CELL BIOCHEMISTRY AND FUNCTION
VOL.
9: 13-21 (1991)
Decarboxylation of Branched-Chain a-Ketoacids in Hepatocytes from Alloxan-Diabetic Rats. The Effect of Insulin ANNA STERNICZUK, ELZBIETA I. WALAJTYS-RODE? AND ANNA B. WOJTCZAKS Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, Pasteura 3, 02-093 Warsaw, Poland
The flux through branched-chain a-ketoacid dehydrogenase and the activity of the branched-chain a-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain a-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain a-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain a-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminshed flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was parallelled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain a-ketoacid oxidation in liver of diabetic rats. K E Y WORDS- Branched-chain ketoacids; diabetes; insulin; hepatocytes.
INTRODUCTION Oxidative decarboxylation of branched-chain a- diminished the amounts of the active and the total ketoacids is catalysed by the mitochondria1 dehydrogenase complex in liver6v7 concomitantly branched-chain a-ketoacid dehydrogenase com- with a decrease of the rate of oxidative plex. In this nonreversible reaction a single complex decarboxylation.' Treatment of rats fed on a lowoxidizes a-keto-derivatives of leucine, valine and protein diet with adrenalin, glucagon or insulin isoleucine. The activity of the complex is rate- increases the activity of the dehydrogenase complex limiting for the metabolism of branched-chain in liver, but no such effect was observed in animals aminoacids, except in liver where transamination is kept on a standard diet.g It is well established that in liver of starved and rate limiting.' Similarly to the pyruvate dehydrogenase complex, the branched-chain a-ketoacid de- diabetic rats the oxidation of pyruvate and the hydrogenase complex is inactivated by activity of the pyruvate dehydrogenase complex are phosphorylation and reactivated by dephosphory- suppressed (reviewed in reference 10). In contrast, lation (for review see reference 2). This regulation there have been only fragmentary and contradichas been shown to occur in enzyme complexes tory observations concerning the activity state of derived from various tissues and animal species.j branched-chain a-ketoacid dehydrogenase in diaMoreover, oxidative decarboxylation of branched- betes. Patston et ~ l . ~ l. 'found a drastic decrease in chain a-ketoacids is regulated by inhibition by the the activity of the complex, whereas Gillim et aL6 end-products, namely NADH and the respective and Paul and Adibi12 observed almost no change acyl-CoA e ~ t e r . It ~ *has ~ been found that alter- under the same conditions. So far, there have been ations of the dietary or hormonal balance induce no systematic studies on the flux through changes in both the rate of the decarboxylation and branched-chain a-ketoacid dehydrogenase and the the activity of the branched-chain ol-ketoacid de- activity of the complex measured in parallel in hydrogenase complex. Low-protein diet strongly livers of diabetic animals. The inhibitory effect of fatty acid oxidation on t Present address: Institute of internal Medicine, Warsaw Medi- the uptake and oxidation of branched-chain aketoacids in hepatocytes isolated from starved rats cal School, 02-097 Warsaw, Poland $Addressee for correspondence. was first described by Williamson et In our 0243 -6484/91/010013-09 $05.00 1991 by John Wiley & Sons, Ltd.
f4 previous rep01-t'~it was shown that the increased oxidation of fatty acids in diabetes could be responsible for diminished rate of oxidation of branchedchain a-ketoacids in liver. The present study extends our investigation over parallel measurements of the rate of decarboxylation of branched-chain a-ketoacids and the activity state of the complex in hepatocytes isolated from rats with alloxan diabetes. MATERIALS AND METHODS Treatment of Animals
Male Wistar rats (200-25Og) were fed on a standard laboratory diet. These animals are further designated as normal fed rats. Starved rats were deprived of food for 18-24 h and had free access to water. Diabetes was induced in normal fed rats by intravenous injection of alloxan, 60 mg kg-' body weight, into the tail vein and the animals were kept for 3-4 days on normal diet and free access to water. Animals with 25-35 mM glucose in blood and ketones in urine of + + to + + + according to Bili-Labstix (Ames Laboratories, Elkhart, IN, USA.) scale were used for experiments. Isolation and Incubation of Hepatocytes
Liver cells were prepared according to the method of Berry and Friend." Cells (5-7 mg dry weight ml- ') were incubated in Krebs-HenseleitI6 bicarbonate medium containing 1 mM CaC1, and 1 per cent defatted and dialysed bovine serum albumin. The concentration of branched-chain a-ketoacids was 2 mM unless indicated otherwise. Incubations were performed in 25 ml Erlenmeyer flasks at 37°C with shaking under an atmosphere of 95 percent 0, and 5 per cent CO,. Viability of the cells was tested with Trypan blue. Preparations containing more than 90 per cent viable cells were used for experiments. 3-Hydroxybutyrate' ' and acetoacetate" were determined by enzymatic methods in neutralized perchloric acid extracts of the whole cell suspension. The rate of ketogenesis was calculated from 20-min incubations after subtracting values for zero-time controls. Determination of the Flux Through BranchedChain a-Ketoacid Dehydrogenase
Hepatocytes (1-2 ml) were preincubated for 10 min without or with insulin (100-200 mU
A. STERNICZUK E T A L
ml-' = 0.65-1.3 PM) under conditions described above; then l-['4C]-labelled a-ketoacid (100150 cpm nmol- ') was added to a final concentration of 2 mM or as indicated in the legends. Flasks were sealed with rubber serum caps fitted with handing centre wells containing filter paper, and the incubation was continued for 20min. The reaction was stopped by injection of 0.5 ml of 2 M H 2 S 0 4to the reaction medium and of 0.2 ml of phenethylamine to the centre well. The flasks were then shaken for 1 h at room temperature. The wells were removed and counted for radioactivity in a liquid scintillation spectrometer. The total radioactivity was determined by counting the amount of the added labelled substrate in 10 ml of the scintillation mixture plus 0.2 ml of phenethylamine.
Assay of the Branched-Chain a-Keioacid Dehydrogenase Complex Activity in Extracts of Hepatocytes
The assay was performed essentially as described by Harris et aL8 with some modifications. Hepatocytes were incubated for 20 min with or without insulin or other additions as indicated in the legends but without a-ketoacids. The volume was 4.0ml. Other conditions were the same as for measurements of the flux. Aliquots of 1-0ml were withdrawn in duplicate and hepatocytes were separated from the medium by a rapid centrifugation for 30 s at 8000 g using a precooled microcentrifuge (Type 320, Unipan, Warsaw, Poland). Pellets were immediately frozen in liquid nitrogen. Cell extracts were prepared by vigorous mixing of the pellet in 0.5 ml of ice-cold solution containing 50 mM Hepes, pH 7.5, 5 mM dithiothreitol, 2 mM EDTA, 2 per cent (v/v) Triton X-100, 0.5 mM N-u-tosyl-L-lysine chlormethylketone and 50 mM NaF for the measurements of the active form of the complex. For determination of the toaI activity of the dehydrogenase the cells were extracted with the same medium, except that NaF was omitted. The activity of the complex was assayed immediately after solubilization of cells. For the determination of the total activity the assay medium contained 30 mM phosphate (pH 7.4), 2 mM MgCl,, 0.4 mM thiamine pyrophosphate, 0.4 mM CoA, 1 mM dithiothreitol, 3 mM NAD, 0.1 per cent Triton X-100, and 2 U lipoamide dehydrogenase in a total volume of 1.0ml. For determination of the active form the medium also contained 50mM NaF. Aliquots of 0.1 ml of the extracts were added in triplicate and
BRANCHED-CHAIN KETOACIDS IN DIABETES
the samples were preincubated for 5 min at 30°C. The reaction was started by the addition of aketo[ l-'4C]isocaproate (200-300 cpm nmol- ') to final concentration of 0.5 mM. For the determination of total activity NaF was added together with the labelled substrate to a final concentration of 50mM in order to keep the same components at identical concentrations. The blanks were run in parallel with the complete system except the cell extract. The test tubes were sealed with caps and further incubated for Smin, then stopped with H 2 S 0 4 , and 14C02was measured as described for the determination of the flux. In accordance with the observation of Harris et a/.' we found that the addition of exogenous phosphoprotein phosphatase did not increase the total activity of the complex in livers of fed and starved rats and therefore phosphatase has not been added in the routine assay procedure. Calculation of the Results
The rates of enzymatic activity were expressed in pmol min-' g-' dry weight of cells. Linearity of ketone body production, and of the flux through and the activity of branched-chain ketoacid dehydrogenase with respect to time and tissue content was established. The average wet to dry weight ratios of 4.5 (n = 15) for cells from fed and alloxandiabetic rats and of 5.0 ( n = 10) for cells from starved animals were estimated. Chemicals
Collagenase was from Boehringer (Mannheim, F.R.G.) or Worthington (Freehold, NJ, U.S.A.); alloxan, bovine serum albumin, L-aminoacid oxidase from Crotalus adamanteus venom and lipoamide dehydrogenase from porcine heart were obtained from Sigma (St. Louis, MO, U.S.A.); enzymes for metabolite determinations were obtained from Boehringer; porcine insulin (40 U ml-l) was from Polfa (Tarchomin, Poland); and inorganic salts from Merck (Darmstadt, F.R.G.). ['4C]-Labelled a-ketoisocaproate and a-ketoisovalerate were prepared from [1-14C]leucine and [1-14C]valine (Amersham International, Amersham, U.K.), respectively, according to Riidiger el a1.19 RESULTS The rates of oxidative decarboxylation of a-ketoisocaproate and a-ketoisolvalerate in hepatocytes
15 isolated from fed, starved and diabetic rats were measured. Higher rates of the decarboxylation of aketoisolvalerate than of a-ketoisocaproate are in agreement with a higher V,,, estimated for a-ketoisolvalerate.8 In accordance with previous observation^,'^ short starvation increased the rate of decarboxylation of branched-chain a-ketoacids. As it is known from previous studies that fatty acid oxidation has a strong inhibitory effect on the metabolism of branched-chain a-ketoacids,' )*14 we tried to differentiate diabetic rats according to the level of endogenous keotgenesis. As expected, the severity of diabetes expressed by potentiated endogenous ketogenesis was correlated with a decrease of the rate of oxidative decarboxylation of branched-chain a-ketoacids. It appeared that the decarboxylation decreased up to about 60 per cent in hepatocytes from highly ketotic animals and to about 40 per cent in those from fatty livers (Table 1). Fatty livers were recognized by a pale appearance of the tissue, the presence of numerous fat droplets in hepatocytes and the highest rate of endogenous ketogenesis. Hepatocytes isolated from livers of rats with highly elevated glucose in blood and urine but moderate ketogenesis, close to that of starved animals, had the same rate of flux through oxidative decarboxylation as in hepatocytes from starved rats (not shown). This observation strongly suggests that the increased oxidation of fatty acids accompanying alloxan-induced diabetes could be a direct cause of the impaired decarboxylation of branched-chain a-ketoacids in liver. The antiketogenic effect of insulin administered in uioo to diabetic rats2' and in perfused liver of starved rats" has already been established. The results in Table 2 show that preincubation with insulin of hepatocytes from high-ketotic diabetic rats results in a lowering of endogenous ketogenesis concomitantly with an increase by about 30 per cent of the decarboxylation of a-ketoisocaproate. The effect of insulin was dose-dependent (Figure 1) and was expressed within a short time. Ten-min preincubation of hepatocytes with insulin appeared to be optimal for the maximal effect on the decarboxylation of branched-chain a-ketoacid. The question thus appeared whether the decreased flux through branched-chain a-ketoacid dehydrogenase in ketotic diabetic rats was caused by a lowered level of the active form of the complex. In order to approach this problem both the flux through and the activity of the branched-chain aketoacid dehydrogenase complex were measured in the same cell preparations. As shown in Table 3, the
