thc liver is responsible.

Urinary excretion of butyrobetaine in the fasted group dccreased to 74.1 nmol/day from the 222-nmol/day control value while the butyrobetaine content of whole body did not significantly decrease (2.85 pmol vs. 3.04 pmol). Urinary excretion of trimethyllysine was also depressed.

The enzymatic steps leading to the synthesis of carnitine from L-lysine and L-methionine are well established [I]. In view of- the present work it is an important detail that the last step, hydroxylation of butyrobetaine to form carnitine in rats takes place exclusively in the liver [2 ] . Relatively few reports have appeared on the regulation of the overall pathway. Con- tributions by Rebouche et al. [3] and by Davis and Hoppel [4] proposed trimethyllysine availability as the regulatory factor for carnitine biosynthcsis. Rcccntly we made a substantial modification on a known method for butyrobetaine determination [5 ] . The modified method [6] enabled us to determine butyrobetaine and to investigate the flux through butyrobetaine hydroxylase, using [3H]butyrobetaine.

Having an essential role in fatty acid oxidation, the metab- olism (biosynthesis, excretion and balance) of carnitine de- serves special attention under conditions when fatty acids are the primary fuel, such as during starvation. Previously, it was reported that urinary excretion of carnitine markedly decreased on the effect of starvation while acid-soluble carnitine (free plus short-chain carnitine esters), in the whole body remained constant [7]. These observations suggest that carnitine biosynthesis proceeds at a reduced rate in the fasting state (keeping in mind that urinary excretion is the only signif- icant way of losing carnitine). In the present work we extend the measurement of carnitine to total carnitine in the whole

Correspondence to A. Sandor, Institute of Biochemistry, Univer-

Enzymed. Carnitine acetyltransferase (EC 2.3.1.7); y-butyro- sity Medical School, SLigeti ul 12, H-7624, POcs, Hungary

betaine hydroxylase (EC 1.14.31.1).

body to establish the carnitine balance and to define whether or not overall biosynthesis slows down. If so, the possibility will be tested whether the flux through butyrobetaine hydroxylase is involved in regulation. We have also made some determinations of trimethyllysine by a currently im- proved method [8]. Combining the above methods we can survey the full pathway of carnitine biosynthesis.

MATERIALS AND METHODS

Chemicals Chemicals and solvents were of reagent grade. Carnitine

acetyltransferase was from Boehringer Mannheim (In- dianapolis, USA) [ l-14C]Acetyl-coenzyme A was from New England Nuclear (Boston, USA). [Me-3H]Butyrobetaine was prepared according to the procedure described for labelled carnitine [9], using 4-dimethylaminobutyric acid (purchased from Aldrich, Milwaukee, USA). Its specific radioactivity was close to 80 mCi/mmol.

Animals

Sprague-Dawley male rats of mass 200 - 220 g were used in all experiments. They were housed in individual wire-bot- tom metabolic cages in a light-controlled room and maintained on a balanced laboratory diet (from Purina) ad libitum. Urine was collected when indicated. Starved animals were deprived of food 48 h prior to the experiment. All experiments were performed bctwcen 0900 and 1200.

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Sample preparations urid unulj

0.5 g tissue (liver, kidney and muscle) was homogenized in 2.0 ml water in a conical glass tube by a motor-driven homogenizer. To this was added 100 p1 10 M KOH then samples were hydrolysed for 1 h at 50°C. After chilling in ice- cold water 200 p1 70% (masslvol.) perchloric acid was added and, following vigorous shaking, the tubes were centrifuged (2000 x g, 10 min). The pellets were washed with 1.0 ml 3% perchloric acid and supernatants were combined. To 1.0 ml plasma was added 50 p1 10 M KOH then the procedure was the same as for tissue. Finally, 3% perchloric acid extracts were neutralized with 10 M KOH to pH 6- 7, frozen, thawed and centrifuged (2000 x g for 10 rnin). The resulting supernatant, the neutralized perchloric acid extract, was used for the following determinations : (a) measurement of total radioactivity; (b) measurement of total carnitine; (c) determination of the distribution of radioactivity between carnitine and butyrobetaine. From separate samples (0.5 g tissue, 1.0 ml plasma) 3% perchloric acid extracts were prepared again using the same volume ratios and procedure as above, with the exception that hydrolysis in KOH was omitted. After neutralization this extract was used for butyrobetaine detcrmi- nation.