16
A. STERNlCZUK ETAL.
Table I. Decarboxylation of branched-chain a-ketoacids and endogenous ketogenesis in hepatocytes isolated from normal fed, starved and diabetic rats. ~
Rats
_
_
a-Ketoacid decarboxylation Endogenous ketone a-Ketoisocaproate a-Ketoisovalerate body production (pmol min - g- dry weight)
'
Fed Starved Diabetic: High ketotic Fatty liver
2.50 f 0.45 (7) 3.38 f 0.42 (9)*
3.00 f 0.33 (4) 4.17 & 0.50 (3)*
1.08 f 0.20 (4) 2.08 f 0.44 (4)+
2.05 f 0.73 (6)* 1.25 f 0.33 (6)t
2.33 f 0.72 (4)* 1.58 f 0.33 (4)t
3.75 f 0.35 (4)t 6.5 f 1.2 (4)t
Hepatocytes were incubated for 20 min with 2 mM ~-keto[l-'~C]acid. Conditions for measuring '4C0, are as described in Materials and Methods. Values are the means & S.D. for the number of different cell preparations shown in parentheses. Endogenous ketogenesis is expressed as the sum of acetoacetate and 3-hydroxybutyrate produced by the hepatocytes without added a-ketoacids. The statistical significance with respect to values from fed rats calculated by the Student's !-test was as follows: * P < 0.05; tP < 0.01.
Table 2. Etfect of insulin on the flux through the branchedchain a-ketoacid dehydrogenase and endogenous ketogenesis in hepatocytes isolated from normal starved and high ketotic diabetic rats. Rats
4.0
1
Decarboxylation of Endogenous ketone a-ketoisocaproate body production (pmol min-' g-' dry weight) No insulin With insulin No insulin With insulin
Starved 3.07 f 0.32 3.43 f 0.43 1.87 f 0.32 1.33 & 0.32 Diabetic 2.17 f 0.23 3.07 f 0.42* 4.15 f 0.33 2.32 f 0.38' Hepatocytes were preincubated for 10min without or with insulin 200 mU mi-'. a-Ket~[I-'~C]isocaproatewas added and the incubation continued for 20 min. The rate of ketone body production was estimated in the absence of a-ketoisocaproate. I4CO2 and ketone bodies were determined as described under Materials and Methods. Values are means f SEM for four hepatocyte preparations in each group. Significancefor the effect of insulin was calculated by the Student's r-test for paired data; * P 4 0.01.
total activity of the complex as well as the amount of the active form were the same in hepatocytes from starved and high ketotic diabetic animals. High level of the active form of the dehydrogenase in starvation, as observed in the present study (Table 3), is in agreement with the results of other a ~ t h o r s . In ~ ~hepatocytes -~~ from acutely diabetic rats with fatty livers the total activity of the complex was diminished by about 20 per cent and the percentage of the active form was also lowered. On the other hand, the flux through branched-chain aketoacid dehydrogenase was diminished in diabetes and restored by insulin in the high ketotic group but not in animals with fatty livers.
1.5 P,
0
100
200
Insulin (mu/ml) Figure 1. Effect of insulin concentration on the decarboxylation of a-ketoisocaproate in isolated rat hepatocytes. Hepatocytes were preincubated, as described under Material and Methods, for 1Omin with insulin, followed by addition of aket~[l-'~C]isocaproateto final concentration of 2 mM, and the incubation was continued for 20 min. t--., Hepatocytes from high ketotic diabetic rats; A - - - A, hepatocytes from normal starved rats (shown for comparison). Similar results were obtained in three separate experiments.
17
BRANCHED-CHAIN KETOAClDS IN DIABETES Table 3. Activity of the branched-chain a-ketoacid dehydrogenase complex and the flux through branched-chain a-ketoacid dehydrogenase in hepatocytes isolated from normal starved and diabetic rats. The effect of insulin. Rats
Starved Diabetic: (a) High Ketotic (b) High ketotic, fatty liver
Activity of the branched-chain a-ketoacid dehydrogenase complex (pmol min-' g-' dry weight) Active Total + insulin
Flux through branched-chain a-ketoacid dehydrogenase
bmol min-' g-' dry weight) + insulin
5.23 & 0.39
5.50 & 0.33
5.81 & 0.45
3.23 & 0.18
3.50 & 0.23
4.83 & 0.80
5.04 _t 0.34
5.25 _+ 0.10
2.20 & 0.03
3.12 & 0.13*
3.59; 3.57
4.65; 3.85
4.64, 4.48
1.33; 1.28
1.83; 1.37
Decarboxylation of a-ketoisocaproate was measured as in the legend to Table 2 and the activity of the branched-chain a-ketoacid dehydrogenase complex was determined as described under Materials and Methods in the absence or presence of insulin, 200 mU m1-I. Results are the means & S.D. for four experiments in each group or two experiments with high ketotic animals with fatty liver are presented. The total activity of the dehydrogenase was the same in the presence and absence of insulin. Hepatocytes from diabetic rats were characterized by the following rates of ketogenesis: high ketotic, about 3.5; and high ketotic with fatty liver, above 5.0 pmol acetoacetate plus 3-hydroxybutyrate min- g- dry weight. Statistical significance calculated according to the Student's t-test for paired data for the effect of insulin: * P < 0001; others not significant.
' '
In order to check whether the enzyme activity changes during incubation of hepatocytes, we determined actual and total activities of the complex in freeze-clamped livers. The extracts were prepared as described by Harris et a1.22and the assay for activity was as used for the isolated cells. The activity of the branched-chain a-ketoacid dehydrogenase complex in fatty livers of diabetic rats amounted to 428 5 23 and 580 5 23 mU g-' liver ( k SEM; n = 4) for the active and the total forms, respectively. Thus it appeared that about 73 per cent of the enzyme existed in the active form in liver of severely diabetic rats. In the liver of starved rats the actual and total activities were 560 k 40 and 580 k 30 mU g-' liver, respectively. These results are in good agreement with those shown in Table 3. Hepatocytes from diabetic fatty livers revealed a strongly depressed flux through the dehydrogenase (Tables I and 3). In that case insulin had a variable effect on both the amount of the active form of the dehydrogenase and the flux (Table 3). A poor response to the hormone might be either due to an intrinsic resistance of the tissue or be a result of some impairment of the receptors during preparations. To differentiate between these possibilities, diabetic fatty livers were preperfused with insulin, then hepatocytes were isolated and the rate of oxidative decarboxylation was measured (Table 4). It appeared that the preperfusion with insulin fully reactivated the flux with both substrates up to the
rates observed in hepatocytes from fed rats (see Table 1). The resistance of hepatocytes from fatty liver with respect to insulin should be therefore ascribed to an increased fragility of the receptors in fatty livers during hepatocyte preparation. The inverse correlation between the rate of decarboxylation of branched-chain a-ketoacids and ketogenesis was also observed in hepatocytes from starved rats incubated in the presence of oleate Table 4. Effect of perfusion with insulin on the decarboxylation of a-ketoisocaproate and a-ketoisovalerate in hepatocytes isolated from high ketotic diabetic rats with fatty liver. Substrate
CO, production (pmol min-' g - ' dry weight) No insulin With insulin
a-Ketoisocaproate a-Ketoisovalerate
0.72 & 0.12 0.97 It 0.20
2.72 & 0.17 3.02 _+ 0.35
Livers were perfused in a closed system for 15-20 min with Krebs-Henseleit buffer without or with a single dose of 100 mU insulin ml-'. Subsequently collagenase was added and heptatocytes were isolated by the usual procedure. Hepatocytes were incubated with 2 mM l-['4CJ-labelled branched-chain a-ketoacids for 20 min and I4CO2 was collected by the procedure described under Materials and Methods. Values are means &S.D. for three livers in each group.
18
A. STERNICZUK ETAL.
Table 5. Effect of oleate and insulin on the flux through branched-chain a-ketoacid dehydrogenase, ketone body production and the activity of the branched-chain a-ketoacid dehydrogenase complex in hepatocytes isolated from normal starved rats. ~
Measurement
a-Ketoisocaproate decarboxylation (pmol min-' g - ' dry weight Ketone body production (ymol min-' g-' dry weight) Activity state of the branched-chain a-ketoacid dehydrogenase complex as % of the total
~~
None
0.5 mM Oleate
Additions 0.5 mM Oleate + insulin
3.37 f 0.35
1.83 rf: 0.28*
2.95 & 0.35t
1.31 rf: 0.20*
1.80
2.53 5 0.17
3.38 rf: 0.08$
2.33 5 0.535
3.85 rf: 0.501
3.35 & 0.475
96; 82
96; 80
96; 80
1.0 mM Oleate
80; 64
1.0 mM Oleate
+ insulin
0.12t
86; 69
Hepatocytes isolated from normal rats starved overnight were incubated under standard conditions in the presence of 2 mM aketoisocaproate for 20 min without or with oleate & 100 mU insulin ml-' as indicated. The rate of ketone body production was estimated in the absence of a-ketoisocaproate. The activity of the branched-chain a-ketoacid dehydrogenase complex was determined as described under Materials and Methods. The total activity of this enzyme was the same in the presence and absence of oleate within the experimental error and amounted to 5.7 and 6.3 pmol min-' g - ' dry weight in two experiments. Results are expressed as mean rf: S.D. for six experiments or results of two experiments are shown. Significance was calculated by the Student's t-test for paired data. *f < 0@01 for the decarboxylation in the presence of oleate versus control without oleate; t P < 0.01 for the decarboxylation in the presence of oleate plus insulin versus oleate; $ P i0.01 for ketone body production in the presence of oleate versus control without oleate; § f i0.02 for ketone body production in the presence of oleate plus insulin versus oleate.
(Table 5). Insulin decreased the oleate-induced ketogenesis in parallel to the increase of the flux through branched-chain a-ketoacid dehydrogenase. The level of the active form of the dehydrogenase was not affected by 0.5 mM oleate but decreased by 20-30 per cent by 1 mM oleate, and insulin had a small protective effect, if any. In all experiments presented so far the flux through branched-chain a-ketoacid dehydrogenase was measured at 2mM concentration of the substrates, i.e., well above the K, which is 20-40 PM for the purified Physiological concentration of branched-chain a-ketoacids in the plasma varies from 16 ,uM in fed rats to 30-40 PM in diabetic ratsz6 Therefore, in further experiments the effect of oleate on the flux through the dehydrogenase was examined over a broad range of aketoacid concentration (Figures 2 A and B). Although the K , for the purified enzyme is micromolar, the maximum rates of the decarboxylation in isolated hepatocytes were attained at about 1 mM for both a-ketoacids. It is noteworthy that the inhibitory effect of oleate was substantial only at higher (above 0.5mM) but not at lower aketoacid concentations. The Hanes plots (Figure 2, inserts) reveal an uncompetitive character of the inhibition in which both the apparent K, and V,,, of the flux are decreased, a picture characteristic for complex systems in which the control point of a reaction sequence is downstream from the enzyme
for which the plot is made. Lowering of the amount of the active form of the enzyme by oleate is unlikely since, as shown in Table 5, a large diminution of the flux was accompanied by only a small decrease, if any, of the active form. Moreover, the amount of the active form was always in excess compared to the flux (Table 3). One of the possibilities is the inhibition of branched-chain a-ketoacid dehydrogenase by NADH (resulting from fatty acid oxidation), as was established for isolated hepatocytes2' and for the purified e n ~ y m e . ~ . ' ~ The effect of insulin on the decarboxylation rate as a function of a-ketoisocaproate concentration in hepatocytes from high ketotic rats is presented in Figure 2C. As shown in previous experiments (Tables 2, 3 and 4), insulin significantly enhanced the rate of decarboxylation at 2 mM substrate concentration but its stimulatory effect decreased at lower concentrations. At 0.1 mM a-ketoisocaproate there was practically no effect of insulin. DISCUSSION The present investigation deals with the relation between the activity state of the dehydrogenase complex and the actual flux through the dehydrogenase in hepatocytes isolated from starved and diabetic rats. As has been reported p r e v i ~ u s l y 'and ~ in the present paper, endogenous ketogenesis is inversely
19
BRANCHED-CHAIN KETOACIDS I N DIABETES
B
A
'I
'r
A
C 1
1
2
2
Branched-chain
a-ketoacid
concentration
(mM)
Figure 2. EKect of oleate and insulin on the oxidation of branched-chain a-ketoacids as a function of substrate concentration. A and B, hepatocytes isolated from starved rats oxidizing a-ketoisocaproate (A) and a-ketoisovalerate (B) in the absence ( 0 )or presence ( 0 )of 1 mM oleate. The kinetic parameters calculated from the Hanes plots (inserts) are: A, apparent K, 044 and 0.14 mM, V , , , 4.4 and 2.3 lcmol min-l g - ' dry weight in the absence and presence of oleate, respectively; B, apparent K, 0.33 and 0.07 mM, V,,, 6.0 and 3.5 pmol min-' g-' dry weight in the absence and presence of oleate, respectively. C, Hepatocytes from high ketotic diabetic rats oxidizing a-ketoisocdproate without (W) and with (0) insulin, 200 mU mi-'.
correlated with the rate of branched-chain a-ketoacid oxidation in hepatocytes from diabetic rats. Also experiments with hepatocytes from non-diabetic starved rats (reference 13 and this paper) confirm that the flux through branched-chain aketoacid dehydrogenase is diminished by concomitant oxidation of added fatty acids. The present results (Tables 3 and 5 ) show that the effect of fatty acids is not due to the interconversion (phosphorylation) of the dehydrogenase complex but is, most likely, exerted by a direct inhibition of the dehydrogenase by NADH and acyl-CoA esters and, possibly, of further steps of the degradation pathway of branched-chain a - k e t ~ a c i d s . ' ~This . ~ ' conclusion is supported by the present observation that the amount of the active form and the total activity of the dehydrogenase complex were only slightly, if at all, diminished in livers and hepatocytes from diabetic rats (Table 3) and also in hepatocytes from starved rats in the presence of 1 mM oleate (Table 5). The fact that the dehydrogenase complex was fully active in diabetic liver was also observed by A Gillim et aL6 but not by Patston et decrease by 2 mbi oleate of the flux through and the amount of the active form of the dehydrogenase in hepatocytes isolated from fed rats were described by Harris et a1.' ~
1
.
~
3
"
It has to be noted that fatty acids exert a dual effect on branched-chain a-ketoacid oxidation: (i) end-product inhibition, (ii) activation of the dehydrogenase complex due to the potent inhibitory effect of fatty acid oxidation products, mainly acetoacetyl-CoA, on branched-chain a-ketoacid dehydrogenase kinase.2s The latter effect is prevailing in tissues where the percentage of the active form of the dehydrogenase complex is low, e.g., in heart and skeletal muscle (for further literature see reference 28). On the other hand, in liver of fed, starved and diabetic rats, where the dehydrogenase is practically fully active, fatty acid oxidation results in a decrease of branched-chain a-ketoacid decarboxylation due to inhibition by NADH and/or acylCoA. The activatory effect of insulin on oxidative decarboxylation of branched-chain a-ketoacids in hepatocytes was correlated with the inhibition by insulin of ketogenesis derived from fatty acid oxidation. This short-term effect of insulin was therefore exerted predominantly on the flux rather than on the actual activity of the branched-chain a-ketoacid dehydrogenase complex since the latter was already practically fully active. This is in contrast to adipose tissue where insulin increases the activity of branched-chain dehydrogenase complex by stimu-