The animal carcasses (the remainder of the body after removal of liver, kidney, stomach and all intestines) were chopped into about ten pieces then dissolved in 500 ml 50% ethanol (by vol.) 20% KOH (massivol.) solution in a Waring blender. The blendcr was turned on for 30 s several times until a homogenous slurry was obtained. (This procedure usually took no more than 10 min.) After measuring the final volume, a 6.0-ml aliquot of slurry was taken out, diluted to 30 ml with water and kept at 50°C for 1 h. After chilling, it was acidified with approximately 2.0 ml 70% perchloric acid, centrifuged, and the pellet was washed with 3.0 ml 3% perchloric acid. The combined supernatant was neutralized with 10 M KOH, frozen overnight and centrifuged following thawing. This neutralized perchloric acid extract was evaporated to dryness under air in a 40°C water bath and the resultant pellet was reconstituted in 6.0 ml 3% perchloric acid. (Note that this acid extract went through alkaline hydrolysis and therefore contains total carnitine). This extract was neutralized with 10 M KOH as above and used for all analyses.

Total carnitine was determined by a radioisotopic method [lo] and applied to small columns as previously described [7].

Distribution of 3H radioactivity between carnitine and butyrobetaine was determined as follows: butyrobetaine and carnitine (together with other trimethylamines) were isolated by small-column, ion-exchange chromatography as described [6]. The resultant effluent (3.0 m12.4 M NH,OH) was evaporated to dryness under a stream of compressed air and reconstituted in 100 pl eluent to be used for high-performance liquid chromatography in order to separate butyrobetaine and carnitine. 1 .O-ml fractions were collected directly into vials for measurement of radioactivity. HPLC conditions were the same as for butyrobetaine determination [6].

Butyrobetaine was determined in a neutralized perchloric acid extract of tissues or 1 .O ml plasma as described previously [61.

Radioactive measurement

Radioactivity in each sample was measured in toluene/ Triton X-100 scintillant (2: 1. by vol.) containing 2,5- diphenyloxazole ( 5 g/l) and 1,4-bis(5-phenyloxazolyl-2)ben-

2 0 0 1 A Total radioactivity

0 - - s B Carnitine radioactivity

"0 15 30 15 60 Time after injection of I3H1 butyrobetaine (min)

Fig. 1. Content tmd distribution of injected [3H]butyrobetaine in rat organs usfunction ($'time. 3.5 x lo6 cprn [311]butyrobetaine was injected intraperitoncally and thc animals wcrc sacrificed at the indicated times. Tissues and plasma were workcd up. tritium content and its distribution between [3H]butyrobetaine and [3H]carnitine were measured as detailed under Materials and Methods. (A) Total radioactivity; (B) radioaclivity present as carnitine. Points represent mean SD for four animals; (0-e) liver; (0-0) kiney; (A-A) serum

zene (200 mg/l) by a scintillation spectrometer (Prias Model, Packard Instruments). When 14C: radioactivity was to be measured in thc presence of tritium (e.g. in carnitine determination) the 3H content was excluded by narrow window set- ting.

RESULTS The conversion of [ 3H]butyrobetaine into carnitine is

shown in Fig. 1 as appearance of [3H]carnitine as a function of time in different organs. Rased on this investigation, the point 30 rnin after the application of isotope was chosen as optimal time to study the conversion (Table I ) because (a) an easily measurable portion of radioactivity is present in carnitine at this time and (b) the amount of total radioactivity does not change dramatically between 15 rnin and 60 min. (Otherwise, Fig. 1 complies well with the fact that, in rat, the liver takes up most of the butyrobetaine, converts it to carnitine and supplies other organs with it [2].)