20
A. STERNICZUK E T A L .
lating its dephosphorylation.2' Similarly, insulin ACKNOWLEDGEMENTS strongly activates pyruvate dehydrogenase in hepatocytes from starved rats by promoting the inter- We wish to thank Barbara Burcan and Maria conversion of this enzyme complex to its Buszkowska for technical assistance. This work dephosphorylated form.30 Insulin was found to was supported by the Polish Academy of Sciences overcome the inhibitory effect of ketone bodies on under grant No. CPBP 04.01. the oxidation of a-ketoisovalerate and a-ketoisocaproate in perfused hindquarters of diabetic rats,31 but it has not been established whether this was due REFERENCES to a change of the activity state of the dehydrogen1. Shinnick, F. L. and Harper, A. E. (1976). Branched-chain ase complex. amino acid oxidation by isolated rat tissue preparations. Biochim. Biophys. Acta, 437,477-486. The collcentration of insulin which produced a 2. Harris, R. A., Paxton, R., Powell, S. M., Goodwin, G. W., significant effect on the Rux through branchedKuntz, M. J. and Han, A. C. (1986). Regulation of chain a-ketoacid dehydrogenase in our experiments branched-chain a-ketoacid dehydrogenase complex by cowas 10 times higher than that used in experiments valent modification. In: Advances in Enzyme Regulation, with hepatocytes from healthy fed animals.32 CulVol. 25, (Weber, G., ed,) Pergamon Press: Oxford, pp. 219-237. tured hepatocytes exhibited even much higher senR., Kuntz, M. J. and Harris, R. A. (1986). Phossitivity towards insulin and g l ~ c a g o n . ~ ~3. . ~Paxton, ~ phorylation sites and inactivation of branched-chain aHowever, in our perfusion experiments (Table 4) ketoacid dehydrogenase isolated from rat heart, bovine the insulin concentration was comparable to that kidney, and rabbit liver, kidney, heart, and skeletal muscle. Arch. Biochem. Biophys., 244,187-201. used by Menahan and Wie1and2l who observed a 4. Parker, P. J. and Randle, P. J. (1978). Branched-chain 2suppressing effect of this hormone on glucagon0x0-acid dehydrogenase complex of rat liver. FEBS Leti., stimulated ketogenesis in perfused liver of starved 90, 183-186. rat. The apparent resistance of diabetic hepatocytes 5. Parker, P. J. and Randle, P. J. (1978). Partial purification towards insulin may be partially artifactual since, and properties of branched-chain 2-0x0 acid dehydrogenase of ox liver. Biochem. J., 171, 751 -757. as we have shown, hepatocytes isolated from fatty 6. Gillim, S. E., Paxton, R., Cook, G. A. and Harris, R. A. liver were refractory towards the hormone (Table (1983). Activity state of the branched chain a-ketoacid 3) in contrast to the perfused liver (Table 4). The dehydrogenase complex in heart, liver, and kidney of poor response may therefore be due to the fragility normal, fasted, diabetic, and protein-starved rats. Biochem. Biophys. Res. Commun., 111, 74-81. of the receptors in diabetic hepatocytes as well as to 7. Patston, P. A., Espinal, J. and Randle, P. J. (1984). Effects the resistance of the diabetic liver, as has already of diet and of alloxan-diabetes on the activity of branchedbeen described.3 5 * chain 2-0x0 acid dehydrogenase complex and of activator The present study shows that the oxidation of protein in rat tissues. Biochem. J., 222,711-719. branched-chain a-ketoacids is strongly depressed 8. Harris, R. A., Paxton, R.,Goodwin, G. W. and Powell, S . M. (1986). Regulation of the branched-chain 2-0x0 acid by fatty acid oxidation at the maximal flux through dehydrogenase complex in hepatocytes isolated from rats the dehydrogenase but only slightly diminished at on a low-protein diet. Biochem. J., 234, 285-294. lower concentrations of these substrates. This is in 9. Block, K. P., Heywood, B. W., Buse, M. G. and Harper, agreement with previous observations of Corkey et A. E. (1985). Activation of rat liver branched-chain 2-0x0 acid dehydrogenase in uiuo by glucagon and adrenaline. aL2' and Harris et a1.' The activatory effect of Biochem. J., 232, 593-597. insulin on decarboxylation of branched-chain a10. Reed, L. J. (1981). Regulation of mammalian pyruvate ketoacids is much higher at the maximal flux dehydrogenase' complex by a phosphorylation-dephosthrough the enzyme than at lower concentrations phorylation cycle. Curr. Top. Cell. Regul., 18, 95-106. of the substrate (Figure 2C). Therefore, it can be 11. Patston, P. A., Espinal, J., Shaw, J. M. and Randle, P. J. (1986). Rat tissue concentrations of branched-chain 2-0x0 concluded that in diabetic liver the oxidation of acid dehydrogenase complex. Re-evaluation by immunoasboth fatty acids and branched-chain a-ketoacids is say and bioassy. Biochem. J., 235, 429--434. maintained at a high rate. The contribution of these 12. Paul, H. S. and Adibi, S. A. (1982). Role of ATP in the substrates to the energy demand depends on their regulation of branched-chain n-keto acid dehydrogenase activity in liver and muscle of fed, fasted, and diabetic rats. supply from peripheral tissues. Insulin administraJ. Biol. Chem., 257,4875-4881. tion in diabetes results in a decrease of fatty acid 13. Williamson, J. R., Watajtys-Rode, E. 1. and Coll, K. E. oxidation in favour of the utilization of branched(1979). Effects of branched chain a-ketoacids on the metachain a-ketoacids, as shown in this study, and of bolism of isolated rat liver. 1. Regulation of branched chain a-ketoacid metabolism. J. Bid. Chem., 254, 11511-1 1520. carbohydrates, as is well-established.
21
BRANCHED-CHAIN KETOACIDS IN DIABETES 14. Watajtys-Rode, E. I., Kietducka, A. and Lenartowicz, E. (1981). Oxidation of branched chain a-ketoacids in hepatocytes from alloxan-diabetic rats. Inhibition by long chain fatty acids. In: Metabolism and Clinical Implications of Branched-Chain Amino and Ketoacids, (Walser, M. and Williamson, J. R., eds.) Elsevier: New York, pp. 129-134. 15. Berry, M. N. and Friend, D. S. (1969). High-yield preparation of isolated rat liver parenchymal cells. A biochemical and fine structural study. J. Cell Biol., 43, 506-520. 16. Krebs, H. A. and Henseleit, K. (1932). Untersuchungen uber die Harnstoflbildung im Tierkorper. Hoppe-Seylers Z. Physiol. Chem., 210, 33-66. 17. Williamson, D. H. and Mellanby, J. (1974). ~-(->3-Hydroxybutyrate. In: Methods of Enzymatic Analysis, Vol. 4, 2nd English edn., (Bergmeyer, H. U., ed.) Academic Press: New York, London, pp. 1836-1839. 18. Mellanby, J. and Williamson, D. H. (1974). Acetoacetate. In: Merhoa3 of Enzymatic Analysis, Vol. 4, 2nd English edn., (Bergmeyer, H. U., ed.) Academic Press: New York, London, pp. 1840- 1843. 19. Rudiger, H. W., Langenbeck, U. andGoedde, H. W. (1972). A simplified method for the preparation of "C-labeled branched chain a-oxoacids. Biochem. J., 126,445-446. 20. Kaloyianni, M. and Freedland, R. A. (1990). Effect of diabetes and time after in uiuo insulin administration on ketogenesis and gluconeogenesis in isolated rat hepatocytes. Int. J. Biochem., 22, 59-164. 21. Menahan, L. A. and Wieland, 0. (1969). Interactions of glucagon and insulin on the metabolism of perfused livers from fasted rats. Eur. J. Biochem., 9, 55-62. 22. Harris, R. A., Powell, S. M., Paxton, R., Gillim, S. E. and Nagae, H. (1985). Physiological covalent regulation of rat liver branched-chain a-ketoacid dehydrogenase. Arch. Biochem. Biophys., 243, 542-555. 23. Solomon, M., Cook, K. G. and Yeaman, S. J. (1987). Effect of diet and starvation on the activity state of branchedchain 2-0x0-acid dehydrogenase complex in rat liver and heart. Biochim. Biophys. Acta, 931, 335-338. 24. Wagenmakers, A. J. M., Schepens, J. T. G. and Veerkamp, J. H. (1984). Erect of starvation and exercise on actual and total activity of the branched-chain 2-0x0 acid dehydrogenase complex in rat tissues. Biochem. J., 223,815-821. 25. Danner, D. J., Lemmon, S. K. and Elsas, L. J. (1978). Substrate specificity and stabilization by thiamine pyrophosphate of rat liver branched-chain a-keto acid dehydrogenase. Biochem. Med., 19,27-38.
26. Hutson, S. M. and Harper, A. E. (1981). Blood and tissue branched-chain amino and a-ketoacid concentrations: Effect of diet, starvation, and disease. Amer. J. Clin. Nutr., 34, 173-1 83. 27. Corkey, B. E., Martin-Requero, A., Walajtys-Rode, E., Williams, R. J. and Williamson, J. R. (1982). Regulation of the branched chain u-ketoacid pathway in liver. J. Biol. Chem., 257,9668-9676. 28. Paxton, R. and Harris, R. A. (1984). Regualtion of branched-chain a-ketoacid dehydrogenase kinase. Arch. Biochem. Biophys., 231, 48-57. 29. Frick, G. P. and Goodman, H. M. (1989). Insulin regulation of the activity and phosphorylation of branched-chain 2-0x0 acid dehydrogenase in adipose tissue. Biochem. J., 258,229-235. 30. Marchington, D. R., Kerbey, A. L., Jones, A. E. and Randle, P. J. (1987). Insulin reverses effects of starvation on the activity of pyruvate dehydrogenase kinase in cultured hepatocytes. Biochem. J., 246,233-236. 31. Zapalowski, C., Hutson, S. M. and Harper, A. E. (1981). Effects of starvation and diabetes on leucine and valine metabolism in the perfused rat hindquarter. In:Metabolism and Clinical Implications of Branched-Chain Amino and Ketoacids, (Walser, M. and Williamson, J. R., eds.) Elsevier: New York, pp. 239-244. 32. Vaartjes, W. J., de Haas, C. G. M. and van den Bergh, S. G. (1985). Differential short-term effects of growth factors on fatty acid synthesis in isolated rat-liver cells. Biochem. Biophys. Res. Commun., 131, 449-455. 33. Geelen, M. J. H. and Gibson, D. M. (1975). Lipgenesis in maintenance cultures of rat hepatocytes. .FEBS Lett., 58, 334-339. 34. Harano, Y., Kosugi, K., Kashiwagi, A., Nakano, T.. Hidaka, H, and Shigeta, Y. (1982). Regulatory mechanism of ketogenesis by glucagon and insulin in isolated cultured hepatocytes. J. Biochem., 91, 1739-1748. 35. Reaven, G. M. and Olefsky, J. M. (1978). Role of insulin resistance in the patogenesis of diabetes mellitus. Ado. Metab. Res., 9, 313-325. 36. Hussin, A. H. and Skett, P. (1988). Lack of effect of insulin in hepatocytes isolated from streptozotocin-diabetic male rats. Biochem. Pharmacol., 37, 1683- 1686. Receioed in reuisedforrn 9 July 1990 Accepted 28 August 1990
VOL.
9: 13-21 (1991)
Decarboxylation of Branched-Chain a-Ketoacids in Hepatocytes from Alloxan-Diabetic Rats. The Effect of Insulin ANNA STERNICZUK, ELZBIETA I. WALAJTYS-RODE? AND ANNA B. WOJTCZAKS Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, Pasteura 3, 02-093 Warsaw, Poland
The flux through branched-chain a-ketoacid dehydrogenase and the activity of the branched-chain a-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain a-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain a-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain a-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminshed flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was parallelled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain a-ketoacid oxidation in liver of diabetic rats. K E Y WORDS- Branched-chain ketoacids; diabetes; insulin; hepatocytes.
INTRODUCTION Oxidative decarboxylation of branched-chain a- diminished the amounts of the active and the total ketoacids is catalysed by the mitochondria1 dehydrogenase complex in liver6v7 concomitantly branched-chain a-ketoacid dehydrogenase com- with a decrease of the rate of oxidative plex. In this nonreversible reaction a single complex decarboxylation.' Treatment of rats fed on a lowoxidizes a-keto-derivatives of leucine, valine and protein diet with adrenalin, glucagon or insulin isoleucine. The activity of the complex is rate- increases the activity of the dehydrogenase complex limiting for the metabolism of branched-chain in liver, but no such effect was observed in animals aminoacids, except in liver where transamination is kept on a standard diet.g It is well established that in liver of starved and rate limiting.' Similarly to the pyruvate dehydrogenase complex, the branched-chain a-ketoacid de- diabetic rats the oxidation of pyruvate and the hydrogenase complex is inactivated by activity of the pyruvate dehydrogenase complex are phosphorylation and reactivated by dephosphory- suppressed (reviewed in reference 10). In contrast, lation (for review see reference 2). This regulation there have been only fragmentary and contradichas been shown to occur in enzyme complexes tory observations concerning the activity state of derived from various tissues and animal species.j branched-chain a-ketoacid dehydrogenase in diaMoreover, oxidative decarboxylation of branched- betes. Patston et ~ l . ~ l. 'found a drastic decrease in chain a-ketoacids is regulated by inhibition by the the activity of the complex, whereas Gillim et aL6 end-products, namely NADH and the respective and Paul and Adibi12 observed almost no change acyl-CoA e ~ t e r . It ~ *has ~ been found that alter- under the same conditions. So far, there have been ations of the dietary or hormonal balance induce no systematic studies on the flux through changes in both the rate of the decarboxylation and branched-chain a-ketoacid dehydrogenase and the the activity of the branched-chain ol-ketoacid de- activity of the complex measured in parallel in hydrogenase complex. Low-protein diet strongly livers of diabetic animals. The inhibitory effect of fatty acid oxidation on t Present address: Institute of internal Medicine, Warsaw Medi- the uptake and oxidation of branched-chain aketoacids in hepatocytes isolated from starved rats cal School, 02-097 Warsaw, Poland $Addressee for correspondence. was first described by Williamson et In our 0243 -6484/91/010013-09 $05.00 1991 by John Wiley & Sons, Ltd.
f4 previous rep01-t'~it was shown that the increased oxidation of fatty acids in diabetes could be responsible for diminished rate of oxidation of branchedchain a-ketoacids in liver. The present study extends our investigation over parallel measurements of the rate of decarboxylation of branched-chain a-ketoacids and the activity state of the complex in hepatocytes isolated from rats with alloxan diabetes. MATERIALS AND METHODS Treatment of Animals
Male Wistar rats (200-25Og) were fed on a standard laboratory diet. These animals are further designated as normal fed rats. Starved rats were deprived of food for 18-24 h and had free access to water. Diabetes was induced in normal fed rats by intravenous injection of alloxan, 60 mg kg-' body weight, into the tail vein and the animals were kept for 3-4 days on normal diet and free access to water. Animals with 25-35 mM glucose in blood and ketones in urine of + + to + + + according to Bili-Labstix (Ames Laboratories, Elkhart, IN, USA.) scale were used for experiments. Isolation and Incubation of Hepatocytes
Liver cells were prepared according to the method of Berry and Friend." Cells (5-7 mg dry weight ml- ') were incubated in Krebs-HenseleitI6 bicarbonate medium containing 1 mM CaC1, and 1 per cent defatted and dialysed bovine serum albumin. The concentration of branched-chain a-ketoacids was 2 mM unless indicated otherwise. Incubations were performed in 25 ml Erlenmeyer flasks at 37°C with shaking under an atmosphere of 95 percent 0, and 5 per cent CO,. Viability of the cells was tested with Trypan blue. Preparations containing more than 90 per cent viable cells were used for experiments. 3-Hydroxybutyrate' ' and acetoacetate" were determined by enzymatic methods in neutralized perchloric acid extracts of the whole cell suspension. The rate of ketogenesis was calculated from 20-min incubations after subtracting values for zero-time controls. Determination of the Flux Through BranchedChain a-Ketoacid Dehydrogenase
Hepatocytes (1-2 ml) were preincubated for 10 min without or with insulin (100-200 mU
A. STERNICZUK E T A L
ml-' = 0.65-1.3 PM) under conditions described above; then l-['4C]-labelled a-ketoacid (100150 cpm nmol- ') was added to a final concentration of 2 mM or as indicated in the legends. Flasks were sealed with rubber serum caps fitted with handing centre wells containing filter paper, and the incubation was continued for 20min. The reaction was stopped by injection of 0.5 ml of 2 M H 2 S 0 4to the reaction medium and of 0.2 ml of phenethylamine to the centre well. The flasks were then shaken for 1 h at room temperature. The wells were removed and counted for radioactivity in a liquid scintillation spectrometer. The total radioactivity was determined by counting the amount of the added labelled substrate in 10 ml of the scintillation mixture plus 0.2 ml of phenethylamine.
Assay of the Branched-Chain a-Keioacid Dehydrogenase Complex Activity in Extracts of Hepatocytes
The assay was performed essentially as described by Harris et aL8 with some modifications. Hepatocytes were incubated for 20 min with or without insulin or other additions as indicated in the legends but without a-ketoacids. The volume was 4.0ml. Other conditions were the same as for measurements of the flux. Aliquots of 1-0ml were withdrawn in duplicate and hepatocytes were separated from the medium by a rapid centrifugation for 30 s at 8000 g using a precooled microcentrifuge (Type 320, Unipan, Warsaw, Poland). Pellets were immediately frozen in liquid nitrogen. Cell extracts were prepared by vigorous mixing of the pellet in 0.5 ml of ice-cold solution containing 50 mM Hepes, pH 7.5, 5 mM dithiothreitol, 2 mM EDTA, 2 per cent (v/v) Triton X-100, 0.5 mM N-u-tosyl-L-lysine chlormethylketone and 50 mM NaF for the measurements of the active form of the complex. For determination of the toaI activity of the dehydrogenase the cells were extracted with the same medium, except that NaF was omitted. The activity of the complex was assayed immediately after solubilization of cells. For the determination of the total activity the assay medium contained 30 mM phosphate (pH 7.4), 2 mM MgCl,, 0.4 mM thiamine pyrophosphate, 0.4 mM CoA, 1 mM dithiothreitol, 3 mM NAD, 0.1 per cent Triton X-100, and 2 U lipoamide dehydrogenase in a total volume of 1.0ml. For determination of the active form the medium also contained 50mM NaF. Aliquots of 0.1 ml of the extracts were added in triplicate and
BRANCHED-CHAIN KETOACIDS IN DIABETES
the samples were preincubated for 5 min at 30°C. The reaction was started by the addition of aketo[ l-'4C]isocaproate (200-300 cpm nmol- ') to final concentration of 0.5 mM. For the determination of total activity NaF was added together with the labelled substrate to a final concentration of 50mM in order to keep the same components at identical concentrations. The blanks were run in parallel with the complete system except the cell extract. The test tubes were sealed with caps and further incubated for Smin, then stopped with H 2 S 0 4 , and 14C02was measured as described for the determination of the flux. In accordance with the observation of Harris et a/.' we found that the addition of exogenous phosphoprotein phosphatase did not increase the total activity of the complex in livers of fed and starved rats and therefore phosphatase has not been added in the routine assay procedure. Calculation of the Results
The rates of enzymatic activity were expressed in pmol min-' g-' dry weight of cells. Linearity of ketone body production, and of the flux through and the activity of branched-chain ketoacid dehydrogenase with respect to time and tissue content was established. The average wet to dry weight ratios of 4.5 (n = 15) for cells from fed and alloxandiabetic rats and of 5.0 ( n = 10) for cells from starved animals were estimated. Chemicals
Collagenase was from Boehringer (Mannheim, F.R.G.) or Worthington (Freehold, NJ, U.S.A.); alloxan, bovine serum albumin, L-aminoacid oxidase from Crotalus adamanteus venom and lipoamide dehydrogenase from porcine heart were obtained from Sigma (St. Louis, MO, U.S.A.); enzymes for metabolite determinations were obtained from Boehringer; porcine insulin (40 U ml-l) was from Polfa (Tarchomin, Poland); and inorganic salts from Merck (Darmstadt, F.R.G.). ['4C]-Labelled a-ketoisocaproate and a-ketoisovalerate were prepared from [1-14C]leucine and [1-14C]valine (Amersham International, Amersham, U.K.), respectively, according to Riidiger el a1.19 RESULTS The rates of oxidative decarboxylation of a-ketoisocaproate and a-ketoisolvalerate in hepatocytes
15 isolated from fed, starved and diabetic rats were measured. Higher rates of the decarboxylation of aketoisolvalerate than of a-ketoisocaproate are in agreement with a higher V,,, estimated for a-ketoisolvalerate.8 In accordance with previous observation^,'^ short starvation increased the rate of decarboxylation of branched-chain a-ketoacids. As it is known from previous studies that fatty acid oxidation has a strong inhibitory effect on the metabolism of branched-chain a-ketoacids,' )*14 we tried to differentiate diabetic rats according to the level of endogenous keotgenesis. As expected, the severity of diabetes expressed by potentiated endogenous ketogenesis was correlated with a decrease of the rate of oxidative decarboxylation of branched-chain a-ketoacids. It appeared that the decarboxylation decreased up to about 60 per cent in hepatocytes from highly ketotic animals and to about 40 per cent in those from fatty livers (Table 1). Fatty livers were recognized by a pale appearance of the tissue, the presence of numerous fat droplets in hepatocytes and the highest rate of endogenous ketogenesis. Hepatocytes isolated from livers of rats with highly elevated glucose in blood and urine but moderate ketogenesis, close to that of starved animals, had the same rate of flux through oxidative decarboxylation as in hepatocytes from starved rats (not shown). This observation strongly suggests that the increased oxidation of fatty acids accompanying alloxan-induced diabetes could be a direct cause of the impaired decarboxylation of branched-chain a-ketoacids in liver. The antiketogenic effect of insulin administered in uioo to diabetic rats2' and in perfused liver of starved rats" has already been established. The results in Table 2 show that preincubation with insulin of hepatocytes from high-ketotic diabetic rats results in a lowering of endogenous ketogenesis concomitantly with an increase by about 30 per cent of the decarboxylation of a-ketoisocaproate. The effect of insulin was dose-dependent (Figure 1) and was expressed within a short time. Ten-min preincubation of hepatocytes with insulin appeared to be optimal for the maximal effect on the decarboxylation of branched-chain a-ketoacid. The question thus appeared whether the decreased flux through branched-chain a-ketoacid dehydrogenase in ketotic diabetic rats was caused by a lowered level of the active form of the complex. In order to approach this problem both the flux through and the activity of the branched-chain aketoacid dehydrogenase complex were measured in the same cell preparations. As shown in Table 3, the