Table 1 shows the amount of radioactivity and its distribution between [3H]carnitine and [3H]butyrobetaine in liver 15 min and 30 min after intraperitoneal injection of [3H]butyrobetaine in the case of fed and fasted rats. Butyrobetaine content was also determined in order to obtain its specific radioactivity because i t is indispensable for calcu- lating the amount of carnitine formed. The animals from the 30-min experiment were subjected to a detailed study

673

'I'able 1. Butyrobetaine content and conversion of [3H]hutyrobvtaine into carnitine ufter 15 min and 30 rnin in the liver o f fed and fusted rats Fed and 48-h fastcd animals were injected with 3.5 x lo6 cpm [3H]butyrobetaine, 15 rnin or 30 rnin prior to sacrifice. In the 15-min group liver mass was 10.0 _+ 1 . I g in the fed and 5.21 g in the fasted animals. The respective values in the 30-min group were 11.9 0.90 g and 6.6 f 0.32 g . Values are mean f SEM for six animals. Numbers in parentheses are radioactivity or butyrobetaine in whole liver

[3H]Butyrobetainc Butyrobetaine Specific Injection Treatment Total [3 HICarnit ine time radioactivity radioactivity

of [3H]butyrobcta~ne

min cpm x 1 0 - ~ i g nmol/g cpm/nmol

15 fed 92.6 5.26 45.3 t 4.50 (451) 47.5 24.50 (475) 8.66 f 1.01 (86.6) 5484 fasted 143 k 7.51h 83 3 7.20b (433) 5Y.6 k7.20" (310) 6.08 5 1.10" (31.6) 9802

30 fed 137 f 5.9 92.6 7.1 (1101) 44.4 f 7.1 (528) 11.6f 1.27 (138) 3827 Pasted 203 & 18.4b 161 12.1 (1062) 42.0 f 8.1 (277) 9.30& 1.19a(61.3) 4516

a p < 0.01. p < 0.001 whcn compared with the Ted value within an experiment.

where extrahepatic tissues and urine were analyzed (Tables 2, 4 and 5).

In parallel with the 1 5-min experiment seen in Table 1 , another 15-min experiment was performed in which the isotope was supplied intravenously. The uptake and distribution of isotope in the liver of fed and fasted rats showed the same pattern as in the case of intraperitoneal application (data not shown). This observation indicates that starvation does not affect the fate of intraperitoneally injected [3H]butyrobetaine by changing its abdominal absorption.

It is seen in Table 1 that whole livers of fed and fasted animals contain about the same amount of [3H]carnitine: 1.101 x lo6 cpin and 1 . 0 6 2 ~ lo6 cpm in the 30-min experiment;4.51 x lo5 cpmand4.33 x lo5 cpminthe15-minexper- iment. To calculate the amount of carnitine synthesized we have to use the specific radioactivity of butyrobetainc, that is, radioactivity of carnitine (cpm)/specific radioactivity of butyrobetaine (cpmjnmol) gives the amount of carnitine formed (nmol). Due to the much lower liver butyrobetaine content, the specific radioactivity of the butyrobetaine pool must be higher in the liver of fasted animals (Table 1). On this basis it is clear that in livers from fasted rats less carnitine was formed.

For the quantitation of carnitine formed during the 30 min we need a butyrobetaine specific radioactivity which is characteristic for the whole 30-min period. As time elapses after injection of [3H]butyrobetaine, the greater part of radioactivity is present in carnitine (Fig. I), that is, less and less in butyrobetaine. The specific radioactivities of butyrobetaine found after 30 rnin (Table 1) are extreme values, characteristic for the terminal part of the period. The relative (fasted/fed) specific radioactivity is 1.18 at this point. Using these butyrobetaine specific radioactivities to calculate the carnitine formed we obtain 287 nmol for fed rats and 235 nmol(81.8%) for fasted rats.

The other extreme condition is the earliest time of the experiment when almost all radioactivity is present in butyrobetaine. For this point, only the relative specific radioactivity can be estimated assuming that the fasted/fed ratio of total rddioactivities, 1.48, is close the same during the experiment. (This value is 1.54 for the 25-min experiment.) According to this, the fasted/fed relative specific radioactivity is: (203 cpm/l37 cpm)/(9.30 nmol/ll.6 nmol) = 1.84. Using this relative specific radioactivity to calculate the relative amount of formed carnitine: (1062 cpm/l101 cpm)/1.84 =

Table 2. Appearance of r3 Hjcarnitinv 30 min ajter intraperitoneal administration of [3H]hutyrohetaine in extrahepatic tissues of f e d and ,fasted rats Animals were the same as in the 30-min experiment in Table 1. Tissues were analyzed for distribution of radioactivity. Values are mean f SEM for six animals. Total plasma volumes were calculated Lo be 5.0 ml in fed and 3.5 ml in fasted animals. Muscle carnitine is included in carcass

Organ Radioactivity in carnitinc

fed fasted

cpm x 10-3/organ

Kidney 61.7 2.87 91.6 & 4.43 Plasma 8.85 7.8 Muscle (1 g) 0.62 1.77 Carcass 143 f 11.6 265 k 8.67

0.52, that is, the carnitine formed (and contained) in the liver of fasted rats is 52% of controls.