16
A. STERNlCZUK ETAL.
Table I. Decarboxylation of branched-chain a-ketoacids and endogenous ketogenesis in hepatocytes isolated from normal fed, starved and diabetic rats. ~
Rats
_
_
a-Ketoacid decarboxylation Endogenous ketone a-Ketoisocaproate a-Ketoisovalerate body production (pmol min - g- dry weight)
'
Fed Starved Diabetic: High ketotic Fatty liver
2.50 f 0.45 (7) 3.38 f 0.42 (9)*
3.00 f 0.33 (4) 4.17 & 0.50 (3)*
1.08 f 0.20 (4) 2.08 f 0.44 (4)+
2.05 f 0.73 (6)* 1.25 f 0.33 (6)t
2.33 f 0.72 (4)* 1.58 f 0.33 (4)t
3.75 f 0.35 (4)t 6.5 f 1.2 (4)t
Hepatocytes were incubated for 20 min with 2 mM ~-keto[l-'~C]acid. Conditions for measuring '4C0, are as described in Materials and Methods. Values are the means & S.D. for the number of different cell preparations shown in parentheses. Endogenous ketogenesis is expressed as the sum of acetoacetate and 3-hydroxybutyrate produced by the hepatocytes without added a-ketoacids. The statistical significance with respect to values from fed rats calculated by the Student's !-test was as follows: * P < 0.05; tP < 0.01.
Table 2. Etfect of insulin on the flux through the branchedchain a-ketoacid dehydrogenase and endogenous ketogenesis in hepatocytes isolated from normal starved and high ketotic diabetic rats. Rats
4.0
1
Decarboxylation of Endogenous ketone a-ketoisocaproate body production (pmol min-' g-' dry weight) No insulin With insulin No insulin With insulin
Starved 3.07 f 0.32 3.43 f 0.43 1.87 f 0.32 1.33 & 0.32 Diabetic 2.17 f 0.23 3.07 f 0.42* 4.15 f 0.33 2.32 f 0.38' Hepatocytes were preincubated for 10min without or with insulin 200 mU mi-'. a-Ket~[I-'~C]isocaproatewas added and the incubation continued for 20 min. The rate of ketone body production was estimated in the absence of a-ketoisocaproate. I4CO2 and ketone bodies were determined as described under Materials and Methods. Values are means f SEM for four hepatocyte preparations in each group. Significancefor the effect of insulin was calculated by the Student's r-test for paired data; * P 4 0.01.
total activity of the complex as well as the amount of the active form were the same in hepatocytes from starved and high ketotic diabetic animals. High level of the active form of the dehydrogenase in starvation, as observed in the present study (Table 3), is in agreement with the results of other a ~ t h o r s . In ~ ~hepatocytes -~~ from acutely diabetic rats with fatty livers the total activity of the complex was diminished by about 20 per cent and the percentage of the active form was also lowered. On the other hand, the flux through branched-chain aketoacid dehydrogenase was diminished in diabetes and restored by insulin in the high ketotic group but not in animals with fatty livers.
1.5 P,
0
100
200
Insulin (mu/ml) Figure 1. Effect of insulin concentration on the decarboxylation of a-ketoisocaproate in isolated rat hepatocytes. Hepatocytes were preincubated, as described under Material and Methods, for 1Omin with insulin, followed by addition of aket~[l-'~C]isocaproateto final concentration of 2 mM, and the incubation was continued for 20 min. t--., Hepatocytes from high ketotic diabetic rats; A - - - A, hepatocytes from normal starved rats (shown for comparison). Similar results were obtained in three separate experiments.
17
BRANCHED-CHAIN KETOAClDS IN DIABETES Table 3. Activity of the branched-chain a-ketoacid dehydrogenase complex and the flux through branched-chain a-ketoacid dehydrogenase in hepatocytes isolated from normal starved and diabetic rats. The effect of insulin. Rats
Starved Diabetic: (a) High Ketotic (b) High ketotic, fatty liver
Activity of the branched-chain a-ketoacid dehydrogenase complex (pmol min-' g-' dry weight) Active Total + insulin
Flux through branched-chain a-ketoacid dehydrogenase
bmol min-' g-' dry weight) + insulin
5.23 & 0.39
5.50 & 0.33
5.81 & 0.45
3.23 & 0.18
3.50 & 0.23
4.83 & 0.80
5.04 _t 0.34
5.25 _+ 0.10
2.20 & 0.03
3.12 & 0.13*
3.59; 3.57
4.65; 3.85
4.64, 4.48
1.33; 1.28
1.83; 1.37
Decarboxylation of a-ketoisocaproate was measured as in the legend to Table 2 and the activity of the branched-chain a-ketoacid dehydrogenase complex was determined as described under Materials and Methods in the absence or presence of insulin, 200 mU m1-I. Results are the means & S.D. for four experiments in each group or two experiments with high ketotic animals with fatty liver are presented. The total activity of the dehydrogenase was the same in the presence and absence of insulin. Hepatocytes from diabetic rats were characterized by the following rates of ketogenesis: high ketotic, about 3.5; and high ketotic with fatty liver, above 5.0 pmol acetoacetate plus 3-hydroxybutyrate min- g- dry weight. Statistical significance calculated according to the Student's t-test for paired data for the effect of insulin: * P < 0001; others not significant.
' '
In order to check whether the enzyme activity changes during incubation of hepatocytes, we determined actual and total activities of the complex in freeze-clamped livers. The extracts were prepared as described by Harris et a1.22and the assay for activity was as used for the isolated cells. The activity of the branched-chain a-ketoacid dehydrogenase complex in fatty livers of diabetic rats amounted to 428 5 23 and 580 5 23 mU g-' liver ( k SEM; n = 4) for the active and the total forms, respectively. Thus it appeared that about 73 per cent of the enzyme existed in the active form in liver of severely diabetic rats. In the liver of starved rats the actual and total activities were 560 k 40 and 580 k 30 mU g-' liver, respectively. These results are in good agreement with those shown in Table 3. Hepatocytes from diabetic fatty livers revealed a strongly depressed flux through the dehydrogenase (Tables I and 3). In that case insulin had a variable effect on both the amount of the active form of the dehydrogenase and the flux (Table 3). A poor response to the hormone might be either due to an intrinsic resistance of the tissue or be a result of some impairment of the receptors during preparations. To differentiate between these possibilities, diabetic fatty livers were preperfused with insulin, then hepatocytes were isolated and the rate of oxidative decarboxylation was measured (Table 4). It appeared that the preperfusion with insulin fully reactivated the flux with both substrates up to the
rates observed in hepatocytes from fed rats (see Table 1). The resistance of hepatocytes from fatty liver with respect to insulin should be therefore ascribed to an increased fragility of the receptors in fatty livers during hepatocyte preparation. The inverse correlation between the rate of decarboxylation of branched-chain a-ketoacids and ketogenesis was also observed in hepatocytes from starved rats incubated in the presence of oleate Table 4. Effect of perfusion with insulin on the decarboxylation of a-ketoisocaproate and a-ketoisovalerate in hepatocytes isolated from high ketotic diabetic rats with fatty liver. Substrate
CO, production (pmol min-' g - ' dry weight) No insulin With insulin
a-Ketoisocaproate a-Ketoisovalerate
0.72 & 0.12 0.97 It 0.20
2.72 & 0.17 3.02 _+ 0.35
Livers were perfused in a closed system for 15-20 min with Krebs-Henseleit buffer without or with a single dose of 100 mU insulin ml-'. Subsequently collagenase was added and heptatocytes were isolated by the usual procedure. Hepatocytes were incubated with 2 mM l-['4CJ-labelled branched-chain a-ketoacids for 20 min and I4CO2 was collected by the procedure described under Materials and Methods. Values are means &S.D. for three livers in each group.
18
A. STERNICZUK ETAL.
Table 5. Effect of oleate and insulin on the flux through branched-chain a-ketoacid dehydrogenase, ketone body production and the activity of the branched-chain a-ketoacid dehydrogenase complex in hepatocytes isolated from normal starved rats. ~
Measurement
a-Ketoisocaproate decarboxylation (pmol min-' g - ' dry weight Ketone body production (ymol min-' g-' dry weight) Activity state of the branched-chain a-ketoacid dehydrogenase complex as % of the total
~~
None
0.5 mM Oleate
Additions 0.5 mM Oleate + insulin
3.37 f 0.35
1.83 rf: 0.28*
2.95 & 0.35t
1.31 rf: 0.20*
1.80
2.53 5 0.17
3.38 rf: 0.08$
2.33 5 0.535
3.85 rf: 0.501
3.35 & 0.475
96; 82
96; 80
96; 80
1.0 mM Oleate
80; 64
1.0 mM Oleate
+ insulin
0.12t
86; 69
Hepatocytes isolated from normal rats starved overnight were incubated under standard conditions in the presence of 2 mM aketoisocaproate for 20 min without or with oleate & 100 mU insulin ml-' as indicated. The rate of ketone body production was estimated in the absence of a-ketoisocaproate. The activity of the branched-chain a-ketoacid dehydrogenase complex was determined as described under Materials and Methods. The total activity of this enzyme was the same in the presence and absence of oleate within the experimental error and amounted to 5.7 and 6.3 pmol min-' g - ' dry weight in two experiments. Results are expressed as mean rf: S.D. for six experiments or results of two experiments are shown. Significance was calculated by the Student's t-test for paired data. *f < 0@01 for the decarboxylation in the presence of oleate versus control without oleate; t P < 0.01 for the decarboxylation in the presence of oleate plus insulin versus oleate; $ P i0.01 for ketone body production in the presence of oleate versus control without oleate; § f i0.02 for ketone body production in the presence of oleate plus insulin versus oleate.
(Table 5). Insulin decreased the oleate-induced ketogenesis in parallel to the increase of the flux through branched-chain a-ketoacid dehydrogenase. The level of the active form of the dehydrogenase was not affected by 0.5 mM oleate but decreased by 20-30 per cent by 1 mM oleate, and insulin had a small protective effect, if any. In all experiments presented so far the flux through branched-chain a-ketoacid dehydrogenase was measured at 2mM concentration of the substrates, i.e., well above the K, which is 20-40 PM for the purified Physiological concentration of branched-chain a-ketoacids in the plasma varies from 16 ,uM in fed rats to 30-40 PM in diabetic ratsz6 Therefore, in further experiments the effect of oleate on the flux through the dehydrogenase was examined over a broad range of aketoacid concentration (Figures 2 A and B). Although the K , for the purified enzyme is micromolar, the maximum rates of the decarboxylation in isolated hepatocytes were attained at about 1 mM for both a-ketoacids. It is noteworthy that the inhibitory effect of oleate was substantial only at higher (above 0.5mM) but not at lower aketoacid concentations. The Hanes plots (Figure 2, inserts) reveal an uncompetitive character of the inhibition in which both the apparent K, and V,,, of the flux are decreased, a picture characteristic for complex systems in which the control point of a reaction sequence is downstream from the enzyme
for which the plot is made. Lowering of the amount of the active form of the enzyme by oleate is unlikely since, as shown in Table 5, a large diminution of the flux was accompanied by only a small decrease, if any, of the active form. Moreover, the amount of the active form was always in excess compared to the flux (Table 3). One of the possibilities is the inhibition of branched-chain a-ketoacid dehydrogenase by NADH (resulting from fatty acid oxidation), as was established for isolated hepatocytes2' and for the purified e n ~ y m e . ~ . ' ~ The effect of insulin on the decarboxylation rate as a function of a-ketoisocaproate concentration in hepatocytes from high ketotic rats is presented in Figure 2C. As shown in previous experiments (Tables 2, 3 and 4), insulin significantly enhanced the rate of decarboxylation at 2 mM substrate concentration but its stimulatory effect decreased at lower concentrations. At 0.1 mM a-ketoisocaproate there was practically no effect of insulin. DISCUSSION The present investigation deals with the relation between the activity state of the dehydrogenase complex and the actual flux through the dehydrogenase in hepatocytes isolated from starved and diabetic rats. As has been reported p r e v i ~ u s l y 'and ~ in the present paper, endogenous ketogenesis is inversely
19
BRANCHED-CHAIN KETOACIDS I N DIABETES
B
A
'I
'r
A
C 1
1
2
2
Branched-chain
a-ketoacid
concentration
(mM)
Figure 2. EKect of oleate and insulin on the oxidation of branched-chain a-ketoacids as a function of substrate concentration. A and B, hepatocytes isolated from starved rats oxidizing a-ketoisocaproate (A) and a-ketoisovalerate (B) in the absence ( 0 )or presence ( 0 )of 1 mM oleate. The kinetic parameters calculated from the Hanes plots (inserts) are: A, apparent K, 044 and 0.14 mM, V , , , 4.4 and 2.3 lcmol min-l g - ' dry weight in the absence and presence of oleate, respectively; B, apparent K, 0.33 and 0.07 mM, V,,, 6.0 and 3.5 pmol min-' g-' dry weight in the absence and presence of oleate, respectively. C, Hepatocytes from high ketotic diabetic rats oxidizing a-ketoisocdproate without (W) and with (0) insulin, 200 mU mi-'.
correlated with the rate of branched-chain a-ketoacid oxidation in hepatocytes from diabetic rats. Also experiments with hepatocytes from non-diabetic starved rats (reference 13 and this paper) confirm that the flux through branched-chain aketoacid dehydrogenase is diminished by concomitant oxidation of added fatty acids. The present results (Tables 3 and 5 ) show that the effect of fatty acids is not due to the interconversion (phosphorylation) of the dehydrogenase complex but is, most likely, exerted by a direct inhibition of the dehydrogenase by NADH and acyl-CoA esters and, possibly, of further steps of the degradation pathway of branched-chain a - k e t ~ a c i d s . ' ~This . ~ ' conclusion is supported by the present observation that the amount of the active form and the total activity of the dehydrogenase complex were only slightly, if at all, diminished in livers and hepatocytes from diabetic rats (Table 3) and also in hepatocytes from starved rats in the presence of 1 mM oleate (Table 5). The fact that the dehydrogenase complex was fully active in diabetic liver was also observed by A Gillim et aL6 but not by Patston et decrease by 2 mbi oleate of the flux through and the amount of the active form of the dehydrogenase in hepatocytes isolated from fed rats were described by Harris et a1.' ~
1
.
~
3
"
It has to be noted that fatty acids exert a dual effect on branched-chain a-ketoacid oxidation: (i) end-product inhibition, (ii) activation of the dehydrogenase complex due to the potent inhibitory effect of fatty acid oxidation products, mainly acetoacetyl-CoA, on branched-chain a-ketoacid dehydrogenase kinase.2s The latter effect is prevailing in tissues where the percentage of the active form of the dehydrogenase complex is low, e.g., in heart and skeletal muscle (for further literature see reference 28). On the other hand, in liver of fed, starved and diabetic rats, where the dehydrogenase is practically fully active, fatty acid oxidation results in a decrease of branched-chain a-ketoacid decarboxylation due to inhibition by NADH and/or acylCoA. The activatory effect of insulin on oxidative decarboxylation of branched-chain a-ketoacids in hepatocytes was correlated with the inhibition by insulin of ketogenesis derived from fatty acid oxidation. This short-term effect of insulin was therefore exerted predominantly on the flux rather than on the actual activity of the branched-chain a-ketoacid dehydrogenase complex since the latter was already practically fully active. This is in contrast to adipose tissue where insulin increases the activity of branched-chain dehydrogenase complex by stimu-