Although the two calculations outlined above represent extremes, both of them led to the conclusion that liver from fasting rats formed less carnitine from butyrobetaine. To find the butyrobetaine specific radioactivity which is most characteristic for the 30-min period we consider the best ap- proach to calculate with specific radioactivity found after I S min, at the mid-point of the 30-min period (Table 1). These calculations are presented in Table 3. The Table 3 shows that the radioactive carnitine found in the liver corresponds to 200 nmol and 108 nmol for fed and fasted rats, respectively.

The considerations made so far refer to newly formed carnitine contained in the liver. To estimate the overall rate of butyrobetaine - carnitine conversion we have to take into account the carnitine formed and released from the liver to extrahepatic tissues during the 30-min experimental period. Tablc 2 shows [3H]carnitine found in plasma, kidney, muscle and whole carcass 30 min after injection of [3H]butyrobetaine. Comparing data from Table 2 with Table 1, it appears that a major part of ['Hlcarnitine after 30 rnin is contained in the liver, so, released carnitine can only slightly modify the quanti- tative conclusion. The summarized [3H]carnitine in the whole body (liver plus extrahepatic tissucs) can bc seen in Tablc 3 . Based on the [3H]carnitine content of the whole body we

674

Table 3. Quantitulion of carnitine ,formed in 30 min in ,fed and fasted rats For calculation of carnitine formed, the specific radioactivities of liver butyrobctaine found in the 15-min experiment (Table 1) were used. Carnitine radioactivity in whole body is thc sum of radioactivity in liver and extrahepatic tissues (Table 2)

Treatment Carnitine radioactivity in Formed carnitine found in

liver body liver body

lo-' x cpin nmol (YO)

Fed 1 .lo1 1.330 200 (100) 242 (100) Fastcd 1.062 1.426 108 (54) 145 (59)

conclude that the rate of carnitine synthesis from butyrobetaine decreases to 59% of control in the 48-h fasted rats (Table 3).

Further analyses of the animals of the 30-min experiment showed that, consistent with earlier findings [7], the total carnitine content of the body was constant (83.3 - 83.5 Fmol), while urinary excretion of carnitine dropped from 2.23 - 0.30 pmol/day due to the effect of 48 h of starvation (data not shown). The fact that despite strong urinary conservation whole body carnitine remains constant suggests a slow-down in carnitine biosynthesis. This study provides evidence that the butyrobetaine - carnitine conversion is the step (or one of the steps) where carnitine synthesis is impaired.

To achieve further insight into the regulation of the biosynthesis of carnitine, measurements were extended to its precur- sors, butyrobetaine and trimethyllysine. Table 4 shows the content of butyrobetaine in different organs and in the whole body and its urinary excretion. While the excretion of butyrobetaine strictly drops by day 2 of fasting, the butyrobetaine content of the whole body tends to decrease (but does not change significantly).

The first special precursor or carnitine byosynthesis is trimethyllysine. In exists as free compound in body fluids and tissues, but the major part of it occurs in a peptide-linked form in tissue [4. 111. A special derivative of trimethyllysine is acetyltrimethyllysine which occurs only in urine [3,4]. Table 5 shows a marked decrease in urinary excretion of trimethyllysine on days 1 and 2 of starvation. The reduction is even more pronounced in the free fraction, so the reduction or the free/total ratio is also impressive. The conservation of trimethyllysine was accompanied by its increased plasma level (Table 5), suggesting that renal handling had changed.

DISCUSSION

Earlier data, including markedly reduced urinary excretion and constant body content of carnitine in fasting rats [7, 121, suggested a reduced synthesis rate for carnitine. While searching for the site of impairment in biosynthesis we assessed the conversion rate of butyrobetaine into carnitine. For fidclity, we confirmed earlier findings 171 and tested butyrobetaine - carnitine conversion on the same animals.