20
A. STERNICZUK E T A L .
lating its dephosphorylation.2' Similarly, insulin ACKNOWLEDGEMENTS strongly activates pyruvate dehydrogenase in hepatocytes from starved rats by promoting the inter- We wish to thank Barbara Burcan and Maria conversion of this enzyme complex to its Buszkowska for technical assistance. This work dephosphorylated form.30 Insulin was found to was supported by the Polish Academy of Sciences overcome the inhibitory effect of ketone bodies on under grant No. CPBP 04.01. the oxidation of a-ketoisovalerate and a-ketoisocaproate in perfused hindquarters of diabetic rats,31 but it has not been established whether this was due REFERENCES to a change of the activity state of the dehydrogen1. Shinnick, F. L. and Harper, A. E. (1976). Branched-chain ase complex. amino acid oxidation by isolated rat tissue preparations. Biochim. Biophys. Acta, 437,477-486. The collcentration of insulin which produced a 2. Harris, R. A., Paxton, R., Powell, S. M., Goodwin, G. W., significant effect on the Rux through branchedKuntz, M. J. and Han, A. C. (1986). Regulation of chain a-ketoacid dehydrogenase in our experiments branched-chain a-ketoacid dehydrogenase complex by cowas 10 times higher than that used in experiments valent modification. In: Advances in Enzyme Regulation, with hepatocytes from healthy fed animals.32 CulVol. 25, (Weber, G., ed,) Pergamon Press: Oxford, pp. 219-237. tured hepatocytes exhibited even much higher senR., Kuntz, M. J. and Harris, R. A. (1986). Phossitivity towards insulin and g l ~ c a g o n . ~ ~3. . ~Paxton, ~ phorylation sites and inactivation of branched-chain aHowever, in our perfusion experiments (Table 4) ketoacid dehydrogenase isolated from rat heart, bovine the insulin concentration was comparable to that kidney, and rabbit liver, kidney, heart, and skeletal muscle. Arch. Biochem. Biophys., 244,187-201. used by Menahan and Wie1and2l who observed a 4. Parker, P. J. and Randle, P. J. (1978). Branched-chain 2suppressing effect of this hormone on glucagon0x0-acid dehydrogenase complex of rat liver. FEBS Leti., stimulated ketogenesis in perfused liver of starved 90, 183-186. rat. The apparent resistance of diabetic hepatocytes 5. Parker, P. J. and Randle, P. J. (1978). Partial purification towards insulin may be partially artifactual since, and properties of branched-chain 2-0x0 acid dehydrogenase of ox liver. Biochem. J., 171, 751 -757. as we have shown, hepatocytes isolated from fatty 6. Gillim, S. E., Paxton, R., Cook, G. A. and Harris, R. A. liver were refractory towards the hormone (Table (1983). Activity state of the branched chain a-ketoacid 3) in contrast to the perfused liver (Table 4). The dehydrogenase complex in heart, liver, and kidney of poor response may therefore be due to the fragility normal, fasted, diabetic, and protein-starved rats. Biochem. Biophys. Res. Commun., 111, 74-81. of the receptors in diabetic hepatocytes as well as to 7. Patston, P. A., Espinal, J. and Randle, P. J. (1984). Effects the resistance of the diabetic liver, as has already of diet and of alloxan-diabetes on the activity of branchedbeen described.3 5 * chain 2-0x0 acid dehydrogenase complex and of activator The present study shows that the oxidation of protein in rat tissues. Biochem. J., 222,711-719. branched-chain a-ketoacids is strongly depressed 8. Harris, R. A., Paxton, R.,Goodwin, G. W. and Powell, S . M. (1986). Regulation of the branched-chain 2-0x0 acid by fatty acid oxidation at the maximal flux through dehydrogenase complex in hepatocytes isolated from rats the dehydrogenase but only slightly diminished at on a low-protein diet. Biochem. J., 234, 285-294. lower concentrations of these substrates. This is in 9. Block, K. P., Heywood, B. W., Buse, M. G. and Harper, agreement with previous observations of Corkey et A. E. (1985). Activation of rat liver branched-chain 2-0x0 acid dehydrogenase in uiuo by glucagon and adrenaline. aL2' and Harris et a1.' The activatory effect of Biochem. J., 232, 593-597. insulin on decarboxylation of branched-chain a10. Reed, L. J. (1981). Regulation of mammalian pyruvate ketoacids is much higher at the maximal flux dehydrogenase' complex by a phosphorylation-dephosthrough the enzyme than at lower concentrations phorylation cycle. Curr. Top. Cell. Regul., 18, 95-106. of the substrate (Figure 2C). Therefore, it can be 11. Patston, P. A., Espinal, J., Shaw, J. M. and Randle, P. J. (1986). Rat tissue concentrations of branched-chain 2-0x0 concluded that in diabetic liver the oxidation of acid dehydrogenase complex. Re-evaluation by immunoasboth fatty acids and branched-chain a-ketoacids is say and bioassy. Biochem. J., 235, 429--434. maintained at a high rate. The contribution of these 12. Paul, H. S. and Adibi, S. A. (1982). Role of ATP in the substrates to the energy demand depends on their regulation of branched-chain n-keto acid dehydrogenase activity in liver and muscle of fed, fasted, and diabetic rats. supply from peripheral tissues. Insulin administraJ. Biol. Chem., 257,4875-4881. tion in diabetes results in a decrease of fatty acid 13. Williamson, J. R., Watajtys-Rode, E. 1. and Coll, K. E. oxidation in favour of the utilization of branched(1979). Effects of branched chain a-ketoacids on the metachain a-ketoacids, as shown in this study, and of bolism of isolated rat liver. 1. Regulation of branched chain a-ketoacid metabolism. J. Bid. Chem., 254, 11511-1 1520. carbohydrates, as is well-established.
21
BRANCHED-CHAIN KETOACIDS IN DIABETES 14. Watajtys-Rode, E. I., Kietducka, A. and Lenartowicz, E. (1981). Oxidation of branched chain a-ketoacids in hepatocytes from alloxan-diabetic rats. Inhibition by long chain fatty acids. In: Metabolism and Clinical Implications of Branched-Chain Amino and Ketoacids, (Walser, M. and Williamson, J. R., eds.) Elsevier: New York, pp. 129-134. 15. Berry, M. N. and Friend, D. S. (1969). High-yield preparation of isolated rat liver parenchymal cells. A biochemical and fine structural study. J. Cell Biol., 43, 506-520. 16. Krebs, H. A. and Henseleit, K. (1932). Untersuchungen uber die Harnstoflbildung im Tierkorper. Hoppe-Seylers Z. Physiol. Chem., 210, 33-66. 17. Williamson, D. H. and Mellanby, J. (1974). ~-(->3-Hydroxybutyrate. In: Methods of Enzymatic Analysis, Vol. 4, 2nd English edn., (Bergmeyer, H. U., ed.) Academic Press: New York, London, pp. 1836-1839. 18. Mellanby, J. and Williamson, D. H. (1974). Acetoacetate. In: Merhoa3 of Enzymatic Analysis, Vol. 4, 2nd English edn., (Bergmeyer, H. U., ed.) Academic Press: New York, London, pp. 1840- 1843. 19. Rudiger, H. W., Langenbeck, U. andGoedde, H. W. (1972). A simplified method for the preparation of "C-labeled branched chain a-oxoacids. Biochem. J., 126,445-446. 20. Kaloyianni, M. and Freedland, R. A. (1990). Effect of diabetes and time after in uiuo insulin administration on ketogenesis and gluconeogenesis in isolated rat hepatocytes. Int. J. Biochem., 22, 59-164. 21. Menahan, L. A. and Wieland, 0. (1969). Interactions of glucagon and insulin on the metabolism of perfused livers from fasted rats. Eur. J. Biochem., 9, 55-62. 22. Harris, R. A., Powell, S. M., Paxton, R., Gillim, S. E. and Nagae, H. (1985). Physiological covalent regulation of rat liver branched-chain a-ketoacid dehydrogenase. Arch. Biochem. Biophys., 243, 542-555. 23. Solomon, M., Cook, K. G. and Yeaman, S. J. (1987). Effect of diet and starvation on the activity state of branchedchain 2-0x0-acid dehydrogenase complex in rat liver and heart. Biochim. Biophys. Acta, 931, 335-338. 24. Wagenmakers, A. J. M., Schepens, J. T. G. and Veerkamp, J. H. (1984). Erect of starvation and exercise on actual and total activity of the branched-chain 2-0x0 acid dehydrogenase complex in rat tissues. Biochem. J., 223,815-821. 25. Danner, D. J., Lemmon, S. K. and Elsas, L. J. (1978). Substrate specificity and stabilization by thiamine pyrophosphate of rat liver branched-chain a-keto acid dehydrogenase. Biochem. Med., 19,27-38.
26. Hutson, S. M. and Harper, A. E. (1981). Blood and tissue branched-chain amino and a-ketoacid concentrations: Effect of diet, starvation, and disease. Amer. J. Clin. Nutr., 34, 173-1 83. 27. Corkey, B. E., Martin-Requero, A., Walajtys-Rode, E., Williams, R. J. and Williamson, J. R. (1982). Regulation of the branched chain u-ketoacid pathway in liver. J. Biol. Chem., 257,9668-9676. 28. Paxton, R. and Harris, R. A. (1984). Regualtion of branched-chain a-ketoacid dehydrogenase kinase. Arch. Biochem. Biophys., 231, 48-57. 29. Frick, G. P. and Goodman, H. M. (1989). Insulin regulation of the activity and phosphorylation of branched-chain 2-0x0 acid dehydrogenase in adipose tissue. Biochem. J., 258,229-235. 30. Marchington, D. R., Kerbey, A. L., Jones, A. E. and Randle, P. J. (1987). Insulin reverses effects of starvation on the activity of pyruvate dehydrogenase kinase in cultured hepatocytes. Biochem. J., 246,233-236. 31. Zapalowski, C., Hutson, S. M. and Harper, A. E. (1981). Effects of starvation and diabetes on leucine and valine metabolism in the perfused rat hindquarter. In:Metabolism and Clinical Implications of Branched-Chain Amino and Ketoacids, (Walser, M. and Williamson, J. R., eds.) Elsevier: New York, pp. 239-244. 32. Vaartjes, W. J., de Haas, C. G. M. and van den Bergh, S. G. (1985). Differential short-term effects of growth factors on fatty acid synthesis in isolated rat-liver cells. Biochem. Biophys. Res. Commun., 131, 449-455. 33. Geelen, M. J. H. and Gibson, D. M. (1975). Lipgenesis in maintenance cultures of rat hepatocytes. .FEBS Lett., 58, 334-339. 34. Harano, Y., Kosugi, K., Kashiwagi, A., Nakano, T.. Hidaka, H, and Shigeta, Y. (1982). Regulatory mechanism of ketogenesis by glucagon and insulin in isolated cultured hepatocytes. J. Biochem., 91, 1739-1748. 35. Reaven, G. M. and Olefsky, J. M. (1978). Role of insulin resistance in the patogenesis of diabetes mellitus. Ado. Metab. Res., 9, 313-325. 36. Hussin, A. H. and Skett, P. (1988). Lack of effect of insulin in hepatocytes isolated from streptozotocin-diabetic male rats. Biochem. Pharmacol., 37, 1683- 1686. Receioed in reuisedforrn 9 July 1990 Accepted 28 August 1990