The present study has supplied evidence that the amount of butyrobetaine converted into carnitine was reduced to 59% of the fed, control value in 48-h fasted rats. In other words, the flux through butyrobetaine hydroxylase slowed down. An obvious reason for this is the markedly reduced butyrobetaine

Table 4. Butyobetnine contetzt in tissues and urinary excrerioii of hutyvohetaine in fed andfasted rats Butyrobetainc content was determined as described in Matcrials and Mcthods. Urine was collccted over two days, thus day 1 indicatcs 24 h, day 2 indicates 4X h of Fasting in the starved group. Values are mean SEM for six animals. Total plasma volumes were calculated to be 5.0 ml in fed and 3.5 ml in fasted animals. Muscle butyrobetaine is included in carcass. Whole body butyrobetaine was calculated by addition. Urinary excretion of butyrobetaine was measured after days 1 and 2 in fed rats as 243 f 31 .I nmol/day and 224 & 24.7 nmol/ day, respcctively, and in fasted rats as 189 24.2 nmol/day and 74.4 & 11.6 nmol/day, respectively

Organ Butyrobctaine content

fed fasted fed fasted

nmol/g nmoliorgan

Liver 11.6 f1 .27 9.30f 1.19 238 61.3" Plasma 6.04 0.8 4.73 f 0.6 30.0 16.5 Kidney 19.1 f 1.4 19.8 k2.6 44.0 36.8

Carcass 13.6 f 0 . 9 15.2 f 1.2 2828 2741 - 3040 2855 Wholebody -

- Muscle (g) 27.2 f 2 . 3 26.5 f 1.4 -

a 1) < 0.001 whcn compared with the fed value.

content in the liver (Table l), in the only organ which is able to convert butyrobetaine into carnitine.

The reason for the depressed liver butyrobetaine in fasting is surely based on the properties of hepatocellular transport. Butyrobetaine, like carnitine, is transported into the liver by an active process [13, 141. However, unlike carnitine, butyrobetaine cannot be concentrated by liver of fasting rats above the fed value. For carnitine, both the release and uptake aspects of transport are controlled in order to increase the hepatic carnitine level in fasting rats [14, 151. This mecha- nism, it seems, does not operate for butyrobetainc. Con- versely, the level of butyrobetaine/g tissue slightly decreased (Table 1) [5] resulting in a severe depression on a whole liver basis. This comparison raises the issue that butyrobetaine and carnitine must have different transport systems.

Using a recently developed method [6], we determined the butyrobetaine balance in the rat by measuring butyrobetaine in urine, different organs and in the whole carcass. Urinary excretion of butyrobetaine dropped from 224 nmol/day (fed rats) to 74.4 nmol by the day 2 of starvation (Table 4), while the whole-body content tended to decrease, but not significantly (Table 4). The fact that despite the increased retention and reduced conversion into carnitine, the whole-body butyrobetaine content remained constant (instead of- rising) in Fasting rats, can be explained by reduced formation from precursor. ('The lack of dietary uptake, 11 nmol/day, was neg- ligible.)

Finally, we should discuss the present results in view of trimethyllysine availability. The free trimethyllysine level in plasma increased by day 2 of starvation (Table 5 ) [4] in liver and in the kidney [4], which possesses the highest trimethyllysine hydroxylase activity. However, as Davis and Hoppel established [4] the efficiency of trimethyllysine entry into carnitine biosynthetic pathway decreases, from 80% to 45%, by day 2 of starvation. In the light of present work, the markedly decreased butyrobetaine availability in liver may be a reason for this decreased efficiency.

675

Table 5. Free and total trimethpllysine in the urine and plasma of fed and fasted rats Trimcthyllysinc was determined as described earlier [8]. Values are mean 5 SEM for number of animals shown in parcnthesis. Total trimethyllysine means trimethyllysine plus acetyltrimethyllysine

Treatment n Trimethyll ysine in

urine plasma (free)

free total freelto t a1

nrnol/day nmol/ml

Fed 6 382 t 5 2 930$_95 0.41 Fasted for 24 h 6 43.5 8.4" 382 f 51 0.12 Fed 6 347 +58 848 _+ 77 0.40 Fasted for 48 h 5 8.9 -1- 1.8" 308 & 37b 0.02

-

- 1.08 5 0.07 1.32 Ifr. 0.1 1

p < 0.01. p < 0.005 when compared with the fed value.

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butyrobetaine availability in liver is a regulatory factor for carnitine biosynthesis in rat : flux...

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